UBC Theses and Dissertations

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UBC Theses and Dissertations

Studies on yeast soluble-ribonucleic acid Millward, Stewart 1967

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The U n i v e r s i t y of B r i t i s h Columbia FACULTY OF GRADUATE STUDIES PROGRAMME OF THE FINAL ORAL EXAMINATION FOR THE DEGREE OF DOCTOR OF PHILOSOPHY OF STEWART MILLWARD B.Sc. (Zoology/Biochemistry), U n i v e r s i t y of B r i t i s h Columbia, 1960 M.Sc. . (Organic Chemistry), U n i v e r s i t y of B r i t i s h Columbia, 1963 WEDNESDAY, JUNE 28, 1967 AT 10:00 A.M. IN ROOM 231, MEDICAL SCIENCES BLOCK A COMMITTEE IN CHARGE Chairman; B.N. Moyls G.G.S. Dutton M. Smith • C T . Beer GM. Tener P.D. Bragg S«H."Zbarsky E x t e r n a l Examiner: R.M. Bock Department of Biochemistry U n i v e r s i t y of Wisconsin Research Supervisor: G.M. Tener STUDIES ON YEAST SOLUBLE-RIBONUCLEIC ACID 1 ' ABSTRACT Part I of the thesis describes attempts to c o n t r o l the extent of hydrolysis of yeast s-RNA with pancreatic ribonuclease. The extent of hydrolysis was shown to be a function of temperature, concentration of magnesium ions and the s-RNA : ribonuclease r a t i o (w/w). Digestion of s-RNA with pancreatic ribonuclease ( r a t i o , 2000:1, w/w)) i n the presence of 0.2 M magnesium and at 0 , followed by chromatographic analysis, indicated that about 75% of the u l t r a v i o l e t absorbing material was of medium to high molecular weight. Analysis of the nucleotide composition of the f r a c t i o n containing the intermediate s i z e oligonucleotides showed that f r a c t i o n to be enriched i n the odd bases ,VjT, 1-methylG, N,N-dimethylG and several other u n i d e n t i f i e d nucleotides. This result.was incompatible with the 'hairpin' models for s-RNA, proposed by McCully and Cantoni (1962). The presence of spermine in the digestion mixture c l e a r l y masked the c a t a l y t i c action of pancreatic ribonuclease, a l b e i t to a lesser extent than magnesium ions,,contrary to a report by Thomas and Hiibst (1963)' i n their- study of the a f f e c t of spermine on the c a t a l y t i c a c t i v i t y of E. c o l i ribonuclease toward E. c o l i ribosomal RNA. Part II of the thesis i s concerned with the f r a c t i o n a t i o n of mixed s^RNA from yeast. Part II-A describes attempts to r e t a i n *"^ C-cysteinyl-s-RNA on an organomercurial-cellulose (Material I) derived from aminoethyl-cellulose. It appeared that .the s a l t concentration required to over-come the ion-exchange properties of M a t e r i a l I pre-cluded any "interaction between the mercury of Material I and the s u l f h y d r y l group of l^C-cysteinyl-s-RNA. This d i f f i c u l t y was overcome by preparing an organomercurial-c e l l u l o s e devoid of ion-exchange properties (Material II)„ Preliminary studies showed that Material II can r e t a i n the r a d i o a c t i v i t y associated'with -^C-cysteinyl-s-RNA. It i s suggested that M a t e r i a l II might f r a c t i o n a t e nucleic acids according to t h e i r base composition. Part II-B describes some of the attempts to fr a c t i o n a t e yeast s-RNA by column p a r t i t i o n chromatography. In general, r e s o l u t i o n of acceptor a c t i v i t i e s of s-RNA, as well as the recovery of b i o l o g i c a l a c t i v i t y , was poor. • P a r t l l G describes some of the author's c o n t r i -butions' to the studies c a r r i e d out in this laboratory on the f r a c t i o n a t i o n of yeast s-RNA on another new chromatographic material, benzoylated-DEAE-cellulose (Material I I I ) . Certain variables (such as, magnesium ion concentration and JDH), which can be manipulated during rechromatography of s-RNA on Material I I I , are discussed i n l i g h t of the chromatographic behaviour of glycine and other acceptor RNA's. The forces respon-s i b l e for th i s f r a c t i o n a t i o n are also discussed. :' Part XI-D describes a chemical procedure for the i s o l a t i o n of glycine s-RNA which, when combined with the chromatographic procedures described i n Part II-C, afforded g l y c i n e - s p e c i f i c s-RNAy 'in high p u r i t y . Observations on the e f f e c t of magnesium ion during enzymatic synthesis of glycyl-s-RNA are discussed. The advantages and possible consequences of using p u r i f i e d aminoacyl-s-RNA synthetase enzymes for the preparation of the aminoacyl-s-RNA 1s are also discussed. Part I I I describes, a simple procedure for the. - • preparation and p a r t i a l p u r i f i c a t i o n of quantities of yeast aminoacyl-s-RNA synthetase enzymes, suitable for charging gram quantities of s-RNA with a given.amino aci d . Two procedures are described for l y s i n g yeast c e l l s . When yeast c e l l s were lysed with toluene at 2>1°, most of the synthetase enzymes were preserved by c o n t r o l l i n g the of the toluene-yeast mixture. When yeast c e l l s were lysed i n toluene containing excess dry i c e , followed by an incubation period at 3-5°, a l l of the aminoacyl-s-RNA synthetase enzymes were' detected i n the c e l l - f r e e preparation.. P a r t i a l p u r i f i c a t i o n of the aminoacyl-s-RNA synthetases was achieved by chromatographing the c e l l - f r e e preparation on hydroxyapatite;at 3-5 and in the presence of 407. gl y c e r o l (v/v). • GRADUATE STUDIES F i e l d of Study: Biochemistry Structure and Function of Proteins Biochemistry of Nucleic Acids Biochemistry of Amino Acids and Proteins Biochemistry of Carbohydrates Biochemistry of L i p i d s Biochemistry of Steroids and Hormones THESES AND PUBLICATIONS I-C »• Gillam, S. Millward, D. Blew, M. von Tigerstrom, E. •Wimmer, and G.M„ Tener Separation of t-RNA on Benzoylated-DEAE-cellulose, (1967), manuscript submitted for p u b l i c a t i o n . G.M. Tener, I.C. Gillam, M. von Tigerstrom, S. Millward and E. Wimmer, Separation of t-RNA on Benzoylated DEAE-Cellulose, Fed. Proc. 25, 519 (1966). A. Rosenthal and S. Millward, Reaction of Benzal-dehyde Semicarbazone and Azine with Carbon Monoxide, Can. J. Chem. 42, 956 (1964) W.J. Polglase G.M. Tener J.F. Richards S.H. Zbarsky W.J. Polglase G.I. Drummond G.M. Tener J.J.R. Campbell P.D. Bragg G.I. Drummond C T . Beer J.F. Richards P.H. J e l l i n c k V . J i . O'Donnell •P.H. J e l l i n c k G.H. Dixon S. Millward, Direct Carbonylation of Aromatic Semicarbazone and Azines, M 0 S c Thesis, U.B.C. 1963 A. Rosenthal and S. Millward, Direct Carbonylation of Benzophenone Semicarbazone and Benzophenone Azine using Dicobalt Octacarbonyl, Can. J. Chem. 41 2504 (1963). A. Rosenthal, M. Weir, and S. Millward, Reactions of Aromatic Phenylhydrazones and Semicarbazones with Carbon Monoxide, A.C.S. Abstract of Papers presented at Chicago, I l l i n o i s , Sept. 1961, p..113Q S T U D I E S O N Y E A S T S O L U B L E - R I B O N U C L E I C A C I D b y > S T E W A R T M I L L W A R D B . S c . ( Z o o l o g y / B i o c h e m i s t r y ) , U n i v e r s i t y of B r i t i s h C o l u m b i a , I960 M . S c . ( O r g a n i c C h e m i s t r y ) , U n i v e r s i t y of B r i t i s h C o l u m b i a , 1963 A T H E S I S S U B M I T T E D I N P A R T I A L F U L F I L M E N T O F T H E R E Q U I R E M E N T S F O R T H E D E G R E E O F D O C T O R O F P H I L O S O P H Y i n the D e p a r t m e n t of B i o c h e m i s t r y . W e a c c e p t t h i s t h e s i s as c o n f o r m i n g to the r e q u i r e d s t a n d a r d T H E U N I V E R S I T Y O F B R I T I S H C O L U M B I A J u n e , 1967 In p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t o f t h e r e q u i r e m e n t s f o r an a d v a n c e d d e g r e e a t t h e U n i v e r s i t y o f B r i t i s h C o l u m b i a , I a g r e e t h a t t h e L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e a n d s t u d y . I f u r t h e r a g r e e t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y p u r p o s e s may be g r a n t e d by t h e Head o f my D e p a r t m e n t o r by h i s r e p r e s e n t a t i v e s . I t i s u n d e r s t o o d t h a t c o p y i n g o r p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l n o t be a l l o w e d w i t h o u t my w r i t t e n p e r m i s s i o n . D e p a r t m e n t o f BIOCHEMISTRY The U n i v e r s i t y o f B r i t i s h C o l u m b i a V a n c o u v e r 8, C a n a d a D a t e June 28th, 1967 A B S T R A C T P a r t I of t h i s t h e s i s d e s c r i b e s the d i g e s t i o n of b a k e r s ' y e a s t s - R N A at 0° a n d at r o o m t e m p e r a t u r e i n the p r e s e n c e of v a r i o u s c o n c e n t r a t i o n s of m a g n e s i u m i o n s a n d at s e v e r a l d i f f e r e n t s - R N A : e n z y m e r a t i o s ( w / w ) . D i g e s t i o n of s - R N A w i t h p a n c r e a t i c r i b o n u c l e a s e ( r a t i o , 2000:1, (w/w) ) i n the p r e s e n c e of 0. 2 M m a g n e s i u m a n d at 0 ° , f o l l o w e d by c h r o m a t o g r -r a p h i c a n a l y s i s , i n d i c a t e d that about 75% of the u l t r a v i o l e t a b s o r b i n g m a t e r i a l w a s of m e d i u m to h i g h m o l e c u l a r w e i g h t . A n a l y s i s of the n u c l e o t i d e c o m p o s i t i o n of the f r a c t i o n c o n t a i n i n g the i n t e r m e d i a t e s i z e o l i g o n u c l e o t i d e s s h o w e d that f r a c t i o n to be e n r i c h e d i n the odd b a s e s 4^  , T , 1 - m e t h y l G , . N , N - d i m e t h y l G a n d s e v e r a l o t h e r u n i d e n t i f i e d n u c l e o t i d e s . T h i s r e s u l t w a s i n c o m p a t i b l e w i t h the ' h a i r p i n ' m o d e l s f o r s - R N A , p r o p o s e d by M c C u l l y a n d C a n t o n i (1962). T h e p r e s e n c e of s p e r m i n e i n the d i g e s t i o n m i x t u r e c l e a r l y m a s k e d the c a t a l y t i c a c t i o n of p a n c r e a t i c r i b o n u c l e a s e , , a l b e i t to a l e s s e r ex tent t h a n m a g n e s i u m i o n s , c o n t r a r y to a r e p o r t by T h o m a s and H u b s t (1963) i n t h e i r s tudy of the a f fect of s p e r m i n e on the c a t a l y t i c a c t i v i t y of E , c o l i r i b o n u c l e a s e t o w a r d E . c o l i r i b o s o m a l R N A . T h e s t u d i e s d e s c r i b e d i n P a r t II o f t h i s t h e s i s a r e c o n c e r n e d w i t h the f r a c t i o n a t i o n of m i x e d s - R N A f r o m y e a s t . - i i -P a r t II-A d e s c r i b e s a new chromatographic m a t e r i a l c o n s i s t i n g of a bifunctional m e r c u r i - d i o x a n e d e r i v a t i v e attached through.a t h i o l group to the c e l l u l o s e m a t r i x . A previous r e p o r t ( E l d j a r n and Je l l u m , 1963) had shown that HS-proteins could be fr a c t i o n a t e d on an o r g a n o m e r c u r i a l -c e l l u l o s e ( M a t e r i a l I) d e r i v e d f r o m a m i n o e t h y l - c e l l u l o s e . The studies reported, here suggest that ^ C - c y s t e i n y l - s - R N A cannot be retai n e d on M a t e r i a l I because the salt concentration r e q u i r e d to overcome its ion-exchange p r o p e r t i e s preclude any i n t e r a c t i o n between the m e r c u r y of M a t e r i a l I and the s u l f h y d r y l group of ^ C - c y s t e i n y l - s - R N A . The present study d e s c r i b e s how this disadvantage was overcome by p r e p a r i n g an o r g a n o m e r c u r i a l - c e l l u l o s e devoid of ion-exchange p r o p e r t i e s ( M a t e r i a l II). P r e l i m i n a r y , studies showed that M a t e r i a l II can r e t a i n the r a d i o -a c t i v i t y a s s o c i a t e d with ^ C - c y s t e i n y l - s - R N A . These studies suggested that M a t e r i a l II might fractionate n u c l e i c acids a c c o r d i n g to their base composition. P a r t II-B d e s c r i b e s some of the attempts to fractionate yeast s-RNA by column p a r t i t i o n chromatography. In general, r e s o l u t i o n of acceptor a c t i v i t i e s of s-RNA, as w e l l as the r e c o v e r y of b i o l o g i c a l activity, was poor. P a r t II-C d e s c r i b e s some of the author's contributions to the studies c a r r i e d out i n this l a b o r a t o r y on the f r a c t i o n a t i o n of yeast s-RNA on another new chromatographic m a t e r i a l , b e n z o y l a t e d - D E A E - c e l l u l o s e — i i i — ( M a t e r i a l III). C e r t a i n v a r i a b l e s ( s u c h as, m a g n e s i u m i o n c o n c e n t r a t i o n and J D H ) , w h i c h c a n be m a n i p u l a t e d d u r i n g r e c h r o m a t o g r a p h y of s-RNA on M a t e r i a l III, a r e d i s c u s s e d i n l i g h t of the c h r o m a t o g r a p h i c b e h a v i o u r of g l y c i n e and other a c c e p t o r RNA's. The f o r c e s r e s p o n s i b l e f o r t h i s f r a c t i o n a t i o n a r e a l s o d i s c u s s e d . P a r t II-D d e s c r i b e s a c h e m i c a l p r o c e d u r e f o r the i s o l a t i o n of g l y c i n e s-RNA w h i c h , when c o m b i n e d w i t h the c h r o m a t o g r a p h i c p r o c e d u r e s d e s c r i b e d i n P a r t I I - C , a f f o r d e d g l y c i n e - s p e c i f i c s-RNA, i n h i g h p u r i t y . O b s e r v a t i o n s on the e f f e c t of m a g n e s i u m i o n d u r i n g e n z y m a t i c s y n t h e s i s of g l y c y l - s - R N A a r e d i s c u s s e d . The advantages and p o s s i b l e con-sequences of u s i n g p u r i f i e d a m i n o a c y l - s - R N A synthetase e n z y m e s f o r the p r e p a r a t i o n of the a m i n o a c y l - s - R N A ' s a r e a l s o d i s c u s s e d . The need to p r e p a r e l a r g e q u a n t i t i e s of a m i n o a c y l - s - R N A synthetases d e r i v e s f r o m t h e i r i n d i s p e n s a b l e r o l e i n the development of c h e m i c a l methods f o r p u r i f y i n g a m i n o a c i d - s p e c i f i c s-RNA's (see P a r t II-D). G r i n d i n g of y e a s t c e l l s w i t h g l a s s beads, w h i c h i s the u s u a l m ethod e m p l o y e d f o r d i s r u p t i n g y e a s t c e l l s , was found to be inadequate. L y s i s of y e a s t c e l l s w i t h toluene at 37° was found to be a p a r t i a l s o l u t i o n to t h i s p r o b l e m . A l t h o u g h p r e v i o u s s t u d i e s showed that m o s t of the synthetase e n z y m e s w e r e d e s t r o y e d by. t h i s t r e a t m e n t (von T i g e r s t r o m - i v -and T e n e r , 1967), i n v e s t i g a t i o n s d e s c r i b e d i n P a r t III of t h i s t h e s i s , v. show that m o s t of the synthetase a c t i v i t i e s can be p r e s e r v e d by c o n t r o l l i n g the pH of the t o l u e n e - y e a s t m i x t u r e . However, t h e r e w e r e s t i l l s e v e r a l synthetase a c t i v i t i e s m i s s i n g i n the c e l l - f r e e e x t r a c t s p r e p a r e d by the w a r m toluene method. P r e p a r a t i o n of l a r g e q u a n t i t i e s of y e a s t c e l l - f r e e e x t r a c t c o n t a i n i n g a l l of the a m i n o a c y l - s - R N A synthetases was f i n a l l y a c h i e v e d by t r e a t m e n t of the y e a s t c e l l s w i t h toluene c o n t a i n i n g e x c e s s d r y i c e o f o l l o w e d by an i n c u b a t i o n p e r i o d at 3-5 . P a r t i a l p u r i f i c a t i o n of the a m i n o a c y l - s - R N A s y n t h e t a s e s was a c h i e v e d by c h r o m a t o g r aphing the c e l l - f r e e p r e p a r a t i o n on h y d r o x y -ap a t i t e . The p r e s e n c e of 4 0 % g l y c e r o l i n the e l u t i n g . b u f f e r s was found to be e s s e n t i a l f o r the p r e s e r v a t i o n of m o s t of the synthetase a c t i v i t i e s . When the c e l l - f r e e p r e p a r a t i o n s w e r e c h r o m a t o g r a p h e d on h y d r o x y -ap a t i t e , m o s t of the p r o t e i n , but only a few of the s y n t h e t a s e s w e r e el u t e d by a l i n e a r g r a d i e n t of phosphate b u f f e r to 0. 20 M. M o s t of the synthetases w e r e eluted only when the c o l u m n was washed w i t h a m m o n i u m s u l f a t e . The s i g n i f i c a n c e of t h i s o b s e r v a t i o n i s d i s c u s s e d . I N D E X P A G E N U M B E R P A R T I . D E G R A D A T I O N O F Y E A S T S O L U B L E - R I B O -N U C L E I C A C I D W I T H P A N C R E A T I C R I B O N U C L E A S E 1 T a b l e of C o n t e n t s 2 L i s t of F i g u r e s 4 L i s t of T a b l e s 6 I n t r o d u c t i o n 7 E x p e r i m e n t a l 11 D i s c u s s i o n 36 B i b l i o g r a p h y 52 P A R T I I . F R A C T I O N A T I O N O F Y E A S T s - R N A 55 T a b l e of C o n t e n t s 56 L i s t of F i g u r e s 61 L i s t of C h a r t s 63 I n t r o d u c t i o n 64 E x p e r i m e n t a l 76 A . A t t e m p t s to I s o l a t e C y s t e i n y l - s - R N A by C h r o m a t o g r a p h y on M e r c u r i -C e l l u l o s e 7 8 B . P a r t i t i o n C h r o m a t o g r a p h y of s - R N A on B i o g e l 97 C . F r a c t i o n a t i o n of s - R N A on B e n z o y l a t e d -D E A E - C e l l u l o s e 103 - v i -P A G E N U M B E R D . A C o m b i n e d C h e m i c a l a n d C h r o m a t o g r a p h i c M e t h o d f o r the I s o l a t i o n o f A m i n o A c i d - S p e c i f i c s - R N A ' s 136 D i s c u s s i o n 154 B i b l i o g r a p h y 190 P A R T III . P R E P A R A T I O N A N D P A R T I A L P U R I F I C A T I O N O F A M I N O A C Y L - s - R N A S Y N T H E T A S E S F R O M B A K E R S ' Y E A S T 195 T a b l e of C o n t e n t s 196 L i s t of F i g u r e s 199 L i s t of T a b l e s a n d C h a r t s 200 I n t r o d u c t i o n 201 E x p e r i m e n t a l 205 D i s c u s s i o n 238 B i b l i o g r a p h y 254 - v i i -A C K N O W L E D G E M E N T S I w o u l d l i k e to e x p r e s s m y t h a n k s a n d a p p r e c i a t i o n to D r . G . M . T e n e r f o r h i s a d v i c e and e n c o u r a g e m e n t t h r o u g h o u t the c o u r s e of t h i s s t u d y . I w o u l d a l s o l i k e to t h a n k M r s . D . B l e w f o r h e r t e c h n i c a l a s s i s t a n c e w i t h the s t u d i e s d e s c r i b e d i n P a r t III, a n d D r . C . T . B e e r f o r h i s a d v i c e d u r i n g the p r e p a r a t i o n of the m a n u s c r i p t . S p e c i a l t h a n k s a r e due I . A . M . w h o spent m a n y , h o u r s t y p i n g a n d p r o o f - r e a d i n g the m a n u s c r i p t . T h e s t u d i e s d e s c r i b e d i n t h i s t h e s i s w e r e s u p p o r t e d f i n a n c i a l l y by the N a t i o n a l R e s e a r c h C o u n c i l (1964-65), i n the f o r m of a S t u d e n t s h i p to the a u t h o r a n d by the M e d i c a l R e s e a r c h C o u n c i l , i n the f o r m of a r e s e a r c h g r a n t to D r . G . M . T e n e r . P A R T I D E G R A D A T I O N O F Y E A S T S O L U B L E - R I B O N U C L E I C A C I D W I T H P A N C R E A T I C R I B O N U C L E A S E - 2 -T A B L E O F C O N T E N T S P A G E N U M B E R L I S T O F F I G U R E S 4 L I S T O F T A B L E S 6 I N T R O D U C T I O N 7 E X P E R I M E N T A L 11 A b b r e v i a t i o n s U s e d 11 M a t e r i a l s 11 M e t h o d s 12 1. P r e p a r a t i o n of the D E A E - C e l l u l o s e C o l u m n 12 2. P u r i f i c a t i o n of C r u d e s - R N A 13 3. D i g e s t i o n of Y e a s t s - R N A b y R i b o n u c l e a s e at V a r i o u s C o n c e n t r a t i o n s of M a g n e s i u m Ion 15 4 . Q u a n t i t a t i v e P r e c i p i t a t i o n . o f s - R N A 15 5. R e l e a s e of A c i d - S o l u b l e N u c l e o t i d e s D u r i n g R i b o n u c l e a s e D i g e s t i o n at R o o m T e m p e r a t u r e 17 6. E f f e c t of P r o n a s e o n the D i g e s t i o n of s - R N A by P a n c r e a t i c R i b o n u c l e a s e 18 7. R e m o v a l of P a n c r e a t i c R i b o n u c l e a s e w i t h C M -C e l l u l o s e 20 A . B e h a v i o u r of P a n c r e a t i c R i b o n u c l e a s e on C M - C e l l u l o s e 20 B. R e m o v a l of R i b o n u c l e a s e f r o m a D i g e s t i o n M i x t u r e 20 - 3 -P A G E N U M B E R 8. R e l e a s e of A c i d - S o l u b l e N u c l e o t i d e s D u r i n g R i b o n u c l e a s e D i g e s t i o n at 0 ° i n the P r e s e n c e of M a g n e s i u m Ions 21 9. R e l e a s e of A c i d - S o l u b l e N u c l e o t i d e s D u r i n g R i b o n u c l e a s e D i g e s t i o n at R o o m T e m p e r a t u r e i n the P r e s e n c e of S p e r m i n e 22 10. C o n t r o l l e d , L a r g e S c a l e D i g e s t i o n of s - R N A w i t h R i b o n u c l e a s e at R o o m T e m p e r a t u r e 22 11. L a r g e S c a l e D i g e s t i o n of s - R N A w i t h R i b o -n u c l e a s e at 0° i n the P r e s e n c e of 0. 2 M M g C ^ 26 12. B a s e A n a l y s i s of F r a c t i o n A 28 13 . D i g e s t i o n of M i x e d s - R N A a n d P a r t i a l l y P u r i f i e d G l y c i n e s - R N A w i t h P a n c r e a t i c R i b o n u c l e a s e 31 A . G-100 S e p h a d e x C h r o m a t o g r a p h y 31 B . D i g e s t i o n w i t h P a n c r e a t i c R i b o n u c l e a s e 34 D I S C U S S I O N 36 B I B L I O G R A P H Y 52 - 4 -L I S T O F F I G U R E S F I G U R E P A G E N U M B E R D E A E - c e l l u l o s e c h r o m a t o g r a p h y of s - R N A f r o m b a k e r s ' y e a s t 14 I n c r e a s e i n o p t i c a l d e n s i t y at 260 rry/L d u r i n g d i g e s t i o n of s - R N A w i t h r i b o n u c l e a s e at r o o m t e m p e r a t u r e i n the p r e s e n c e a n d a b s e n c e of M g . s - R N A : r i b o n u c l e a s e r a t i o , 2. 3:1 (w/w) 16 R e l e a s e of a c i d - s o l u b l e u l t r a v i o l e t a b s o r b i n g m a t e r i a l d u r i n g d i g e s t i o n of s - R N A w i t h p a n c r e a t i c r i b o n u c l e a s e at r o o m t e m p e r a t u r e i n the p r e s e n c e a n d a b s e n c e of M g . s - R N A : r i b o n u c l e a s e r a t i o , 16:1 (w/w) 19 R e l e a s e of a c i d - s o l u b l e u l t r a v i o l e t a b s o r b i n g m a t e r i a l d u r i n g d i g e s t i o n of s - R N A w i t h p a n c r e a t i c r i b o n u c l e a s e at 0° i n the p r e s e n c e a n d a b s e n c e of M g . s - R N A : r i b o n u c l e a s e r a t i o , 50:1 (w/w) , 23 R e l e a s e of a c i d - s o l u b l e u l t r a v i o l e t a b s o r b i n g m a t e r i a l d u r i n g d i g e s t i o n of s - R N A w i t h r i b o n c u l e a s e at 0° i n the p r e s e n c e a n d a b s e n c e of M g . s - R N A : r i b o n u c l e a s e r a t i o , 500:1 (w/w) 24 I n c r e a s e i n a c i d - s o l u b l e u l t r a v i o l e t a b s o r b i n g m a t e r i a l d u r i n g d i g e s t i o n . o f s - R N A w i t h p a n c r e a t i c r i b o n u c l e a s e i n the p r e s e n c e a n d a b s e n c e of s p e r m i n e . s - R N A : r i b o n u c l e a s e r a t i o , 500:1 (w/w) 25 D E A E - c e l l u l o s e c h r o m a t o g r a p h y of s - R N A a f t e r d i g e s t i o n w i t h r i b o n u c l e a s e at r o o m t e m p e r a t u r e i n the p r e s e n c e of 0. 2 M M g C l ^ -s - R N A : r i b o n u c l e a s e r a t i o , 500:1 (w/w) 27 - 5 -F I G U R E 8 9. i o : 11. 12 P A G E N U M B E R D E A E - c e l l u l o s e c h r o m a t o g r a p h y of s - R N A a f t e r d i g e s t i o n w i t h r i b o n u c l e a s e at 0° i n the p r e s e n c e of 0. 2 M M g C l 2 « s - R N A : r i b o n c u l e a s e r a t i o , 2000:1 (w/w) 29 R e c h r o m a t o g r a p h y of F r a c t i o n II ( F i g . 8) on D E A E - c e l l u l o s e 30 A . C h r o m a t o g r a p h y of a n a l k a l i n e d i g e s t of F r a c t i o n A ( F i g . 9) on D E A E - c e l l u l o s e . B . C o m p o s i t e d i a g r a m of c h r o m a t o g r a m s o b t a i n e d a f t e r p a p e r c h r o m a t o g r a p h y of F r a c t i o n s A to I i n F i g u r e 1 0 - A . 32 G-100 S e p h a d e x c h r o m a t o g r a p h y of: A , m i x e d s - R N A ; B , p a r t i a l l y p u r i f i e d g l y c i n e s - R N A ; C , r e c h r o m a t o g r a p h y of p e a k II f r o m F i g u r e 11-B. 33 D i g e s t i o n of m i x e d s - R N A ( -) a n d p a r t i a l l y p u r i f i e d g l y c i n e s - R N A ( ) i n the p r e s e n c e a n d a b s e n c e of 0 . 2 M M g C l 2 35 -6-L I S T O F T A B L E S T A B L E P A G E NUMBER Comparison of the Extent of Hydrolysis of 42 s-RNA with Pancreatic Ribonuclease under Various Conditions II Base Composition of Fraction A 46 - 7 -• I N T R O D . U C T I O N E v i d e n c e that the s - R N A f r a c t i o n s o b t a i n e d f r o m o r g a n i s m s i s c o m p o s e d of a p o p u l a t i o n , o f p o l y n u c l e o t i d e c h a i n s , e a c h s p e c i f i c f o r a p a r t i c u l a r a m i n o a c i d , o b t a i n s f r o m a v a r i e t y of b i o l o g i c a l e x p e r i m e n t s ( C o h e n a n d G r o s , I960; B e r g , 1961; S i m p s o n , 1962) as w e l l as the p h y s i c a l s e p a r a t i o n of s - R N A p r e p a r a t i o n s w i t h a c c e p t o r s p e c i f i c i t y t o w a r d p a r t i c u l a r a m i n o a c i d s ( T a n a k a , 1966). A t t e m p t s to e l u c i d a t e the m o l e c u l a r b a s i s of the u n i q u e f u n c t i o n and s p e c i f i c i t y of s - R N A r e q u i r e a k n o w l e d g e of the c o m p l e t e n u c l e o t i d e s e q u e n c e of the s p e c i f i c s - R N A m o l e c u l e s . U n t i l r e c e n t l y , (1965), the o n l y a p p r o a c h to d e t e r m i n i n g n u c l e o t i d e s e q u e n c e s of l a r g e o l i g o r i b o -n u c l e o t i d e s was. to s p l i t t h e m w i t h e n z y m e s i n t o f r a g m e n t s w h i c h a r e a m e n a b l e to s e q u e n c e a n a l y s i s . O n l y two e n d o n u c l e a s e s of w e l l d e f i n e d s p e c i f i c i t y a r e a v a i l a b l e f o r t h i s p u r p o s e . One of t h e s e , p a n c r e a t i c r i b o n u c l e a s e , s p l i t s p o l y r i b o n u c l e o t i d e s at the p y r i m i d i n e - 3 ' e s t e r l i n k a g e g i v i n g r i s e to b l o c k s of p u r i n e s t e r m i n a t e d i n a p y r i m i d i n e - 3 ' p h o s p h a t e ( u r i d i n e - or c y t i d i n e - 3 ' p h o s p h a t e ) . H o w e v e r , b e c a u s e m i x e d s - R N A c o n t a i n s 4 7 % u r i d i n e p l u s c y t i d i n e ( B e l l et a l . , (1964), c o m p l e t e d i g e s t i o n of s - R N A w i t h p a n c r e a t i c r i b o n u c l e a s e g i v e s a l a r g e n u m b e r of s m a l l f r a g m e n t s ( I n g r a m a n d P i e r c e , 1962). T h e w o r k r e p o r t e d i n t h i s p a r t of the t h e s i s w a s c a r r i e d out i n 1963 to d e t e r m i n e i f the a c t i o n of p a n c r e a t i c r i b o --8-nuclease could be r e s t r i c t e d to a s m a l l number of s p e c i f i c sites, y i e l d i n g large, well-defined.oligonucleotide fragments. P r e v i o u s work ( P e n n i s t o n a n d Doty, 1963; N i s h i m u r a and N o v e l l i , 1963; Zubay and M a r c i e l l o , 1963) established that m a g n e s i u m ions s t a b i l i z e d the secondary s t r u c t u r e of s-RNA. These w o r k e r s a l s o showed that m a g n e s i u m ions inhibit the degradation of s-RNA by a number of ribonueleases and.phosphodiesterases. F r e s c o et al . , (I960) attempted to d e s c r i b e the s t r u c t u r e of s-RNA in t e r m s of a base sequence model. These authors a ssumed a random base sequence and i n order to r e c o n c i l e the r e l a t i v e l y high double-h e l i c a l content of s-RNA with a random base sequence they proposed a "looping-out" of non-paired bases between double-stranded regions. A l e s s d e t a i l e d model for s-RNA, co n s i s t i n g of a chain folding back upon itsel f , was proposed by Brown and Zubay (I960), to explain X - r a y d i f f r a c t i o n patterns and the effect of r a p i d cooling on the sedimentation c h a r a c t e r i s t i c s of s-RNA. The latte r model was further r e f i n e d by M c C u l l y and Cantoni (1962) and r e c e i v e d the most favour until the complete sequence of a l a n i n e - s p e c i f i c s-RNA was r e p o r t e d by Ho l l e y e t a l . (1965). The additional or odd bases were confined, i n both models, to the loop regions. It would follow f r o m the foregoing d i s c u s s i o n that digestion of s-RNA by pa n c r e a t i c r i b o n u c l e a s e i n the p r esence of a - 9 -c o n c e n t r a t i o n , o f m a g n e s i u m i o n s s u f f i c i e n t to s t a b i l i z e the b a s e - p a i r e d , d o u b l e - h e l i c a l r e g i o n s s h o u l d g i v e r i s e to s m a l l f r a g m e n t s e n r i c h e d i n the odd b a s e s a n d l a r g e o l i g o n u c l e o t i d e f r a g m e n t s , c o n s i s t i n g of the d o u b l e - h e l i c a l r e g i o n s , w i t h a r e d u c e d content of odd b a s e s . The i n i t i a l s tudy r e p o r t e d h e r e w a s d e s i g n e d to t e s t t h i s h y p o t h e s i s . In a d d i t i o n t o m a g n e s i u m , the e f fect of o t h e r m u l t i v a l e n t c a t i o n s on n u c l e i c a c i d s h a s b e e n , s t u d i e d . T h e a l i p h a t i c p o l y a m i n e s h a v e b e e n k n o w n f o r m a n y y e a r s ( A c k e r m a n , 1952, 1955) as n o r m a l b i o l o g i c a l c o n s t i t u e n t s a l t h o u g h t h e i r s p e c i f i c f u n c t i o n i s s t i l l u n k n o w n . N u m e r o u s s t u d i e s have be en p u b l i s h e d on. t h e i r b i n d i n g to n u c l e i c a c i d s ( T a b o r et a l . , 1961). F e l s e n f e l d a n d H u a n g (I960) s h o w e d that t h e r e w a s a s t r o n g i n t e r a c t i o n b e t w e e n s i n g l e s t r a n d e d p o l y u r i d y l i c a c i d and p o l y a m i n e s ; s p e r m i n e h a v i n g a p a r t i c u l a r l y h i g h a f f i n i t y . T h e s e w o r k e r s a l s o d e m o n s t r a t e d a h y p o c h r o m i c ef fect w h e n the d i a m i n e s , 1, 4 - d i a m i n o -butane, 1, 5 - d i a m i n o p e n t a n e o r 1, 10 - d i a m i n o d e c a n e w e r e a d d e d to a s o l u t i o n of p o l y a d e n y l i c . a n d p o l y u r i d y l i c a c i d . A l l the d i a m i n e s w e r e e q u a l l y e f f e c t i v e a n d o n l y s l i g h t l y l e s s e f f e c t i v e t h a n m a g n e s i u m . C a n t o n i (I960) d e m o n s t r a t e d that a p a r t i a l f r a c t i o n a t i o n of s - R N A c o u l d be o b t a i n e d by. d i f f e r e n t i a l p r e c i p i t a t i o n w i t h s p e r m i n e u n d e r c a r e f u l l y c o n t r o l l e d c o n d i t i o n s of p H a n d i o n i c s t r e n g t h . It w a s s h o w n b y Z i l l i g etjal_. (1959), that p o l y a m i n e s p r e s e n t i n E . c o l i r i b o s o m e s c o m p e t i t i v e l y i n h i b i t e d a l a t e n t r i b o n u c l e a s e a l s o p r e s e n t i n the r i b o s o m e s , a l t h o u g h T h o m a s a n d H u b s t (1963) s h o w e d that t h e s e p o l y a m i n e s m a i n t a i n e d the - 1 0 -i n t e g r i t y of the r i b o s o m e s but w e r e u n a b l e to p r o t e c t the R N A f r o m d e g r a d a t i o n by the p u r i f i e d r i b o n u c l e a s e . T h e s tudy r e p o r t e d h e r e d e m o n s t r a t e s that s p e r m i n e c a n i n h i b i t the a c t i o n of p a n c r e a t i c r i b o n u c l e a s e o n s - R N A . -11-E X P E R I M E N T A L A B B R E V I A T I O N S U S E D s - R N A : s o l u b l e r i b o n u c l e i c a c i d a n d i s e q u i v a l e n t to t r a n s f e r o r a d a p t e r r i b o n u c l e i c a c i d . D E A E - c e l l u l o s e : d i e t h y l a m i n o e t h y l c e l l u l o s e . C M - c e l l u l o s e : c a r b o x y m e t h y l c e l l u l o s e . • O . D . : o p t i c a l d e n s i t y . T h e f o l l o w i n g a b b r e v i a t i o n s w e r e u s e d f o r the n u c l e o s i d e - 2 ' ( 3 ' ) p h o s p h a t e s a n d t h e i r d e r i v a t i v e s : A : a d e n o s i n e - 2 ' ( 3 ' ) p h o s p h a t e , C : c y t i d i n e - 2 ' ( 3 1 ) p h o s p h a t e , G : g u a n o s i n e - 2 ! (3') phosphate ' , U : u r i d i n e - 2 ' ( 3 ' ) p h o s p h a t e , ^P: p s e u d o u r i d i n e - 2 ' ( 3 ') p h o s p h a t e , T : t h y m i n e r i b o s i d e - 2 ' ( 3 ' ) p h o s p h a t e . M A T E R I A L S T h e s - R N A u s e d t h r o u g h o u t t h i s s tudy w a s p r e p a r e d f r o m f r e s h l y g r o w n b a k e r s ' y e a s t o b t a i n e d f r o m S t a n d a r d B r a n d s L i m i t e d . D E A E - c e l l u l o s e a n d C M - c e l l u l o s e w e r e p u r c h a s e d f r o m B r o w n C o . , B e r l i n , N . H . -12-P a n c r e a t i c r i b o n u c l e a s e w a s o b t a i n e d f r o m W o r t h i n g t o n B i o -c h e m i c a l C o r p . , F r e e h o l d , N . J . • T r i s ( c h l o r i d e ) b u f f e r w a s p r e p a r e d by t i t r a t i n g a s o l u t i o n of T r i s ( h y d r o x y m e t h y l ) a m i n o m e t h a n e to the d e s i r e d p_H w i t h h y d r o c h l o r i c a c i d . P r o n a s e ( R e s e a r c h G r a d e ) w a s p u r c h a s e d f r o m the C a l i f o r n i a C o r p o r a t i o n f o r B i o c h e m i c a l R e s e a r c h . S p e r m i n e w a s p u r c h a s e d f r o m A l d r i c h C h e m i c a l C o . I n c . , M i l w a u k e e . O p t i c a l d e n s i t y m e a s u r e m e n t s w e r e m a d e u s i n g a C a r y M o d e l 11 s p e c t r o p h o t o m e t e r . O n e o p t i c a l d e n s i t y (O. D . ) u n i t i s d e f i n e d as that a m o u n t of m a t e r i a l p e r m l w h i c h i n a 1 - c m l i g h t p a t h , at 260 mp. g i v e s a s p e c t r o p h o t o m e t r i c r e a d i n g of one. M E T H O D S 1. P r e p a r a t i o n of the D E A E - C e l l u l o s e C o l u m n D E A E - c e l l u l o s e w a s f r e e d . o f f i n e s by r e p e a t e d s e t t l i n g a n d d e c a n t a t i o n f r o m a l a r g e v o l u m e of w a t e r . T h e s e t t l e d m a t e r i a l w a s m a d e i n t o a t h i n s l u r r y by the a d d i t i o n of 2 M N a C l a n d p o u r e d i n t o a c h r o m a t o g r a p h i c c o l u m n (90 x 3. 5 c m d i a m . . ) , h a l f f i l l e d w i t h 2 M N a C l . A i r p r e s s u r e of about 3 p s i w a s u s e d t o p a c k t h e c o l u m n to the d e s i r e d h e i g h t of 75 c m . The c o l u m n w a s w a s h e d w i t h 2 M N a C l u n t i l the O . D . at 260 m u w a s z e r o , t h e n w i t h d i s t i l l e d w a t e r u n t i l the e l u a t e w a s f r e e -13-f r o m c h l o r i d e i o n ( A ' g N 0 3 t e s t ) a n d f i n a l l y w i t h 1 l i t r e of 0. 10 M N a C l , 0. 005 M T r i s ( c h l o r i d e ) , p H 7. 8. 2. P u r i f i c a t i o n of C r u d e s - R N A C r u d e s - R N A (14. 5 g), p r e p a r e d f r o m f r e s h l y g r o w n b a k e r s ' y e a s t by D r . G . M . T e n e r a c c o r d i n g to the m e t h o d of B e l l et a l . (1964), w a s s u s p e n d e d i n 400 m l of 0.10 M N a C l f o r 1. 5 h r . T h e i n s o l u b l e r e s i d u e w a s r e m o v e d b y c e n t r i f u g a t i o n at 12, 000 r p m f o r 30 m i n i n a S o r v a l l , T y p e S S - 1 c e n t r i f u g e . The s u p e r n a t e w a s a p p l i e d to a D E A E - c e l l u l o s e c o l u m n (75 x 3. 5 c m d i a m . ) p r e p a r e d as d e s c r i b e d a b o v e . T h e c o l u m n w a s d e v e l o p e d u s i n g an 8 l i t r e l i n e a r g r a d i e n t f r o m 0. 10 M to 1. 5 M N a C l , both, v e s s e l s b u f f e r e d at jgH 7. 8 w i t h 0. 005 M T r i s ( c h l o r i d e ) . The f l o w r a t e w a s 3. T m l p e r m i n a n d 20 m l f r a c t i o n s w e r e c o l l e c t e d . F r a c t i o n s 70-180 w e r e c o m b i n e d (2200 m l ) and c o n c e n t r a t e d u n d e r r e d u c e d p r e s s u r e at 30° to a v o l u m e of 500 m l . T h e s o l u t i o n w a s a d j u s t e d to p H 5 w i t h g l a c i a l a c e t i c a c i d , c o o l e d to 0° and the s - R N A p r e c i p i t a t e d w i t h 2. 5 v o l u m e s of i c e - c o l d e t h a n o l a n d a l l o w e d to s e t t l e at 0° f o r 4 h r . T h e p r e c i p i t a t e w a s c o l l e c t e d b y d e c a n t a t i o n a n d c e n t r i f u g a t i o n . T h e r e s u l t i n g p e l l e t w a s d i s s o l v e d i n 80 m l of 0. 10 M N a C l , 0. 005 M T r i s ( c h l o r i d e ) , p H 7. 8, a n d the s o l u t i o n d i s t i l l e d u n d e r r e d u c e d p r e s s u r e at 30° to r e m o v e t r a c e s of e t h a n o l . T h e s o l u t i o n w a s t h e n c h r o m a t o g r a p h e d u n d e r the c o n d i t i o n s d e s c r i b e d a b o v e . T h e e l u t i o n p r o f i l e i s s h o w n i n F i g u r e 1. F r a c t i o n s 145-220 w e r e c o m b i n e d , the p H a d j u s t e d to 7 w i t h a c e t i c a c i d 3 4 fi FRACTION No. FIGURE 1: DEAE-cellulose chromatography of s-RNA from bakers' yeast. -15-a n d c o n c e n t r a t e d u n d e r r e d u c e d . p r e s s u r e at 30° to 500 m l . T h e c o n c e n t -r a t e w a s a d j u s t e d to p_H 5 w i t h a c e t i c a c i d a n d the s o l u t i o n c o o l e d to 0 ° . I c e - c o l d e t h a n o l (2. 5 v o l u m e s ) w a s a d d e d a n d the s o l u t i o n l e f t o v e r n i g h t at 0 ° . T h e p e l l e t , o b t a i n e d by d e c a n t a t i o n a n d c e n t r i f u g a t i o n , w a s w a s h e d w i t h c o l d e t h a n o l , t h e n w i t h e t h e r a n d d r i e d i n a v a c u u m d e s i c c a t o r o v e r P 2 P 5 to y i e l d . 1 . 005 g of y e a s t s - R N A . 3. D i g e s t i o n of Y e a s t s - R N A by R i b o n u c l e a s e at V a r i o u s C o n c e n t r a t i o n s of M a g n e s i u m I o n T h e r e a c t i o n m i x t u r e ( f i n a l v o l u m e 3. 0 m l ) c o n t a i n e d p e r m l of f i n a l v o l u m e , 10 > J m o l e s T r i s ( c h l o r i d e ) , p_H 7. 3, 35 fJ-g y e a s t s - R N A , M g C l 2 at c o n c e n t r a t i o n s b e t w e e n 0 a n d |'Q0 jJ - m o l e s and. p a n c r e a t i c r i b o -n u c l e a s e . T h e T r i s b u f f e r , s - R N A a n d M g C ^ (or d i s t i l l e d w a t e r i n the c a s e of the c o n t r o l ) w e r e m i x e d i n a 1 - c m - c u v e t t e a n d the o p t i c a l d e n s i t y m e a s u r e d at 260 m j j . . T h e p a n c r e a t i c r i b o n u c l e a s e , c o n t a i n e d i n 0. 10 m l , w a s a d d e d at z e r o t i m e . T h e O . D . w a s c o r r e c t e d f o r t h i s d i l u t i o n a n d the r e a c t i o m w a s f o l l o w e d b y the h y p e r c h r o m i c s h i f t m e a s u r e d at 260 mjx.. D i g e s t i o n s w e r e c a r r i e d out at 0. 0 M , 0. 02 M , 0. 05 M a n d 0. 10 M M g C ^ i n the p r e s e n c e of 15 j i g p e r m l of p a n c r e a t i c r i b o n u c l e a s e at r o o m t e m p e r a t u r e ( F i g . 2). 4 . Q u a n t i t a t i v e P r e c i p i t a t i o n of s - R N A T o 0. 50 m l of a s o l u t i o n at r o o m t e m p e r a t u r e c o n t a i n i n g 240 jug 0.16 c -; ~-M9 —^002 MMg 0.05 M Mg O.IOMMg 10 12 14 TIME (min) 24 F I G U R E 2: I n c r e a s e i n o p t i c a l d e n s i t y at 260 mjx d u r i n g d i g e s t i o n of s - R N A w i t h r i b o n u c l e a s e at r o o m t e m p e r a t u r e i n the p r e s e n c e a n d a b s e n c e of M g . s - R N A : r i b o n u c l e a s e r a t i o , 2. 3:1 ( w / w ) . -17-of s - R N A , 4 / . imoles of T r i s ( c h l o r i d e ) , . p H 7 . 3 , a n d 10 ^ m o l e s of M g C l 2 w a s a d d e d 0. 50 m l of a n i c e - c o l d s o l u t i o n c o n s i s t i n g of 5 0 % aqueous e t h a n o l , 2 N i n H C 1 . T h e s o l u t i o n w a s m i x e d t h o r o u g h l y , c o o l e d i n i c e f o r 10 m i n t h e n . c e n t r i f u g e d at 11, 500 r p m i n a M i s c o M o d e l 5500 c e n t r i f u g e . A b o u t 2% of the O . D . r e m a i n e d i n the s u p e r n a t e . 5. R e l e a s e of A c i d - S o l u b l e N u c l e o t i d e s D u r i n g R i b o n u c l e a s e D i g e s t i o n  at R o o m T e m p e r a t u r e A 4 . 00 m l s o l u t i o n c o n t a i n i n g 2. 32 m g of y e a s t s - R N A (46. 4 O . D . u n i t s ) , 40 j U m o l e s of T r i s ( c h l o r i d e ) , p_H 7. 3 and M g C l 2 ( e x c e p t e x p e r i -m e n t 1, w h i c h c o n t a i n e d no 1 M g C ^ ) w a s p r e p a r e d f o r e a c h e x p e r i m e n t . T o a 0. 40 m l a l i q u o t w a s a d d e d 0. 10 m l of T r i s ( c h l o r i d e ) , ; p_H 7. 3, a n d 0 . 5 0 m l of i c e - c o l d 50% aqueous e t h a n o l , 2 N i n . H C l . A f t e r c o o l i n g i n i c e a n d c e n t r i f u g i n g (as d e s c r i b e d . i n S e c t i o n 4) , a 0. 15 m l a l i q u o t of the s u p e r n a t e w a s d i l u t e d to 1. 0 m l w i t h 0. 01 M T r i s ( c h l o r i d e ) b u f f e r p H 7. 3, and the O . D . m e a s u r e d at 260 m p . . T h i s a f f o r d e d a v a l u e f o r the r e s i d u a l , n o n - p r e c i p i t a b l e O . D . w h i c h v a r i e d d e p e n d i n g on the c o n c e n t r a t i o n of m a g n e s i u m i o n s p r e s e n t . T o the r e m a i n i n g ; 3. 60 m l of s o l u t i o n . w a s a d d e d 0. 90 m l of p a n c r e a t i c r i b o n u c l e a s e (150jJ-g p e r m l ) at z e r o t i m e . T h u s the r e a c t i o n m i x t u r e ( f i n a l v o l u m e 4 . 5 m l ) c o n t a i n e d , p e r m l f i n a l v o l u m e , 480 p-g s - R N A , 10 j i m o l e s T r i s ( c h l o r i d e ) , : p H 7. 3, 30 )xg p a n c r e a t i c r i b o n u c l e a s e a n d M g C l 2 at c o n c e n t r a t i o n s of 0. 00 M , 0. 05 M , 0. 10 M a n d 0. 20 M . A l i q u o t s of 0. 50 m l w e r e r e m o v e d at v a r i o u s t i m e i n t e r v a l s , , p r e c i p i t a t e d a n d c e n t r i f u g e d as d e s c r i b e d i n S e c t i o n 4 . A 0. '15 m l a l i q u o t of the s u p e r n a t e w a s d i l u t e d to 1. 00 m l w i t h 0. 01 M T r i s -( c h l o r i d e ) b u f f e r , . p H 7. 3 a n d the O . D . at 260 mp.. w a s r e c o r d e d . T h e r e a d i n g s o b t a i n e d w e r e c o r r e c t e d f o r the r e s i d u a l , n o n - p r e c i p i t a b l e O . D . p r e v i o u s l y d e t e r m i n e d . E a c h c o r r e c t e d r e a d i n g w a s t h e n e x p r e s s e d i n t e r m s of p e r c e n t b y c o m p a r i n g i t to the r e a d i n g o b t a i n e d . w h e n the s - R N A w a s c o m p l e t e l y d i g e s t e d (100% v a l u e ) . V a l u e s f o r 100% d i g e s t i o n w e r e o b t a i n e d b y c a r r y i n g out the d i g e s t i o n . f o r 30 m i n at r o o m t e m p e r a t u r e i n the a b s e n c e of M g C l 2 , t h e n a d d i n g M g C l 2 to the d e s i r e d c o n c e n t r a t i o n f o l l o w e d by p r e c i p i t a t i o n a n d c e n t r i f u g a t i o n i n the u s u a l w a y . T h e r e s u l t s of f i v e e x p e r i m e n t s c a r r i e d out at d i f f e r e n t c o n c e n -t r a t i o n s of M g C l 2 a r e s h o w n i n F i g u r e 3. 6. E f f e c t of P r o n a s e o n the D i g e s t i o n of s - R N A by P a n c r e a t i c R i b o -n u c l e a s e T h e r e a c t i o n m i x t u r e ( f i n a l v o l u m e 1. 00 m l ) c o n t a i n e d p e r m l of f i n a l v o l u m e l O ^ x m o l e s T r i s ( c h l o r i d e ) , p H 7 . 3 , 50 j j i m o l e s M g C l 2 , 85 jxg s - R N A (1. 70 O . D . u n i t s ) , 0 . 5 jxg p a n c r e a t i c r i b o n u c l e a s e a n d P r o n a s e at 10 ^ p-g a n d 10"^ J^-g. T h e c o n t r o l c o n t a i n e d d i s t i l l e d w a t e r i n p l a c e of P r o n a s e . T h e T r i s b u f f e r , M g C l 2 a n d s - R N A w e r e m i x e d t o g e t h e r i n a m i c r o - c u v e t t e a n d r i b o n u c l e a s e w a s a d d e d at z e r o t i m e . T h e r e a c t i o n 0.01 M Mg • 0.05 MMg —-O/IOMMg 0.20M Mg 2 4 6 8 10 12 14 16 18 TIME (min) 20 22 24 26 28 30 F I G U R E 3: R e l e a s e of a c i d - s o l u b l e u l t r a v i o l e t a b s o r b i n g m a t e r i a l d u r i n g d i g e s t i o n of s - R N A w i t h p a n c r e a t i c r i b o n u c l e a s e at r o o m t e m p e r a t u r e i n the " p r e s e n c e and a b s e n c e of M g . s - R N A : r i b o n u c l e a s e r a t i o , 16:1 ( w / w ) . - 2 0 -w a s f o l l o w e d by the h y p e r c h r o m i c s h i f t at 260 m u . and w a s a l l o w e d to p r o c e e d f o r s e v e r a l m i n b e f o r e a d d i n g the P r o n a s e ( c o n t a i n e d i n . a v o l u m e of 0. 10 m l ) . W h e n P r o n a s e a l o n e (10"^ y.g) w a s a d d e d to the s - R N A i n the a b s e n c e of r i b o n u c l e a s e no h y p e r c h r o m i c i t y w a s o b s e r v e d , o v e r 2 5 m i n at r o o m t e m p e r a t u r e . N o i n h i b i t i o n of r i b o n u c l e a s e w a s o b s e r v e d w h e n the c o n c e n t r a t i o n of P r o n a s e w a s i n c r e a s e d to 10 ^ig p e r m l . 7. R e m o v a l of P a n c r e a t i c R i b o n u c l e a s e w i t h C M - C e l l u l o s e A . B e h a v i o u r of P a n c r e a t i c R i b o n u c l e a s e o n . C M - C e l l u l o s e A 0. 10 m l a l i q u o t of a s o l u t i o n c o n t a i n i n g 0. 5 jj.g of r i b o n u c l e a s e w a s a d d e d to a C M - c e l l u l o s e ( a m m o n i u m ) c o l u m n (4. 8 x 0. 5 c m d i a m . ) p r e v i o u s l y e q u i l i b r a t e d w i t h . 0 . 01 M T r i s ( c h l o r i d e ) , pJH 7. 3 a n d . t h e c o l u m n w a s w a s h e d w i t h s e v e r a l c o l u m n v o l u m e s of the s a m e b u f f e r . A n a l i q u o t of the e l u a t e w a s - i n c u b a t e d w i t h a k n o w n a m o u n t of s - R N A a n d the O . D . at 260 m^x r e c o r d e d . N o r i b o n u c l e a s e w a s d e t e c t e d i n the T r i s e l u a t e . R i b o n u c l e a s e w a s e l u t e d by w a s h i n g the c o l u m n w i t h 0. 2 M M g C ^ . 0. 01 M T r i s ( c h l o r i d e ) , : p H 7. 3. B . R e m o v a l of R i b o n u c l e a s e f r o m a D i g e s t i o n M i x t u r e T h e d i g e s t i o n m i x t u r e ( f i n a l v o l u m e 3. 00 m l ) c o n t a i n i n g p e r m l f i n a l v o l u m e , 1 0 y i t m o l e s T r i s ( c h l o r i d e ) , p H 7. 3, 75 jj-g s - R N A (1. 5 O . D . u n i t s ) , 200 J x m o l e s M g C ^ , a n d 0. 5 |xg p a n c r e a t i c r i b o n u c l e a s e w a s m a d e u p as f o l l o w s : a 2 . 7 0 m l s o l u t i o n c o n t a i n i n g the T r i s b u f f e r , s - R N A and -21-M g C l 2 w a s c o o l e d to 0° t h e n 1. 5 u.g of r i b o n u c l e a s e ( i n a v o l u m e of 0. 30 m l ) w a s a d d e d . T h e i n c u b a t i o n w a s c a r r i e d out at 0° f o r 25 m i n . N o h y p e r c h r o m i c i t y w a s d e t e c t e d d u r i n g t h i s t i m e . T h e m i x t u r e w a s t h e n a l l o w e d to w a r m t o r o o m t e m p e r a t u r e d u r i n g 25 m i n t h e n q u e n c h e d , i n a n , i c e b a t h . T h e e x p e c t e d i n c r e a s e i n O. D . w a s o b s e r v e d . T h e m i x t u r e w a s d i l u t e d to 0. 02 M M g C l 2 b y the a d d i t i o n of 27 m l of i c e - c o l d 0. 01 M T r i s ( c h l o r i d e ) , : p H 7. 3,. b u f f e r a n d i m m e d i a t e l y a p p l i e d to a C M - c e l l u l o s e ( a m m o n i u m ) c o l u m n : ( 1 5 - x . l . 7 c m d i a m . ). T h e c o l u m n w a s w a s h e d w i t h • 0. 01 M T r i s ( c h l o r i d e ) , p_H 7. 3 a n d the e l u a t e c o l l e c t e d i n 5. 0 m l f r a c t i o n s . R e c o v e r y of the a p p l i e d m a t e r i a l w a s q u a n t i t a t i v e . A n a l i q u o t of the e l u a t e w a s h e a t e d to 7 0 ° f o r a few m i n u t e s t h e n c o o l e d r a p i d l y . T h e o b s e r v e d h y p e r c h r o m i c i t y at 260 m u . w a s 19 .5%. A n o t h e r a l i q u o t , t r e a t e d w i t h . r i b o n u c l e a s e at r o o m t e m p e r a t u r e , s h o w e d a 10% i n c r e a s e i n O . D . w i t h i n one m i n u t e . B o t h t e s t s s u g g e s t e d that the R N A h a d not b e e n d e g r a d e d by r i b o n u c l e a s e d u r i n g the c h r o m a t o g r a p h y on C M - c e l l -u l o s e . 8. R e l e a s e of A c i d - S o l u b l e N u c l e o t i d e s D u r i n g R i b o n u c l e a s e D i g e s t i o n  at 0° i n the P r e s e n c e of M a g n e s i u m Ions T h e r e a c t i o n c o n d i t i o n s w e r e s i m i l a r to t h o s e d e s c r i b e d . i n S e c t i o n 5 e x c e p t that the d i g e s t i o n ( f i n a l v o l u m e 4 . 5 m l ) w a s c a r r i e d out at 0 ° . i n the p r e s e n c e of 9. 6 juug p e r m l of p a n c r e a t i c r i b o n u c l e a s e a n d M g C l 2 at c o n c e n t r a t i o n s of 0. 00 M , 0. 01 M , 0. 05 M , 0. 10 M a n d 0. 20 M . The r e s u l t s a r e s h o w n i n F i g u r e 4 . T h e d i g e s t i o n s w e r e r e p e a t e d u s i n g 0.96 yog p e r m l of r i b o n u c l e a s e at M g C l 2 c o n c e n t r a t i o n s of 0. 0 0 M , 0. 05 M a n d 0. 20 M . T h e s e r e s u l t s a r e s h o w n i n F i g u r e 5. 9. R e l e a s e of A c i d - S o l u b l e N u c l e o t i d e s D u r i n g R i b o n u c l e a s e D i g e s t i o n at R o o m T e m p e r a t u r e i n the P r e s e n c e of S p e r m i n e T h i s e x p e r i m e n t w a s c a r r i e d out i n a m a n n e r a n a l o g o u s to that d e s c r i b e d i n S e c t i o n 5 e x c e p t that , i n p l a c e of M g C l 2 , s p e r m i n e h y d r o -c h l o r i d e , p_H 7. 3, w a s a d d e d to e a c h . r e a c t i o n m i x t u r e at c o n c e n t r a t i o n s of 0. 00 M , O . l O m M , 0. 50 m M , 1. 0 m M , 5. 0 m M a n d 10. 0 m M . D i g e s t i o n s c a r r i e d out at s p e r m i n e c o n c e n t r a t i o n s of 1. 0 m M a n d a b o v e c o n t a i n e d , , i n a d d i t i o n to the r e a g e n t s l i s t e d above , . 0. 10 M N a C l to p r e v e n t p r e c i p i t a t i o n of the s - R N A . T h e r e s u l t s a r e s h o w n i n F i g u r e 6. 10. C o n t r o l l e d , L a r g e S c a l e D i g e s t i o n of s - R N A w i t h R i b o n u c l e a s e at R o o m T e m p e r a t u r e T h e i n c u b a t i o n m i x t u r e c o n t a i n e d 50 yumoles of T r i s ( c h l o r i d e ) , p H 7. 3, 20 m g of s - R N A (400 O . D . u n i t s ) , . 1 m m o l e of M g C l 2 a n d 40p^g of p a n c r e a t i c r i b o n u c l e a s e i n a t o t a l v o l u m e of 5. 0 m l . The d i g e s t i o n w a s c a r r i e d out f o r 25 m i n at r o o m t e m p e r a t u r e , q u e n c h e d . i n i c e a n d d i l u t e d to 0. 02 M M g C l 2 ' b y the a d d i t i o n . o f 45 m l of i c e - c o l d , 0. 01 M T r i s ( c h l o r i d e ) , p H 7. 3 a n d . a p p l i e d . i m m e d i a t e l y to a C M - c e l l u l o s e ( a m m o n i u m ) c o l u m n (20 x 2. 0 c m d i a m . ) as d e s c r i b e d i n S e c t i o n 7 - B . . 100 90 o CO 70 CVJ 6 60 50 Uj40 < 30 y 20 • — M g -O.OIMMg 0.05M Mg 0.1 OM Mg O.20M Mg 20 40 60 80 100 140 TIME ( m i n ) 180 220 260 F I G U R E 4: R e l e a s e of a c i d - s o l u b l e u l t r a v i o l e t a b s o r b i n g m a t e r i a l d u r i n g d i g e s t i o n of s - R N A w i t h p a n c r e a t i c r i b o n u c l e a s e at 0° i n the p r e s e n c e and a b s e n c e of M g . s - R N A : r i b o n u c l e a s e r a t i o , 50:1 (w/w). -24-_100 £ o CD 8 0 ci d 2 60 LU (/) 2 40 (J •-Mg / / O.OSMMg O.IOMMg •0.20MMg 120 . 160 TIME (min) 200 240 F I G U R E 5: R e l e a s e of a c i d - s o l u b l e u l t r a v i o l e t a b s o r b i n g m a t e r i a l d u r i n g d i g e s t i o n of s - R N A w i t h r i b o n u c l e a s e at 0° i n the p r e s e n c e a n d a b s e n c e of M g . s - R N A : r i b o n u c l e a s e r a t i o , 500:1 ( w / w ) . - 2 5 -0.80 h TIME (min) F I G U R E 6: I n c r e a s e i n a c i d - s o l u b l e u l t r a v i o l e t a b s o r b i n g m a t e r i a l d u r i n g d i g e s t i o n of s - R N A w i t h p a n c r e a t i c r i b o n u c l e a s e i n the p r e s e n c e a n d a b s e n c e of s p e r m i n e . s - R N A : r i b o n u c l e a s e r a t i o , 500:1 ( w / w ) . The e x p e c t e d r e c o v e r y w a s 360 O . D . u n i t s ( m e a s u r e d : i n m a g n e s i u m s o l u t i o n ) i n the a b s e n c e of any d i g e s t i o n b y r i b o n u c l e a s e , o r 4 3 0 O . D . u n i t s i f the d i g e s t i o n w a s c o m p l e t e . T h e a c t u a l r e c o v e r y w a s 380 O . D . u n i t s s u g g e s t i n g that the R N A w a s 2 5 - 3 0 % d e g r a d e d . A b o u t h a l f of the e l u a t e (71 m l ) f r o m the C M - c e l l u l o s e c o l u m n , c o n t a i n i n g 192 O . D . u n i t s w a s a p p l i e d to a D E A E - c e l l u l o s e ( c h l o r i d e ) c o l u m n (47 x 2 c m d i a m . ) , p r e v i o u s l y e q u i l i b r a t e d w i t h 0. 01 M T r i s ( c h l o r i d e ) , p_H 7. 8. T h e c o l u m n w a s w a s h e d w i t h about 50 m l of e q u i l i b r a t i n g : b u f f e r - a n d t h e n s u b j e c t e d to a l i n e a r g r a d i e n t of s o d i u m c h l o r i d e 0. 0 - 0. 3 M ( t o t a l v o l u m e , 8 l i t r e s ) c o n t a i n i n g 0. 01 M T r i s ( c h l o r i d e ) , p_H 1. 8. T h e f l o w r a t e w a s 1 m l p e r m i n a n d 10 m l f r a c t i o n s w e r e c o l l e c t e d . T o e n s u r e c o m p l e t e r e m o v a l of the O . D . the l i n e a r g r a d i e n t of s o d i u m c h l o r i d e w a s e x t e n d e d to 1. 0 M ( t o t a l v o l u m e 1; l i t r e ) . T h e e l u t i o n . p r o f i l e i s shown, i n F i g u r e 7. 11. L a r g e S c a l e D i g e s t i o n of s - R N A w i t h R i b o n u c l e a s e at 0° i n . t h e P r e s e n c e Of 0. 2 M M g C l 2 P a n c r e a t i c r i b o n u c l e a s e (20 yixg) w a s a d d e d to a n - i c e - c o l d s o l u t i o n c o n t a i n i n g 200 j u m o l e s of T r i s ( c h l o r i d e ) , . p_H 1. 3, 40 m g of s - R N A a n d 4 m m o l e s of M g C ^ i n - a f i n a l v o l u m e of 20 m l . A f t e r 30 m i n i n c u b a t i o n at 0° the r e a c t i o n m i x t u r e w a s p o u r e d . i n t o a 200 m l s l u r r y of C M - c e l l -u l o s e , 0. 01 M T r i s ( c h l o r i d e ) , p_H 7. 3, at 3 - 5 ° a n d f i l t e r e d r a p i d l y t h r o u g h a C M - c e l l u l o s e c o l u m n (27 x 4 c m d i a m . ). T e n m l f r a c t i o n s w e r e c o l l e c t e d . T h e f r a c t i o n s c o n t a i n i n g u l t r a v i o l e t a b s o r b i n g m a t e r i a l FRACTION No.(10ml.) F I G U R E 7: D E A E - c e l l u l o s e c h r o m a t o g r a p h y of s - R N A af ter d i g e s t i o n w i t h r i b o n u c l e a s e at r o o m t e m p e r a t u r e i n the p r e s e n c e of 0. 2 M M g C l 2 . s - R N A : r i b o n u c l e a s e r a t i o , 500:1 ( w / w ) . - 2 8 -w e r e c o m b i n e d (520 m l , 705 O. D . u n i t s ) a n d a p p l i e d to a D E A E - c e l l u l o s e ( c h l o r i d e f o r m ) c o l u m n (92 x 1. 5 c m d i a m . ),. p r e v i o u s l y e q u i l i b r a t e d w i t h 0. 01 M T r i s ( c h l o r i d e ) , r g H 7 . 3 . T h e c o l u m n w a s t h e n s u b j e c t e d t o a l i n e a r g r a d i e n t of l i t h i u m c h l o r i d e 0. 0 5 - 1 . 5<M ( t o t a l v o l u m e , 4 l i t r e s ) - c o n t a i n i n g 0. 01 M T r i s ( c h l o r i d e ) , p_H 7. 3. T h e e l u t i o n p r o f i l e i s s h o w n i n F i g u r e 8. T u b e s 50-115 ( F r a c t i o n II) w e r e c o m b i n e d (1100 rr i l ) , d i l u t e d to 13 l i t r e s w i t h d i s t i l l e d w a t e r a n d l o a d e d onto' a D E A E - c e l l u l o s e c o l u m n (90 x . l . 5 c m d i a m . ). T h e c o l u m n . w a s t h e n s u b j e c t e d to a l i n e a r g r a d i e n t of l i t h i u m c h l o r i d e , 0 . - 0 . 8 M ( t o t a l v o l u m e 4 . 1 i t r e s ) c o n t a i n i n g ; 0. 01 M T r i s (ch lor ide) , . r p_H 7. 3. T h e e l u t i o n . p r o f i l e i s s h o w n i n F i g u r e 9. T u b e s 8 0 - 140 ( F r a c t i o n A ) w e r e c o m b i n e d , d i l u t e d to I l i t r e w i t h . d i s t i l l e d w a t e r and l o a d e d o n a D E A E - c e l l u l o s e ( c a r b o n a t e ) c o l u m n (53. 5 x 1. 5 c m diairw ). T h e c o l u m n w a s w a s h e d s u c c e s s i v e l y - w i t h w a t e r (500 m l ) , 0. 01 M (NH 4 ) 2 C03 (500 m l ) a n d 2 M (NH4) 2 C0 3 . T h e a m m o n i u m c a r -bonate w a s r e m o v e d by r e p e a t e d e v a p o r a t i o n at 30° u n d e r r e d u c e d p r e s s u r e . 12. B a s e A n a l y s i s of F r a c t i o n A T h e n o n - v o l a t i l e r e s i d u e (5 mg), r e m a i n i n g a f t e r r e m o v a l of the a m m o n i u m c a r b o n a t e f r o m f r a c t i o n A w a s d i g e s t e d w i t h 0. 3 N KOH f o r 18 h r at 3 7 ° ( M a r k h a m a n d S m i t h , 1952). T h e d i g e s t w a s n e u t r a l i z e d w i t h D o w e x 50 ( h y d r o g e n ) r e s i n a n d a d d e d to a D E A E - c e l l u l o s e ( c a r b o n a t e ) c o l u m n (14 x 1. 7 c m d i a m . ) ( B e l l et a l . , 1964). T h e c o l u m n w a s s u b j e c t e d - 2 9 -FRACTION 1-120 1.00 0.80 0.60 < 0.40 0.20 L»CI(M) -11.0' 40 60 80 FRACTION No. F I G U R E 8: D E A E - c e l l u l o s e c h r o m a t o g r a p h y of s - R N A a f t e r d i g e s t i o n w i t h r i b o n u c l e a s e at 0° i n the p r e s e n c e of 0 . 2 M M g C l £ . s - R N A : r i b o n u c l e a s e r a t i o , 2000:1 ( w / w ) . to a l i n e a r g r a d i e n t of a m m o n i u m c a r b o n a t e , 0. 0 - 0. 3 - M ( t o t a l v o l u m e , 2 l i t r e s ) , p H 7. 3. T h e e l u t i o n , p r o f i l e i s s h o w n i n F i g u r e 1 0 - A . T h e f l o w r a t e w a s 0. 5 m l p e r m i n , a n d f r a c t i o n s of 2. 3; - 2. 5 m l e a c h w e r e c o l l e c t e d . F r a c t i o n s w e r e c o m b i n e d (A - I) as s h o w n i n F i g u r e 1 0 - A , l y o p h i l i z e d , a p p l i e d to t w o 20 c m s h e e t s of W h a t m a n .40 p a p e r - p r e v i o u s l y w a s h e d w i t h a s o l v e n t c o m p o s e d . o f i s o b u t y r i c a c i d : : c o n e . N H 3 , (66:33:1), a n d d e v e l o p e d f o r 1 8 h r i n . t h e s a m e s o l v e n t ( F i g . 1 0 - B ) . T h e u l t r a v i o l e t - a b s o r b i n g a r e a s w e r e cut out, e l u t e d q u a n t i t a t i v e l y a n d m a d e up to 1. 0 m l w i t h w a t e r . T h e s p e c t r a of the e l u a t e s w e r e r e c o r d e d u n d e r n e u t r a l , a c i d i c a n d a l k a l i n e c o n d i t i o n s . T h e r e s u l t i n g , n u c l e o t i d e c o m p o s i t i o n ( m o l e p e r c e n t ) w a s as f o l l o w s : A , 14. 0; G , . 10. 7; C , . 21. 0; U , 2 7 . 7 ; V , 8. 8; T , 4 . 8; 1 - m e t h y l - G , . 1. 7; 2 - N , N - d i m e t h y l - G , 2. 0; and u n i d e n t i f i e d - u l t r a v i o l e t a b s o r b i n g c o m p o u n d s , 9 . 3 . 13. D i g e s t i o n of M i x e d s - R N A a n d P a r t i a l l y P u r i f i e d G l y c i n e s - R N A w i t h  P a n c r e a t i c R i b o n u c l e a s e A . G-100 S e p h a d e x C h r o m a t o g r a p h y M i x e d y e a s t s - R N A (50 m g - C a l b i o c h e m ) w a s d i s s o l v e d i n 3. 0 m l of 1. 0 M N a C l and a d d e d to the t o p of a G-100 S e p h a d e x c o l u m n (115 x 1. 5 c m d i a m . ), p r e v i o u s l y e q u i l i b r a t e d w i t h 1. 0 M N a C l . T h e f l o w r a t e w a s 0 . 4 5 m l p e r m i n a n d 5. 4 m l f r a c t i o n s w e r e c o l l e c t e d ( F i g . 11-A). The m a j o r p e a k (II) c o n t a i n e d 81. 3% of the O. D . . a n d w a s c o l l e c t e d by p r e c i p i t a t i o n w i t h e t h a n o l at 0 ° . F I G U R E 10: A - C h r o m a t o g r a p h y of an a l k a l i n e d i g e s t of F r a c t i o n A ( F i g . 9) on D E A E - c e l l u l o s e . B - C o m p o s i t e d i a g r a m of c h r o m a t o g r a m s o b t a i n e d a f t e r p a p e r c h r o m a t o g r a p h y of F r a c t i o n s A to I i n F i g u r e 1 0 - A . T J Q ~J o zr ~j O 3 o TJ z r A | B | C | D | E F I G | H | I —^ c2> — s*- 7^ ^ C (J J> C M e ( T } C A J (10-B) > "5 s C. i——:—J C D I O J ro i 1 0 0 1 2 0 FRACTION No. 2 2 0 26 24 *—s £22 O C D 2 0 CM w 1 ' 8 >-to U J 1 4 Q 12 _j10 < y 8 \~ O 6 4 2 0 B U 4 * O 10 20 30 40 50 60 0 10 20 30 40 50 60 0 FRACTION No. c o X <D Q CD m 10 20 30 40 50 60 F I G U R E 11: G-100 S e p h a d e x c h r o m a t o g r a p h y of: A -C-m i x e d s - R N A , p a r t i a l l y p u r i f i e d g l y c i n e s - R N A , r e c h r o m a t o g r a p h y of p e a k II f r o m F i g u r e 11-B. - 3-4-G l y c i n e s - R N A (50 m g ) , w h i c h w a s p u r i f i e d 1 2 - f o l d b y c h r o m a -t o g r a p h y on b e n z o y l a t e d D E A E - c e l l u l o s e ( T e n e r et a l . , 1966), w a s c h r o m a t o g r a p h e d on G-100 S e p h a d e x as d e s c r i b e d a b o v e ( F i g . 11-B). The m a j o r p e a k (II) c o n t a i n e d 9 3 . 5% of the O . D . a n d w a s c o l l e c t e d by e t h a n o l p r e c i p i t a t i o n at 0 ° . R e c h r o m a t o g r a p h y of p e a k II (10 m g ) u n d e r the s a m e c o n d i t i o n s gave a s y m m e t r i c a l e l u t i o n p r o f i l e (F ig . . - 11-C) . B . D i g e s t i o n w i t h P a n c r e a t i c R i b o n u c l e a s e S o l u t i o n s ( f i n a l v o l u m e 1. 5 m l ) of the m i x e d a n d p a r t i a l l y p u r i f i e d s - R N A f r o m p e a k II ( F i g s 11-A a n d 11-B), c o n t a i n e d p e r m l of f i n a l v o l u m e , 1 0 y u m o l e s T r i s ( c h l o r i d e ) , p H 7. 5, 0 . 5 O . D . u n i t s of s - R N A , 0 o r 10 y u m o l e s M g C l 2 a n d 0. 33 yUg p a n c r e a t i c r i b o n u c l e a s e . T h e T r i s b u f f e r , s - R N A a n d M g C l 2 ( o r d i s t i l l e d w a t e r - i n the c a s e of the c o n t r o l s ) w e r e m i x e d i n a 1 - c m m i c r o - c u v e t t e a n d the O . D . m e a s u r e d at 260 m u . . T h e p a n c r e a t i c r i b o n u c l e a s e , c o n t a i n e d . i n 0. 10 m l , w a s a d d e d at z e r o t i m e . T h e a b s o r b a n c e at 260 ncy*. w a s c o r r e c t e d f o r t h i s d i l u t i o n a n d the h y p e r c h r o m i c s h i f t w a s r e c o r d e d . T h e r e s u l t s a r e s h o w n i n F i g u r e 12 as p e r c e n t i n c r e a s e i n a b s o r b a n c e o v e r the a b s o r b a n c e at z e r o t i m e i n the p r e s e n c e a n d a b s e n c e of m a g n e s i u m . - 3 5 -cn i I 2 Y cn 2 \ ^ C\J o 00 CO Nf (rtuu092) 'Q'O Nl 3SV3UDNI %> OJ O CO CO O J C\J OJ T — OJ CO ro *r ro OJ ro O ro CO OJ CD OJ OJ O J ^ UJ £ 2 O J p CO CD OJ •o o < rt I 10 <D c • rH U I—I W) CD SH PH (TJ r - H •43 O . » H bJO rt ^  PH ^ rt oo < i u C <D c o rt •g s > * H m o o , c « .2 PH t o c u bo +-> S .S w rt o D I S C U S S I O N B o v i n e p a n c r e a t i c r i b o n u c l e a s e i s not m a r k e d l y i n h i b i t e d by-d i v a l e n t c a t i o n s s u c h as m a g n e s i u m i o n w h e n e n z y m a t i c a c t i v i t y i s d e t e r m i n e d by m e a s u r i n g , the i n c r e a s e i n a c i d - s o l u b l e m a t e r i a l u s i n g h i g h m o l e c u l a r w e i g h t y e a s t R N A as s u b s t r a t e ( D i c k m a n et a l . , 1956; N i s h i m u r a , I 9 6 0 ) . H o w e v e r w h e n s - R N A w a s u s e d as s u b s t r a t e a m a r k e d i n h i b i t i o n of p a n c r e a t i c r i b o n u c l e a s e w a s o b s e r v e d , as m e a s u r e d by the h y p e r c h r o m i c s h i f t i n a b s o r b a n c e at 260 m j _ t ( F i g . 2). I n the a b s e n c e of m a g n e s i u m the h y d r o l y s i s w a s e s s e n t i a l l y c o m p l e t e i n 3 m i n w h i l e i n the p r e s e n c e of 0. 05 M m a g n e s i u m the h y d r o l y s i s w a s o n l y 55% c o m p l e t e i n . 20 m i n a n d . i n the p r e s e n c e of 0. 1 0 M m a g n e s i u m , 27% of the m a x i m u m h y p e r c h r o m i c i t y w a s o b s e r v e d . i n 20 m i n . T o e n s u r e that the h y p e r c h r o m i c s h i f t at 260 m p _ w a s a r e a s o n a b l e i n d i c a t i o n of the e x t e n t of h y d r o l y s i s , s - R N A w a s d i g e s t e d w i t h p a n c r e a t i c r i b o n u c l e a s e a n d the r e l e a s e of a c i d - s o l u b l e , u l t r a v i o l e t a b s o r b i n g m a t e r i a l w a s m e a s u r e d . Q u a n t i t a t i v e p r e c i p i t a t i o n of the s - R N A w a s a c h i e v e d b y adding; to the s o l u t i o n , a n . e q u a l v o l u m e of 50% aqueous e t h a n o l , 2 N i n H C 1 at 0 ° f o l l o w e d by c e n t r i f u g a t i o n . In the a b s e n c e of p a n c r e a t i c r i b o n u c l e a s e , l e s s t h a n 2% of the u l t r a v i o l e t a b s o r b i n g m a t e r i a l r e m a i n e d i n the s u p e r n a t e . F i g u r e 3 s h o w s the r e l e a s e of a c i d - s o l u b l e u l t r a v i o l e t a b s o r b i n g m a t e r i a l w h e n s - R N A i s d i g e s t e d w i t h , p a n c r e a t i c r i b o n u c l e a s e at r o o m t e m p e r a t u r e i n the p r e s e n c e of d i f f e r e n t c o n c e n t r a t i o n s of m a g n e s i u m i o n s . T h e r e s u l t s a r e e x p r e s s e d as p e r c e n t of the a c i d - s o l u b l e m a t e r i a l r e l e a s e d by r i b o n u c l e a s e d u r i n g 30 m i n at r o o m t e m p e r a t u r e i n the a b s e n c e of m a g n e s i u m i o n s . In the a b s e n c e of m a g n e s i u m , the h y d r o l y s i s a g a i n a p p e a r s c o m p l e t e i n t h r e e m i n , but i n the p r e s e n c e of 0. 05 M and 0. 10 M m a g n e s i u m the h y d r o l y s i s w a s o n l y 34% a n d 23% c o m p l e t e , r e s p e c t i v e l y . H o w e v e r , w h e n t h e h y p e r c h r o m i c s h i f t at 260 my. w a s u s e d to m e a s u r e the extent of h y d r o l y s i s , v a l u e s of 55% and 27% ( i n 0. 05 M a n d 0. 10 M m a g -n e s i u m , r e s p e c t i v e l y ) w e r e o b t a i n e d . A t t a c k by the e n z y m e on the l o o p r e g i o n s of s - R N A w o u l d c a u s e a h y p e r c h r o m i c s h i f t due to c l e a v a g e of the p h o s p h o d i e s t e r , l i n k a g e w h i c h c o u l d be at l e a s t p a r t i a l l y of fset by an. i n c r e a s e i n the s t r e n g t h of the i n t e r a c t i o n s i n the b a s e p a i r e d r e g i o n s a d j a c e n t to the l o o p . T h i s e f fect w o u l d l o w e r the h y p e r c h r o m i c s h i f t . O n the o t h e r h a n d , i f l a r g e f r a g -m e n t s w e r e r e l e a s e d t h e s e w o u l d be p r e c i p i t a t e d u n d e r the c o n d i t i o n s u s e d to m e a s u r e the extent of h y d r o l y s i s by the r e l e a s e of a c i d - s o l u b l e m a t e r i a l s i n c e m a g n e s i u m i s k n o w n to e n h a n c e the p r e c i p i t a t i o n of p o l y n u c l e o t i d e s by e t h a n o l ( R a z z e l l , . 1963). S i n c e b o t h e f f e c t s w o u l d t e n d to g i v e l e s s t h a n the t r u e v a l u e f o r the extent of h y d r o l y s i s i t s e e m s - 3 8 -p r o b a b l e that the h y p e r c h r o m i a s h i f t at 260 m j x i s c l o s e s t to the t r u e e x p r e s s i o n f o r the extent of h y d r o l y s i s . B o t h m e t h o d s f o r m e a s u r i n g the extent of h y d r o l y s i s s h o w a n i n i t i a l l y r a p i d r a t e w h i c h d e c r e a s e s e x p o n e n t i a l l y as the m a g n e s i u m i o n c o n c e n t r a t i o n i n c r e a s e s . T h e m o s t p r o b a b l e e x p l a n a t i o n f o r t h i s o b s e r v a t i o n i s that the m a g n e s i u m i o n s s t a b i l i z e the s e c o n d a r y s t r u c t u r e of s - R N A a n d thus p r o t e c t the r i b o n u c l e a s e s e n s i t i v e s i t e s w h i c h a r e p r e s e n t i n h e l i c a l r e g i o n s . In t h i s c o n n e c t i o n i t s h o u l d be p o i n t e d out that c o m p l e m e n t a r y R N A b e c o m e s r e s i s t a n t t o ' p a n c r e a t i c r i b o n u c l e a s e a f t e r i t a s s u m e s a d o u b l e - s t r a n d e d s t r u c t u r e ( G e i d u s c h e k et a l . , 1962). B i n d i n g of m a g n e s i u m i o n s t o the p h o s p h a t e g r o u p s w o u l d t e n d to n e u t r a l i z e o r s h i e l d the r e p u l s i v e f o r c e s b e t w e e n , p h o s p h a t e g r o u p s thus d e c r e a s i n g a n y t e n d e n c y t o w a r d s t r a n d s e p a r a t i o n . If t h i s i s t r u e f o r s - R N A t h e n the s e c o n d a r y s t r u c t u r e of c o m p l e m e n t a r y r e g i o n s w o u l d r e m a i n i n t a c t , e v e n i n m o l e c u l e s w h i c h have s u f f e r e d a few e n z y m a t i c c l e a v a g e s . W h e n m a g n e s i u m i s a b s e n t f r o m the r e a c t i o n m i x t u r e the s i t e s i n the n o n - h e l i c a l r e g i o n s w h i c h a r e s u s c e p t i b l e to a t t a c k b y p a n c r e a t i c r i b o n u c l e a s e m i g h t s t i l l be c l e a v e d f i r s t . F o l l o w i n g t h e s e c l e a v a g e s h o w e v e r , the s e c o n d a r y s t r u c t u r e m i g h t be s u f f i c i e n t l y u n s t a b l e to r e n d e r o t h e r ' b o n d s s u s c e p t i b l e , p a r t i c u l a r l y i f the r i b o n u c l e a s e i t s e l f s t i l l r e t a i n s s o m e a f f i n i t y f o r the d o u b l e - s t r a n d e d s t r u c t u r e . R e l a t i v e - 3 9 -to t h i s p o s t u l a t e i s the s t u d y c a r r i e d out b y F e l s e n f e l d et a l . (1963) i n w h i c h t h e y s h o w e d that r i b o n u c l e a s e h a d a p r o n o u n c e d d e s t a b i l i z i n g ef fect on the h e l i c a l s t r u c t u r e of D N A . I n l i g h t of the a b o v e o b s e r v a t i o n s that s - R N A , i n the a b s e n c e of m a g n e s i u m , i s s u s c e p t i b l e to p a n c r e a t i c r i b o n u c l e a s e to the extent that a l a r g e n u m b e r of s m a l l o l i g o n u c l e o t i d e f r a g m e n t s a r e r e l e a s e d , ( I n g r a m a n d P i e r c e , 1962) w h i l e c o m p l e m e n t a r y R N A i s not s u s c e p t i b l e , i t i s u n l i k e l y that l o n g c o m p l e m e n t a r y r e g i o n s e x i s t i n s - R N A as s u g g e s t e d by C a n t o n i ' s m o d e l ( M c C u l l y a n d C a n t o n i , . 1962). It s e e m s r a t h e r m o r e p r o b a b l e that the c o m p l e m e n t a r y r e g i o n s a r e i n t e r r u p t e d by l o o p s of n o n -h e l i c a l s e q u e n c e s a l o n g the l e n g t h of the m o l e c u l e as s u g g e s t e d by F r e s c o et a l . (I960). T h e c o m p l e t e s e q u e n c e s of f i v e s - R N A m o l e c u l e s have b e e n e l u c i d a t e d s i n c e t h i s s t u d y w a s c a r r i e d out a n d t h e i r m o s t p r o b a b l e s t r u c t u r e s e e m s t o c o n f o r m to a c l o v e r l e a f a r r a n g e m e n t c o n s i s t i n g of t h r e e m a j o r l o o p s a n d a m i n o r l o o p ( H o l l e y _et a l . ,. 1965; M a d i s o n e t a l . , 1966; Z a c h a u et a l . , 1966; R a j B h a n d a r y et a l . ,. 1967). W h e n l o s s of a m i n o a c i d a c c e p t o r f u n c t i o n of s - R N A w a s t a k e n as a m e a s u r e of h y d r o l y s i s , N i s h i m u r a a n d N o v e l l i (1963), s h o w e d that m a g n e s i u m w a s u n a b l e to p r o t e c t l e u c i n e s - R N A . T h e y s u g g e s t e d that the - C C A a m i n o a c i d a c c e p t o r e n d d i d not p a r t i c i p a t e i n h e l i x f o r m a t i o n and w a s t h e r e f o r e e a s i l y h y d r o l y z e d by p a n c r e a t i c r i b o n u c l e a s e . In s u p p o r t of t h i s h y p o t h e s i s t h e s e w o r k e r s s h o w e d that B . s u b t i l i s - 4 0 -r i b o n u e l e a s e , w h i c h p r o d u c e s m a i n l y 3 ' - p u r i n e n u c l e o t i d e s a n d t h e r e f o r e cannot a t t a c k the - C C A e n d , w a s m a r k e d l y i n h i b i t e d by m a g n e s i u m i o n s . In a d d i t i o n t h e s e w o r k e r s s h o w e d a s t r i k i n g s h i f t i n the m e l t i n g c u r v e of s - R N A i n the p r e s e n c e of m a g n e s i u m i o n s , w h i c h w a s not p a r a l l e l to the o r i g i n a l c u r v e as e x p e c t e d by i n c r e a s i n g the s a l t c o n c e n t r a t i o n ( T i s s i e r e s , 1959). T h e r e s u l t s r e p o r t e d . i n t h i s t h e s i s a r e c o n s i s t e n t w i t h o t h e r e v i d e n c e i n s u p p o r t of the h y p o t h e s i s that m a g n e s i u m i o n s a f fect the p h y s i c a l s tate of s - R N A , c a u s i n g i t to a s s u m e a m o r e r i g i d s e c o n d a r y s t r u c t u r e a n d t h e r e b y r e n d e r i n g i t m o r e r e s i s t a n t to a t t a c k b y p a n c r e a t i c r i b o n u c l e a s e . H a v i n g e s t a b l i s h e d the f e a s i b i l i t y of c o n t r o l l i n g the e x t e n t of h y d r o l y s i s of s - R N A w i t h p a n c r e a t i c r i b o n u c l e a s e by c a r r y i n g out t h e d i g e s t i o n . i n the- p r e s e n c e of m a g n e s i u m i o n s , i t b e c a m e n e c e s s a r y to r e m o v e the e n z y m e at the e n d of the s p e c i f i e d r e a c t i o n t i m e . I n i t i a l l y , e n z y m a t i c d i g e s t i o n of r i b o n u c l e a s e w a s a t t e m p t e d u s i n g P r o n a s e , a b r o a d s p e c t r u m p r o t e a s e i s o l a t e d f r o m S t r e p t o m y c e s g r i s e u s ( N o m o t o a n d N a r a h a s h i , . 1959; N o m o t o et a l . , I960) . P r o n a s e f a i l e d to s u p p r e s s the c a t a l y t i c a c t i v i t y of p a n c r e a t i c r i b o n u c l e a s e u n d e r the c o n d i t i o n s s t u d i e d . A r e c e n t r e p o r t on the s u p p r e s s i o n of r i b o n u c l e a s e a c t i v i t y by P r o n a s e ( H u p p e r t a n d S e m m e l , . 1965) s h o w e d that a t w e n t y f o l d e x c e s s of P r o n a s e a n d an i n c u b a t i o n p e r i o d of 2 h r at 3 7 ° w a s r e q u i r e d to s u p p r e s s 9 9 . 8% of r i b o n u c l e a s e a c t i v i t y . In the p r e s e n t w o r k , the r e m o v a l of r i b o n u c l e a s e f r o m d i g e s t i o n m i x t u r e s w a s r e a d i l y a c h i e v e d - 4 1 -by d i l u t i n g the d i g e s t w i t h i c e - c o l d b u f f e r t h e n f i l t e r i n g i t t h r o u g h a c o l u m n of C M - c e l l u l o s e . T h e f i l t r a t e c o n t a i n e d no d e t e c t a b l e r i b o -n u c l e a s e a c t i v i t y . N o s i g n i f i c a n t d e g r a d a t i o n of s - R N A o c c u r r e d d u r i n g the d i l u t i o n a n d s u b s e q u e n t f i l t r a t i o n s teps as d e t e r m i n e d by (i) the h y p e r c h r o m i c i t y at 260 m p . w h e n an a l i q u o t of the f i l t r a t e w a s h e a t e d a n d r a p i d l y c o o l e d a n d ( i i ) the o b s e r v e d h y p e r c h r o m i c i t y at 260 m p . w h e n r i b o n u c l e a s e w a s a d d e d to a n a l i q u o t of the f i l t r a t e . D u r i n g , a t t e m p t s to r e m o v e the r i b o n u c l e a s e f r o m r e a c t i o n m i x t u r e s a s o l u t i o n . c o n t a i n i n g s - R N A a n d m a g n e s i u m w a s p r e p a r e d a n d c o o l e d to 0° p r i o r to the a d d i t i o n of r i b o n u c l e a s e to d e t e r m i n e i f the c a t a l y t i c a c t i v i t y c o u l d be q u e n c h e d . W h e n , r i b o n u c l e a s e w a s a d d e d ( i n a r a t i o , of 5:1 ( w / w ) , s - R N A : R N a s e ) at 0° a n d the d i g e s t i o n m i x t u r e m a i n t a i n e d at 0 ° , no h y p e r c h r o m i c i t y at 260 m p _ w a s o b s e r v e d . S t u d i e s at r o o m t e m p e r a t u r e , i n w h i c h the r a t i o of s - R N A to r i b o n u c l e a s e w a s 7:1, s h o w e d a r e a d i l y d e t e c t a b l e h y p e r c h r o m i c s h i f t at 260 myj. . T h i s s u g g e s t e d that b e s i d e s the r a t i o of s - R N A to r i b o n u c l e a s e a n d the m a g n e s i u m i o n c o n c e n t r a t i o n , a t h i r d p a r a m e t e r n a m e l y t e m p e r a t u r e , c o u l d be i n t r o d u c e d to c o n t r o l the extent of h y d r o l y s i s . A c c o r d i n g l y , a s tudy of the d i g e s t i o n of s - R N A by p a n c r e a t i c o r i b o n u c l e a s e i n the p r e s e n c e of m a g n e s i u m i o n s w a s c a r r i e d out at 0 . A l t h o u g h the s t u d y s h o w n i n F i g u r e 3,. w h i c h w a s c a r r i e d out at r o o m t e m p e r a t u r e , c o n t a i n e d t h r e e t i m e s the a m o u n t of r i b o n u c l e a s e u s e d i n - 4 2 -the s t u d y s h o w n i n F i g u r e 4 , the d e c r e a s e i n the extent of h y d r o l y s i s w h e n the d i g e s t i o n . w a s c a r r i e d out at the l o w e r t e m p e r a t u r e w a s too l a r g e to a t t r i b u t e o n l y to the d e c r e a s e d r i b o n u c l e a s e c o n c e n t r a t i o n . T h e extent of h y d r o l y s i s i n 20 m i n at r o o m t e m p e r a t u r e (about 23°) and at 0 ° , at s e v e r a l c o n c e n t r a t i o n s of m a g n e s i u m i s c o m p a r e d i n T a b l e I. 1 T A B L E I C o m p a r i s o n . o f the E x t e n t of H y d r o l y s i s of s - R N A w i t h . P a n c r e a t i c R i b o n u c l e a s e u n d e r V a r i o u s C o n d i t i o n s C o n c e n t r a t i o n of M a g n e s i u m Ions E X T E N T O F H Y D R O L Y S I S (%) I N 20 M I N U T E S R o o m T e m p e r a t u r e 0 0 16:1 a 50:1 a 500:1 a 0 100 92 47 0 . 0 1 71 33 0. 05 34 19 8 0. 10 23 11 5 0. 20 13 8. 5 2. 5 a R a t i o of s - R N A : R N a s e (w/w) It i s i m m e d i a t e l y a p p a r e n t that the r a t i o of s - R N A to r i b o n u c l e a s e , the c o n c e n t r a t i o n of m a g n e s i u m i o n s a n d the t e m p e r a t u r e , a r e a l l i m p o r t a n t p a r a m e t e r s w h i c h c a n be c o n t r o l l e d f o r the l i m i t e d h y d r o l y s i s of s - R N A . - 4 3 -T h e e x p o n e n t i a l d e c r e a s e i n the extent of h y d r o l y s i s w i t h i n c r e a s -i n g c o n c e n t r a t i o n of m a g n e s i u m at r o o m t e m p e r a t u r e , as s h o w n i n the s e c o n d c o l u m n of T a b l e I , has b e e n d i s c u s s e d p r e v i o u s l y . A s i m i l a r r e l a t i o n s h i p e x i s t s f o r the d i g e s t i o n s c a r r i e d out at 0° ( c o l u m n s 3 a n d 4 of T a b l e I). A l s o a p p a r e n t f r o m c o l u m n s 3 a n d 4 i s that a t e n - f o l d d e c r e a s e i n the r e l a t i v e c o n c e n t r a t i o n of p a n c r e a t i c r i b o n u c l e a s e a p p r o x i m a t e l y d o u b l e s the p r o t e c t i v e i n f l u e n c e at a g i v e n m a g n e s i u m c o n c e n t r a t i o n ( F i g s 4 a n d 5). In the p r e s e n c e of 0. 20 M m a g n e s i u m , a 500:1 r a t i o (w/w) of o s - R N A to r i b o n u c l e a s e a n d 0 , the extent of h y d r o l y s i s w a s 2. 5%. T h e c l e a v a g e of t w o n u c l e o t i d e s f r o m the - C C A e n d of a m o l e c u l e c o n s i s t i n g of 80 n u c l e o t i d e s w o u l d c o r r e s p o n d to 2. 5% r e l e a s e of a c i d - s o l u b l e m a t e r i a l . T h e h y p e r c h r o m i c s h i f t at 260 mju., w h e n s - R N A i s d i g e s t e d by p a n c r e a t i c r i b o n u c l e a s e i n the p r e s e n c e of s p e r m i n e i s s h o w n i n F i g u r e 6. T h e p r e s e n c e of s p e r m i n e c l e a r l y m a s k s the c a t a l y t i c a c t i o n of r i b o n u c l e a s e a l t h o u g h a p r e v i o u s r e p o r t s h o w e d that s p e r m i n e d i d not i n h i b i t the c a t a l y t i c a c t i v i t y of E. c o l i r i b o n u c l e a s e on E. c o l i r i b o s o m a l R N A ( T h o m a s a n d H u b s t , 1963). T h u s s p e r m i n e p r o b a b l y a c t s on the s - R N A s u b s t r a t e r a t h e r t h a n the r i b o n u c l e a s e m o l e c u l e . C o m p l e x i n g b e t w e e n n u c l e i c a c i d s a n d p o l y a m i n e s h a s b e e n the s u b j e c t of a l a r g e n u m b e r of s t u d i e s ( T a b o r et a l . , 1961). - 4 4 -S p e r m i n e c o n c e n t r a t i o n s of 1. 0 m M and h i g h e r c a u s e d the s - R N A t o p r e c i p i t a t e u n l e s s the i o n i c s t r e n g t h of the s o l u t i o n w a s i n c r e a s e d . T h i s i n t e r d e p e n d e n c e b e t w e e n . i o n i c s t r e n g t h of the s o l u t i o n , a n d the s o l u b i l i t y of the s p e r m i n e - s - R N A c o m p l e x w a s d e s c r i b e d by C a n t o n i (I960). C o m p a r i s o n of F i g u r e s 5 a n d 6 i n w h i c h the e n z y m e t o s - R N A r a t i o s a r e b o t h 1:500 (w/w) c l e a r l y s h o w s that s p e r m i n e i s l e s s e f f e c t i v e t h a n m a g n e s i u m as a n i n h i b i t o r of p a n c r e a t i c r i b o n u c l e a s e . A l t h o u g h the m e c h a n i s m of the i n h i b i t i o n of p a n c r e a t i c r i b o n u c l e a s e b y s p e r m i n e i s -p r o b a b l y a t t r i b u t a b l e to n e u t r a l i z a t i o n of the p h o s p h a t e g r o u p s i n the s - R N A w i t h a c o n s e q u e n t i n c r e a s e i n the e f f e c t i v e n e s s of h y d r o g e n • b o n d i n g , o t h e r e x p l a n a t i o n s s u c h - a s c r o s s l i n k a g e , c a n n o t be c o m p l e t e l y e x c l u d e d . It w a s i n f a c t h o p e d that c o m p l e x i n g b e t w e e n the s - R N A a n d s p e r m i n e w o u l d r e n d e r s u s c e p t i b l e s i t e s i n the s - R N A cWain u n a v a i l a b l e to r i b o n u c l e a s e . T h i s e x p e c t a t i o n . w a s not r e a l i z e d a n d b e c a u s e of the m o r e p r o n o u n c e d e f fec ts of m a g n e s i u m , the l a r g e s c a l e d i g e s t i o n s w e r e c a r r i e d . o u t i n the- p r e s e n c e of 0. 2 M m a g n e s i u m . W h e n s - R N A w a s d i g e s t e d at r o o m t e m p e r a t u r e f o r 25 m i n i n 0. 2 M m a g n e s i u m w i t h a n s - R N A t o r i b o n u c l e a s e r a t i o of 5 0 0 : 1 , the r e c o v e r y of O . D . u n i t s s u g g e s t e d that the s - R N A w a s 2 5 - 3 0 % d e g r a d e d . T h i s w a s the e x p e c t e d o r d e r of m a g n i t u d e . H o w e v e r w h e n the d i g e s t w a s c h r o m a t o g r a p h e d on D E A E - c e l l u l o s e m o r e t h a n 75% of the O . D . u n i t s w e r e e l u t e d b e f o r e the s a l t g r a d i e n t h a d r e a c h e d 0. 13 M N a C l ( F i g . 7). T h u s a l a r g e n u m b e r of s m a l l o l i g o n u c l e o t i d e f r a g m e n t s w e r e r e l e a s e d even-, i n the p r e s e n c e of 0. 2 M m a g n e s i u m . T h i s r e s u l t s u g g e s t s that u n d e r the c o n d i t i o n s u s e d . i n t h i s e x p e r i m e n t the r i b o n u c l e a s e h a d c l e a v e d m a n y m o r e l i n k a g e s t h a n w a s i n d i c a t e d by the h y p e r c h r o m i c s h i f t . T h e e a s i e s t e x p l a n a t i o n f o r t h i s o b s e r v a t i o n . i s that w h e n the l i n k a g e s a long, the c h a i n . a r e c l e a v e d the s - R N A m o l e c u l e s t i l l r e t a i n s s o m e s e m b l a n c e of o r d e r due to the s t a b i l i z i n g i n f l u e n c e of m a g n e s i u m . L o w e r i n g , the t e m p e r a t u r e to 0 ° a n d i n c r e a s i n g the s - R N A : r i b o n u c l e a s e ratio;: to 2000:1 (w/w) i n the p r e s e n c e of 0. 20 M m a g n e s i u m gave the d e s i r e d s e l e c t i v e h y d r o l y s i s . F i g u r e 8 s h o w s the e l u t i o n p r o f i l e of the d i g e s t i o n p r o d u c t s f r o m D E A E - c e l l u l o s e c h r o m a t o g r a p h y . A b o u t 75% of the t o t a l d i g e s t i o n . p r o d u c t s w e r e of m e d i u m to h i g h m o l e c u l a r w e i g h t ( f r a c t i o n II) . T h i s i s i n c l o s e a g r e e m e n t w i t h the v a l u e of about 65% o b t a i n e d by L i t t and I n g r a m , ( 1 9 6 4 ) f o r the d i g e s t i o n of s - R N A i n the p r e s e n c e of 0. 1 0 : M m a g n e s i u m at 0 9 . R e c h r o m a t o g r a p h y of f r a c t i o n II o n D E A E - c e l l u l o s e ( F i g . 9) s h o w e d that 71% of the O . D . i n f r a c t i o n II c o n s i s t e d of i n t e r m e d i a t e m o l e c u l a r w e i g h t m a t e r i a l ( f r a c t i o n s A a n d B ) w h i l e the r e m a i n i n g 29% w a s e l u t e d by a s a l t c o n c e n t r a t i o n c o r r e s p o n d i n g . t o u n d e g r a d e d s - R N A ( f r a c t i o n C ) . It m u s t be e m p h a s i z e d at t h i s p o i n t , that u n t i l the s p e c i f i c i t y of t h i s r e a c t i o n h a s b e e n e s t a b l i s h e d . i t cannot be s t a t e d u n e q u i v o c a l l y that the h y d r o l y s i s has b e e n - 4 6 -c o m p l e t e l y s e l e c t i v e . T h e ef fect of t e m p e r a t u r e o n the r a t e of h y d r o l y s i s and the d e c r e a s e d a m o u n t of e n z y m e u s e d . i n t h i s e x p e r i m e n t h a v e not b e e n e x c l u d e d as a l t e r n a t i v e e x p l a n a t i o n s f o r the o b s e r v e d , l i m i t e d h y d r o l y s i s . T h e d i s t r i b u t i o n of m a j o r a n d m i n o r b a s e s i n F r a c t i o n A (30% of the O . D . b e f o r e d i g e s t i o n ) w a s d e t e r m i n e d by the m e t h o d of B e l l et a l . (1964). T h e r e s u l t s a r e l i s t e d i n T a b l e I I . T A B L E II B a s e C o m p o s i t i o n of F r a c t i o n A ( F i g . 10) N u c l e o t i d e F r a c t i o n A U n d e g r a d e d A 14. 0 16 .7 U 27. 7 18. 3 G 10. 7 26. 9 C 21. 0 28. 5 8. 8 3. 15 T 4 . 8 0. 9 1 - M e G 1. 7 1. 15 N , N - d i M e G 2. 0 0. 6 U n i d e n t i f i e d 9. 3 V a l u e s f o r u n d e g r a d e d s - R N A f r o m B e l l et a l . (1964) - 4 7 -T h e p r o t e c t i v e i n f l u e n c e of m a g n e s i u m i s p a r t i c u l a r l y e v i d e n t f r o m the e n r i c h m e n t of U i n the i n t e r m e d i a t e m o l e c u l a r w e i g h t m a t e r i a l . F r a c t i o n A i s a l s o e n r i c h e d i n the odd b a s e s , \K (2. 8x) , T ( 5 . 3x), N , N - d i m e t h y l G (3 . 3x) , 1 - m e t h y l G (1. 5x) . S i n c e t h i s s tudy w a s c a r r i e d out, r e p o r t s h a v e b e e n . p u b l i s h e d i n c o n s i d e r a b l e d e t a i l by I n g r a m a n d c o - w o r k e r s ( L i t t a n d I n g r a m , 1964; A r m s t r o n g et a l . ,. 1964; W a g n e r and. I n g r a m , 1966; A r m s t r o n g et a l . , 1966), w h o s e r e s u l t s a r e i n q u a l i t a t i v e a g r e e m e n t w i t h t h o s e r e p o r t e d h e r e . F u r t h e r s t u d y on m i x e d s - R N A w a s d i s c o n t i n u e d w h e n L i t t a n d I n g r a m (1964), p u b l i s h e d the r e s u l t s of t h e i r s tudy o n t h e c o n t r o l l e d d e g r a d a t i o n of s - R N A w i t h p a n c r e a t i c r i b o n u c l e a s e i n the p r e s e n c e of m a g n e s i u m at 0 ° . E f f o r t s w e r e d i r e c t e d : i n s t e a d t o w a r d the f r a c t i o n a t i o n of m i x e d s - R N A s i n c e o n l y on p u r i f i e d s - R N A c o u l d the s p e c i f i c i t y of t h e s e r e a c t i o n s be e s t a b l i s h e d . If s o m e s p e c i e s of s - R N A w e r e m o r e s u s c e p t i b l e to r i b o n u c l e a s e t h a n o t h e r s , e i t h e r b e c a u s e of d i f f e r e n c e s i n t h e i r s e c o n d a r y s t r u c t u r e a n d for b a s e c o m p o s i t i o n , t h e n i t i s p o s s i b l e that c e r t a i n s - R N A m o l e c u l e s w o u l d be l a r g e l y d e g r a d e d w h i l e o t h e r s r e m a i n l a r g e l y i n t a c t . S u c h a s i t u a t i o n has b e e n s u g g e s t e d b y N i s h i m u r a and N o v e l l i (1963) b a s e d on the r e s u l t s of a s tudy of the r e s i s t a n c e of s-R N A t o r i b o n u c l e a s e s i n the p r e s e n c e of m a g n e s i u m as m e a s u r e d by the l o s s of a m i n o a c i d a c c e p t o r f u n c t i o n s . T h u s the s - R N A ' s w i t h the m o s t - 4 8 -odd b a s e s m i g h t have a l e s s s t a b l e s e c o n d a r y s t r u c t u r e a n d as a r e s u l t , be m o r e s u s c e p t i b l e to r i b o n u c l e a s e . O p t i c a l r o t a t o r y d i s p e r s i o n s t u d i e s r e v e a l e d e v i d e n c e p e r t a i n i n g to d i f f e r e n c e s i n the s e c o n d a r y s t r u c t u r e a m o n g p u r i f i e d s a m p l e s of y e a s t s - R N A (Sarin_et a l . , 1966). D i f f e r e n c e s i n the b a s e c o m p o s i t i o n of f r a c t i o n a t e d s - R N A have a l s o b e e n r e p o r t e d ( B r o w n , 1963). E v i d e n c e r e l a t i n g to d i f f e r e n c e s i n the s e c o n d a r y s t r u c t u r e b e t w e e n m i x e d s - R N A a n d a p a r t i a l l y p u r i f i e d f r a c t i o n w a s o b t a i n e d . i n the p r e s e n t w o r k b y a s tudy of the a b i l i t y of m a g n e s i u m t o i n h i b i t the a c t i o n of p a n c r e a t i c r i b o n u c l e a s e . M i x e d s - R N A , a n d a f r a c t i o n e n r i c h e d 1 2 - f o l d . i n g l y c i n e a c c e p t o r a c t i v i t y b y c h r o m a t o g r a p h y o n b e n z o y l a t e d D E A E - c e l l u l o s e ( T e n e r et a l . , 1966), w e r e c h e c k e d f o r h o m o g e n e i t y w i t h r e s p e c t to m o l e c u l a r s i z e ( F i g s 11-A, B and. C ) , t h e n d i g e s t e d w i t h p a n c r e a t i c r i b o n u c l e a s e i n the p r e s e n c e a n d a b s e n c e of m a g n e s i u m ( F i g . 12). In the p r e s e n c e of m a g n e s i u m the p a r t i a l l y p u r i f i e d s a m p l e s h o w e d a n 8.1% h y p e r c h r o m i c s h i f t c o m p a r e d to 1 2 . 4 % h y p e r c h r o m i c i t y f o r m i x e d s - R N A o v e r a p e r i o d of 30 m i n . T h u s the p r o t e c t i v e i n f l u e n c e of m a g n e s i u m w a s g r e a t e r f o r the p a r t i a l l y p u r i f i e d g l y c i n e s - R N A t h a n f o r the m i x e d s - R N A . T h e d i f f e r e n t s u s c e p t i b i l i t i e s of the t w o s - R N A f r a c t i o n s to r i b o n u c l e a s e i n the p r e s e n c e of m a g n e s i u m w a s p r o b a b l y not due to c o n t a m i n a t i o n of the m i x e d s - R N A s a m p l e b y n o n s - R N A ( w h i c h - 4 9 -r n i g h t not be p r o t e c t e d b y m a g n e s i u m a g a i n s t r i b o n u c l e a s e a t t a c k ) s i n c e the m i x e d s - R N A f r a c t i o n h a d b e e n f r e e d of o t h e r c o m p o n e n t s by p a s s a g e t h r o u g h a G-100 S e p h a d e x c o l u m n p r i o r to d i g e s t i o n . In the a b s e n c e of m a g n e s i u m , the p a r t i a l l y , p u r i f i e d s a m p l e ( f r o m • p e a k . I , . F i g . 11-C) s h o w e d 54% m o r e h y p e r c h r o m i c i t y t h a n the m i x e d s-R N A s a m p l e ( f r o m p e a k II, F i g . 11- A ) . T h i s i s a s i g n i f i c a n t d i f f e r e n c e w h i c h c a n be a t t r i b u t e d e i t h e r to a g r e a t e r n u m b e r of s u s c e p t i b l e s i t e s , a n d t h e r e f o r e a g r e a t e r p y r i m i d i n e c o n t e n t i n the p a r t i a l l y p u r i f i e d g l y c i n e s - R N A c o m p a r e d to the a v e r a g e s - R N A m o l e c u l e , o r to a m o r e r i g i d s e c o n d a r y s t r u c t u r e t h a n the a v e r a g e s - R N A m o l e c u l e , w h i c h u p o n d i s r u p t i o n w o u l d l e a d to a l a r g e r h y p e r c h r o m i c e f fect . T h e l a r g e r h y p e r c h r o m i c i t y a s s o c i a t e d w i t h d i g e s t i o n of the p a r t i a l l y p u r i f i e d f r a c t i o n i s b e l i e v e d t o be due to i t s m o r e r i g i d s e c o n d a r y s t r u c t u r e f o r the f o l l o w i n g r e a s o n s : 1) i f the odd b a s e s i n s - R N A a r e to be c o n s i d e r e d i n d i s p e n s i b l e , t h e n a n . i n c r e a s e i n . p y r i m i d i n e content above that of the a v e r a g e s - R N A m o l e c u l e , m u s t be at the e x p e n s e of the p u r i n e content ; 2) s i n c e the U a n d C of m i x e d s - R N A (18. 3% a n d 28. 5% r e s p e c t i v e l y ) a l r e a d y e x c e e d s the A a n d G c o n t e n t (16. 7% a n d 26. 9% r e s p e c t i v e l y ) ( B e l l et a l . , 1964), i t i s d i f f i c u l t t o r e c o n c i l e a n i n c r e a s e d p y r i m i d i n e c o n t e n t w i t h i n c r e a s e d d o u b l e - h e l i c a l r e g i o n s ; 3) i t f o l l o w s f r o m (2) that a n i n c r e a s e d p y r i m i d i n e c o n t e n t l e a d s to a d e c r e a s e d d o u b l e -h e l i c a l content ( c o m p a r e d to the a v e r a g e i n m i x e d s - R N A ) a n d t h e r e f o r e , - 5 0 -the p r o t e c t i o n a f f o r d e d by m a g n e s i u m a g a i n s t r i b o n u c l e a s e a t t a c k s h o u l d be d i m i n i s h e d . H o w e v e r the r e s u l t s i n F i g u r e 12 c l e a r l y s h o w t h i s not to be the c a s e . It m u s t be e m p h a s i z e d h e r e that i n . t h e a b o v e d i s c u s s i o n the c o n c e p t of s e c o n d a r y s t r u c t u r e h a s b e e n v i e w e d i n the c l a s s i c a l t e r m s of h y d r o g e n b o n d i n g a n d b a s e s t a c k i n g i n t h o s e r e g i o n s a l o n g : t h e l e n g t h of the m o l e c u l e w h i c h a r e c a p a b l e of f o r m i n g a d o u b l e h e l i x . O n the b a s i s of h y d r o g e n . e x c h a n g e s t u d i e s , E n g l a n d e r a n d E n g l a n d e r (1965) h a v e c o n c l u d e d that about 8 0 % of the n u c l e o t i d e s i n a t y p i c a l s - R N A m o l e c u l e a r e h y d r o g e n b o n d e d . T h e r m a l d e n a t u r a t i o n s t u d i e s ( F r e s c o et a l . , 1963; F e l s e n f e l d a n d C a n t o n i , 1964) have l e d to the c o n c l u s i o n that as m u c h as 75% of the b a s e s i n u n f r a c t i o n a t e d s - R N A c o u l d be p a i r e d i n h y d r o g e n b o n d e d d o u b l e h e l i c e s of the D N A t y p e . K e s e l e v et a l . (1964) s t u d i e d the r a t e of l i b e r a t i o n of a c i d -s o l u b l e n u c l e o t i d e s f r o m m i x e d y e a s t s - R N A by a t t a c k w i t h . p a n c r e a t i c r i b o n u c l e a s e at 20° i n the a b s e n c e of m a g n e s i u m ; t h e y c o n c l u d e d that about o n e - q u a r t e r of the m i x e d s - R N A ' m o l e c u l e ' w a s c o m p l e t e l y f r e e of h y d r o g e n b o n d i n g , about o n e - h a l f w a s f u l l y h y d r o g e n b o n d e d a n d the r e m a i n d e r ^ w a s ' i m p e r f e c t l y ' h y d r o g e n b o n d e d a n d s l o w l y l i b e r a t e d by the a c t i o n of the e n z y m e s . T h e p r o p o s e d c o n f o r m a t i o n s of the s - R N A s p e c i e s w h o s e , p r i m a r y s t r u c t u r e s h a v e b e e n e l u c i d a t e d s h o w that about 50% of the c o n s t i t u e n t n u c l e o t i d e s c o u l d f o r m b a s e p a i r s of the W a t s o n -- 5 1 -C r i c k t y p e . It t h e r e f o r e s e e m s l i k e l y that w e s h a l l h a v e to r e v i s e o u r t h i n k i n g i n r e l a t i o n to the s e c o n d a r y s t r u c t u r e of s - R N A . In t h i s r e g a r d , i t i s not u n l i k e l y that the. p r o t e c t i v e i n f l u e n c e of m a g n e s i u m , i o n s d e s c r i b e d i n t h i s s t u d y , i s due to the a b i l i t y of m a g n e s i u m (and s p e r m i n e ) to s t a b i l i z e a f o l d e d c o n f o r m a t i o n . o f s - R N A i n w h i c h t w o of the m a j o r l o o p s a r e i n j u x t a p o s i t i o n , s u c h that h y d r o g e n b o n d i n g a n d / o r h y d r o p h o b i c i n t e r a c t i o n s c o u l d o c c u r b e t w e e n the b a s e s i n the l o o p s . M a s k i n g of the r e p u l s i v e , n e g a t i v e c h a r g e s on the p h o s p h a t e g r o u p s w o u l d c l e a r l y t e n d to e n h a n c e the s t r e n g t h of t h e s e i n t e r a c t i o n s , p a r t i c u l a r l y i f the i n t e r a c t i o n s a r e p r i m a r i l y of the s h o r t r a n g e , h y d r o p h o b i c t y p e . S u c h a c o n f o r m a t i o n c o u l d a c c o u n t f o r the o b s e r v a t i o n s m a d e i n t h i s s t u d y as w e l l as t h o s e of F r e s c o et a l . , (1963); F e l s e n f e l d a n d C a n t o n i , (1964); K e s e l e v et a l . , (1964); a n d E n g l a n d e r a n d E n g l a n d e r , (1965). B I B L I O G R A P H Y A c k e r m a n , D . (1952), Z . P h y s i o l . C h e m . 291, 169. A c k e r m a n , D . (1955), Z . P h y s i o l . C h e m . 302, 87. A r m s t r o n g , A . , H a g o p i a n , H . , I n g r a m , V . M . , S j o q u i s t , I . , a n d S j o q u i s t , J . (1964),. B i o c h e m . 3, 1194. A r m s t r o n g , A . , H a g o p i a n , H . , I n g r a m , V . M . , a n d W a g n e r , E . K . (1966), B i o c h e m . 5, 3 0 2 7 . B e l l , D . , T o m l i n s o n , R . V . , a n d T e n e r , G . M . (1964), B i o c h e m . 3, 317. B e r g , P . (1961), A n n . R e v . B i o c h e m . 30, 293. B r o w n , G . L . (1963), . . in P r o g r e s s i n N u c l e i c A c i d R e s e a r c h , v o l . 2, D a v i d s o n , J . N . , a n d C o h n , W . E . , e d s . , N e w Y o r k , A c a d e m i c , . p . 290. B r o w n , . G . L . , a n d Z u b a y , G . (I960),. J . M o l . B i o l . 2, 287. C a n t o n i , G . L . (I960), N a t u r e 188,. 300. C o h e n , G . N . , a n d G r o s , F . (I960), A n n . R e v . B i o c h e m . 29, 525. D i c k m a n , S. R . , A r a s k a r , J . P . , a n d K r o p f , R . B . (1956), • B i o c h i m . B i o p h y s . A c t a 21, 539. E n g l a n d e r , F. W . , a n d E n g l a n d e r , j . J . (1965), P r o c . 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R . , and Z a m i r , A . (1965), S c i e n c e  147, 1462. H u p p e r t , J. , a n d S e m m e l , M . (1965), B i o c h i m . B i o p h y s . A c t a 108, 501. I n g r a m , V . M . , a n d P i e r c e , J. G . (19 62), B i o c h e m . 1, 580. K e s e l e v , L . L . , F r o v o l a , L . Y . , B o r i s o v a , O . F . , and K u k h a n o v a , M . K . (1964), B i o k h i m i y a 29, 116. L i t t , M . , a n d I n g r a m , V . M . (1964), B i o c h e m . 3, 560. M a d i s o n , J. T. , E v e r e t t , G . A . , a n d K u n g , H . (1966), S c i e n c e 153, 531. M a r k h a m , R . , a n d S m i t h , J. D . (1952), B i o c h e m . J. 52, 552. M c C u l l y , K . S. , a n d C a n t o n i , G . L . (1962), J. M o l . B i o l . _5, 4 9 7 . N i s h i m u r a , S. (I960), B i o c h i m . B i o p h y s . A c t a 4 5 , 15. N i s h i m u r a , S. , a n d N o v e l l i , G . D . (1963), B i o c h e m . B i o p h y s . R e s . C o m m u n . 11, 161. N o m o t o , M . , a n d N a r a h a s h i , Y . (1959), J. B i o c h e m . 4 6 , 653. N o m o t o , M . , N a r a h a s h i , Y . , a n d M u r a k a m i , M . (I960), J. B i o c h e m . 4 8 , 5 9 3 . P e n n i s t o n , J. T. , a n d D o t y , P . (1963), B i o p o l y m e r s , _1, 145. R a j B h a n d a r y , U. L . , C h a n g , S. H . , S t u a r t , A . , F a u l k n e r , R. D . , H o s k i n s o n , R . M . , a n d K h o r a n a , H . G . (1967), P r o c . N a t l . A c a d . S c i .  U. S. 57, 751. R a z z e l l , W . E . (1963), J. B i o l . C h e m . 238, 3053. S a r i n , P . S. , Z a m e c n i k , P . C . , B e r g q u i s t , P . L . , a n d S c o t t , J. F . (1966), P r o c . N a t l . A c a d . S c i . U. S. 5J5, 579. S i m p s o n , M . V . (1962), A n n . R e v . B i o c h e m . 31, 333 . - 5 4 -T a b o r , H . , T a b o r , C . W . , a n d R o s e n t h a l , S. M . (1961), A n n . R e v . B i o c h e m . 30, 579. T a n a k a , K . (1966), i n P r o c e d u r e s i n N u c l e i c A c i d R e s e a r c h , C a n t o n i , G . L . , and D a v i e s , D . R . , e d s . , N e w Y o r k , H a r p e r a n d R o w , p. 4 6 6 . T e n e r , G . M . , G i l l a m , I. , v o n T i g e r s t r o m , M . , M i l l w a r d , S. , and W i m m e r , E . (1966), F e d . P r o c . 25, 519. T h o m a s , G . H . , a n d H u b s t , E . J . (1963), F e d . P r o c . 22, 349. T i s s i e r e s , A . (1959), J . M o l . B i o l . 1_, 365. W a g n e r , E . K . , a n d - I n g r a m , : V . M . (1966), B i o c h e m . 5, 3019. Z a c h a u , H . G . , D u t t i n g , D . , a n d F e l d m a n n , H . (1966), A n g e w a n d t e  C h e m i e 5, 4 2 2 . Z i l l i g , W . , K r o n e , W . , a n d A l b e r s , M . (1959), Z . P h y s i o l . C h e m . 317, 131. Z u b a y , G . , and M a r c i e l l o , R. (1963), B i o c h e m . B i o p h y s . R e s . C o m m u n , 11, 7 9 . P A R T II F R A C T I O N A T I O N O F Y E A S T s - R N A T A B L E O F C O N T E N T S P A G E N U M B E R L I S T O F F I G U R E S 61 L I S T O F C H A R T S 63 I N T R O D U C T I O N 64 E X P E R I M E N T A L 7 6 A b b r e v i a t i o n s U s e d 7 6 M a t e r i a l s 77 M e t h o d s 7 8 P A R T I I - A . A T T E M P T S T O I S O L A T E C Y S T E I N Y L -s - R N A B Y C H R O M A T O G R A P H Y O N M E R C U R I -C E L L U L O S E 78 1. T i t r a t i o n of A E - C e l l u l o s e w i t h A l k a l i 78 2. T h i o l a t i o n of A E - C e l l u l o s e w i t h N - A c e t y l h o m o -c y s t e i n e T h i o l a c t o n e ( H T L ) • 79 A . T h i o l a t i o n of A E - C e l l u l o s e w i t h H T L i n D i o x a n e 79 B . T h i o l a t i o n of A E - C e l l u l o s e w i t h H T L i n A q u e o u s M e d i u m i n the P r e s e n c e of S i l v e r N i t r a t e 79 3. T i t r a t i o n of H S A E - C e l l u l o s e w i t h I o d o a c e t i c A c i d ( I A A ) 80 4 . L a r g e S c a l e T h i o l a t i o n of A E - C e l l u l o s e w i t h N -A c e t y l h o m o c y s t e i n e T h i o l a c t o n e 81 •5. S y n t h e s i s of b i s - 3 , 6 - ( A c e t a t o m e r c u r i m e t h y l ) -D i o x a n e 84 - 5 7 -P A G E N U M B E R 6. P r e p a r a t i o n of the M e r c u r i - C e l l u l o s e ( H g S A E -C e l l u l o s e ) C o l u m n 85 7. B e h a v i o u r of s - R N A on H g S A E - C e l l u l o s e 86 8. P r e p a r a t i o n of A m i n o a c y l - s - R N A S y n t h e t a s e s f r o m Y e a s t 87 9. P r e p a r a t i o n , of 1 4 C - C y s t e i n y l - s - R N A 87 10. C h r o m a t o g r a p h y of ^ C - C y s t e i n y l - s - R N A on H g S A E - C e l l u l o s e 88 11. A c e t y l a t i o n , a n d D e a c e t y l a t i o n of C e l l u l o s e 88 12. P r e p a r a t i o n of T o s y l - C e l l u l o s e 89 A . R e c r y s t a l l i z a t i o n of T o s y l c h l o r i d e 89 B . T o s y l a t i o n of D e a c e t y l a t e d C e l l u l o s e A c e t a t e 90 13. C h a r a c t e r i z a t i o n of T o s y l - C e l l u l o s e 91 14. P r e p a r a t i o n of S - A c e t y l - C e l l u l o s e 92 15. H y d r o l y s i s of S - A c e t y l - C e l l u l o s e 93 • A . H y d r o l y s i s , i n 0 . 0 2 N S o d i u m M e t h o x i d e 93 B . H y d r o l y s i s i n 0.10 N S o d i u m H y d r o x i d e 93 16. L a r g e S c a l e P r e p a r a t i o n of H S - C e l l u l o s e 94 17. C h r o m a t o g r a p h y of s - R N A on H S - C e l l u l o s e 95 18. C o n v e r s i o n of H S - C e l l u l o s e to M e r c u r i - C e l l u l o s e 96 19. C h r o m a t o g r a p h y of 1 4 C - C y s t e i n y l - s - R N A on H g S - C e l l u l o s e 96 - 5 8 -P A G E N U M B E R P A R T I I - B . P A R T I T I O N C H R O M A T O G R A P H Y O F s - R N A O N B I O G E L 97 1. P a r t i t i o n C h r o m a t o g r a p h y of s - R N A on B i o g e l P - 1 0 u s i n g the S y s t e m of W a r n e r a n d V a i m b e r g (1958) 97 A . D e t e r m i n a t i o n of the D i s t r i b u t i o n C o e f f i c i e n t 99 i B . P a r t i t i o n C h r o m a t o g r a p h y of s - R N A : E x p e r i m e n t 1 99 C . P a r t i t i o n C h r o m a t o g r a p h y of s - R N A : E x p e r i m e n t 2 99 2. P a r t i t i o n C h r o m a t o g r a p h y of s - R N A o n B i o g e l P-10 U s i n g the S y s t e m of Z a c h a u et a l . (1961) , 100 A . P r e p a r a t i o n of the T r i b u t y l a m m o n i u m S a l t of s - R N A 100 B . P r e p a r a t i o n of the B i o g e l P-10 f o r P a r t i t i o n C h r o m a t o g r a p h y 101 C . P a r t i t i o n C h r o m a t o g r a p h y of s - R N A 101 D . A s s a y of the S a m p l e s O b t a i n e d f r o m P a r t i t i o n C h r o m a t o g r a p h y 102 P A R T I I - C . F R A C T I O N A T I O N O F s - R N A O N B E N Z O Y L A T E D - D E A E - C E L L U L O S E 103 1. P r e p a r a t i o n of B e n z o y l a t e d - D E A E - C e l l u l o s e 103 2. P r e p a r a t i o n of the B D E A E - C e l l u l o s e C o l u m n 104 3. C h r o m a t o g r a p h y of M o n o n u c l e o t i d e s o n B D E A E -C e l l u l o s e 105 4 . R e c o v e r y of s - R N A f r o m C o l u m n E l u a t e s 107 5. P r e p a r a t i o n of Y e a s t A m i n o a c y l - s - R N A S y n t h e t a s e E n z y m e s 109 6. A s s a y , of C o l u m n F r a c t i o n s f o r S p e c i f i c A m i n o A c i d A c c e p t o r A c t i v i t y 110 - 5 9 -P A G E N U M B E R C h r o m a t o g r a p h y of Y e a s t s - R N A on B D E A E -C e l l u l o s e u n d e r C o n d i t i o n s of V a r y i n g N a C l a n d / o r M g C l 2 113 A . N a C l G r a d i e n t i n the P r e s e n c e of 0.10 M M g C l 2 > p H 5 . 0 113 B . N a C l G r a d i e n t i n the P r e s e n c e of 0 . 0 2 M M g C l 2 , p H 5 . 0 113 C . N a C l G r a d i e n t i n the P r e s e n c e of 0. 005 M M g C l 2 , p H 5. 0 115 3 D . N a C l G r a d i e n t i n the P r e s e n c e of 10 M E D T A , p H 5. 0 117 E . M g C l 2 G r a d i e n t i n , t h e P r e s e n c e of 0. 10 M N a C l , p H 5. 0 119 F . P u l s e G r a d i e n t of N a C l i n the P r e s e n c e of 0. 005 M M g C l 2 , p H 5. 0 122 (i) C h r o m a t o g r a p h i c M e t h o d 122 ( i i ) i D e t e r m i n a t i o n of the M e l t i n g C u r v e s 124 ( i i i ) G-100 S e p h a d e x C h r o m a t o g r a p h y of F r a c t i o n s X V a n d X V I 126 G . P r e p a r a t i v e C o l u m n C h r o m a t o g r a p h y of s - R N A on B D E A E - C e l l u l o s e 126 H . R e c h r o m a t o g r a p h y of the G l y c i n e - I F r a c t i o n •. 128 (i) ; C h r o m a t o g r a p h y at p H 3. 5 i n . t h e P r e s e n c e of 0. 05 M M g C l 2 128 ( i i ) C h r o m a t o g r a p h y at p H 4. 0 i n . t h e P r e s e n c e of 10-3 M E D T A 130 J . R e c h r o m a t o g r a p h y of the G l y c i n e - I I F r a c t i o n ,130 (i) C h r o m a t o g r a p h y at p H 5 i n the P r e s e n c e of 0. 01 M M g C l 2 130 ( i i ) t C h r o m a t o g r a p h y at JDH 3. 5 , i n the P r e s e n c e of l O " 3 M E D T A 133 - 6 0 -P A G E N U M B E R P A R T I I - D . A C O M B I N E D C H E M I C A L A N D C H R O M A T O G R A P H I C M E T H O D F O R T H E I S O L A T I O N O F A M I N O A C I D - S P E C I F I C s - R N A ' s 136 1. P r e p a r a t i o n of the A c y l a t i n g A g e n t 136 2. E x p e r i m e n t I 136 A . C o n d i t i o n s f o r M a x i m u m C h a r g i n g of s - R N A w i t h G l y c i n e 136 B . P r e p a r a t i o n , o f 1 4 C - G l y c y l - s - R N A 136 C . D e r i v a t i z a t i o n . o f 1 4 C - G l y c y l - s - R N A 138 D . C h r o m a t o g r a p h y of the N - ( P h e n o x y a c e t y l ) -1 4 C - g l y c y l - s - R N A on* B D E A E - C e l l u l o s e 139 3. E x p e r i m e n t II 142 • A . C o n d i t i o n s f o r M a x i m u m C h a r g i n g of s - R N A w i t h G l y c i n e u s i n g a P a r t i a l l y P u r i f i e d G l y c y l - s - R N A S y n t h e t a s e 142 B . I s o l a t i o n of G l y c i n e - S p e c i f i c s - R N A 146 S t e p 1. R e m o v a l of P h e n y l a l a n i n e A c c e p t o r s - R N A f r o m m i x e d s - R N A 146 S t e p 2. " S h a m " P h e n o x y a c e t y l a t i o n 147 S t e p ' 3 . P r e p a r a t i o n of 1 4 C - G l y c y l - s - R N A 147 S t e p 4 . P h e n o x y a c e t y l a t i o n of ^ 4 C - G l y c y l -s - R N A 148 S t e p 5. I s o l a t i o n of the N - ( P h e n o x y a c e t y l ) -1 4 C - g l y c y l - s - R N A f r o m the M i x t u r e 149 Step 6. C h r o m a t o g r a p h y of G l y c i n e - s - R N A on B D E A E - C e l l u l o s e 152 • D I S C U S S I O N 154 B I B L I O G R A P H Y 190 - 6 1 -L I S T O F F I G U R E S F I G U R E P A G E N U M B E R 1- A , B , C T i t r a t i o n c u r v e s f o r H S A E - c e l l u l o s e 82 2- A , B C o r r e c t e d t i t r a t i o n c u r v e s f o r H S A E - c e l l u l o s e 83 2- c A p l o t of _gH v s a l k a l i c o n s u m e d f o r the t i t r a t i o n of H S A E - c e l l u l o s e w i t h I A A 83 3 C h r o m a t o g r a p h y , of ^ 4 C - c y s t e i n y l - s - R N A on H g S - c e l l u l o s e 98 4 C h r o m a t o g r a p h y of m o n o r i b o n u c l e o t i d e s on B D E A E - c e l l u l o s e 106 5 C h r o m a t o g r a p h y of m o n o r i b o n u c l e o t i d e s on B D E A E - c e l l u l o s e 108 6 F r a c t i o n a t i o n of b a k e r s ' y e a s t s - R N A on • B D E A E - c e l l u l o s e u s i n g a N a C l g r a d i e n t . i n . t h e p r e s e n c e of 0.10 M M g C l 2 , p H 5. 0 114 7 F r a c t i o n a t i o n . o f b a k e r s ' y e a s t s - R N A on B D E A E - C e l l u l o s e u s i n g a N a C l g r a d i e n t i n the p r e s e n c e of 0. 02 M : M g C l 2 , p H 5. 0 116 8 F r a c t i o n a t i o n of b a k e r s ' y e a s t s - R N A o n B D E A E - c e l l u l o s e u s i n g a N a C l g r a d i e n t i n . t h e p r e s e n c e of 0. 005 M M g C l 2 , - p H 5. 0 118 9 F r a c t i o n a t i o n of b a k e r s ' y e a s t s - R N A o n B D E A E - c e l l u l o s e u s i n g a N a C l g r a d i e n t i n . t h e p r e s e n c e of 10" 3 M E D T A , , p H 5. 0 120 10 F r a c t i o n a t i o n of b a k e r s ' y e a s t s - R N A on B D E A E - c e l l u l o s e u s i n g a M g C l 2 g r a d i e n t i n the p r e s e n c e of 0.10 M N a C l , . p H 5 . 0 121 11 A p p a r a t u s f o r s u p p l y i n g p u l s e g r a d i e n t s d u r i n g c o l u m n c h r o m a t o g r a p h y 123 - 6 2 -F I G U R E P A G E N U M B E R 12 P u l s e g r a d i e n t e l u t i o n of b a k e r s ' y e a s t s - R N A f r o m B D E A E - c e l l u l o s e . N a C l g r a d i e n t i n the p r e s e n c e of 0. 005 M M g C l 2 , p H 5 . 0 125 13 •>:Gel ' ' , f i l trat ion.- ; oi:Fract ions: X V ( F i g . ; 1 3 - A ) . : ?: r a n d - X V I (Fig.^13-B):on^Sephadex^G-lOO: 127 14 P r e p a r a t i v e c o l u m n c h r o m a t o g r a p h y of 5 g of b r e w e r s ' y e a s t s - R N A on B D E A E - c e l l u l o s e . 1 2 9 15 .. C h r o m a t o g r a p h y of G l y c i n e - I f f a c t i o n . o n B D E A E - c e l l u l o s e i n ; the- p r e s e n c e ' o f ;.y r: 0 . 0 5 M M g C l 2 , £ H 3. 5 131 16 C h r o m a t o g r a p h y of F r a c t i o n I ( F i g . 15) on B D E A E - c e l l u l o s e i n the p r e s e n c e of 10" 3 M E D T A , jpH 4 . 0 132 17 ? C h r o m a t o g r a p h y of G l y c i n e - I I f r a c t i o n on B D E A E - c e l l u l o s e i n the p r e s e n c e of 0. 01 M M g C l 2 , _p_H 5. 0 134 18 C h r o m a t o g r a p h y of F r a c t i o n I ( F i g . 17) on B D E A E - c e l l u l o s e i n the p r e s e n c e of 10"3 M E D T A , p H 3. 5 135 19 C h r o m a t o g r a p h y of N-.(Phenoxyacetyl)-^-4c-g l y c y l - s - R N A on B D E A E - c e l l u l o s e . 140 20 • E f f e c t of m a g n e s i u m i o n s o n the s y n t h e s i s of . H C - g l y c y l - s - R N A ( R a n g , 3. 2 to 103yu-moles M g C l 2 ) 144 21 E f f e c t of m a g n e s i u m i o n s o n the s y n t h e s i s of 1 4 c - g l y c y l - s - R N A ( R a n g e , 0 to 28 ya.rnoles M g C l 2 ) 145 22 ' C h r o m a t o g r a p h y . o f ' N - ( p h e n o x y a c e t y l ) - ^ C -••• g l y c y l - s - R N A on B D E A E - c e l l u l o s e 151 23 C h r o m a t o g r a p h y of g l y c i n e a c c e p t o r R N A on B D E A E - c e l l u l o s e 153 - 6 3 -L I S T O F C H A R T S C H A R T P A G E N U M B E R I F r a c t i o n a t i o n of G l y c i n e - I A c c e p t o r R N A o n . B D E A E - C e l l u l o s e 173 II F r a c t i o n a t i o n of G l y c i n e - I I A c c e p t o r R N A 1 s on B D E A E - C e l l u l o s e 176 III S y n t h e s i s of N - ( P h e n o x y a c e t y l ) - a m i n o a c y l -s - R N A 181 I V I s o l a t i o n of G l y c i n e - I s - R N A f r o m W h o l e s - R N A ( P a r t I I - D , S e c t i o n 3 - B ) 185 - 6 4 -I N T R O D U C T I O N S i n c e H o a g l a n d et a l . (1958) f i r s t o b s e r v e d the e s s e n t i a l r o l e of s - R N A i n p r o t e i n s y n t h e s i s , c o n s i d e r a b l e e f f o r t has b e e n e x p e n d e d i n a t t e m p t s to e l u c i d a t e the u n i q u e f u n c t i o n a n d s p e c i f i c i t y of t h i s r e l a t i v e l y l o w m o l e c u l a r w e i g h t R N A . T h e t e r m s o l u b l e R N A ( s - R N A ) , d e r i v e s f r o m the f a c t that t h i s f a m i l y of r i b o n u c l e i c a c i d s e x i s t s i n . t h e s u p e r n a t e of a c e l l - f r e e e x t r a c t f o l l o w i n g u l t r a c e n t r i f u g a t i o n of a c e l l u l a r h o m o g e n a t e . T e r m s e q u i v a l e n t to s - R N A , w h i c h a r e i n c o m m o n u s e , a r e t r a n s f e r R N A ( t - R N A ) , a c c e p t o r R N A a n d a d a p t o r R N A , and t h e s e d e r i v e f r o m the b i o l o g i c a l f u n c t i o n . o f t h i s f a m i l y of r i b o n u c l e i c a c i d s . A m i n o a c y l - s - R N A s y n t h e t a s e s c a t a l y z e the f o r m a t i o n of a n e n z y m e b o u n d a m i n o a c y l - a d e n y l a t e c o m p l e x w h i c h i n t e r a c t s w i t h a s p e c i f i c s - R N A m o l e c u l e r e s u l t i n g i n the t r a n s f e r of the a m i n o a c i d f r o m A M P to the a d e n o s i n e t e r m i n a l of the s - R N A m o l e c u l e . T h e a m i n o -a c y l - s - R N A s y n t h e t a s e s a r e e s s e n t i a l l y s p e c i f i c f o r a g i v e n a m i n o a c i d as w e l l as f o r a g i v e n s - R N A m o l e c u l e . T h e r e a c t i o n s a r e as f o l l o w s : a m i n o a c i d + A T P + e n z y m e a m i n o a c y l - A M P - e n z y m e 4- P P j a m i n o a c y l - A M P - e n z y m e + s - R N A ^ i a m i n o a c y l - s - R N A + A M P + e n z y m e s u m : a m i n o • a c i d + A T P + s - R N A ^± a m i n o a c y l - s - R N A + A M P + P P i - 6 5 -H a v i n g a c c e p t e d the a m i n o a c i d , the s - R N A l e a v e s the e n z y m e s u r f a c e a n d t r a n s f e r s the a m i n o - a c i d to the p r o t e i n - s y n t h e s i z i n g p o l y s o m e c o m p l e x ( B r o w n , 1963). T h e u n i q u e r o l e p l a y e d by s - R N A i n the p r o t e i n s y n t h e s i z i n g m a c h i n e r y of the c e l l has a t t r a c t e d the a t t e n t i o n of a l a r g e n u m b e r of i n v e s t i g a t o r s . A l t h o u g h s t u d i e s o n u n f r a c t i o n a t e d s - R N A h a v e r e s u l t e d i n the a c c u m u l a t i o n of c o n s i d e r a b l e i n f o r m a t i o n , the i s o l a t i o n of s - R N A m o l e c u l e s s p e c i f i c f o r a g i v e n a m i n o a c i d i s e s s e n t i a l f o r the e l u c i d a t i o n of the p r i m a r y s t r u c t u r e as w e l l as the u n i q u e f u n c t i o n . a n d s p e c i f i c i t y of s - R N A ' s . P r e p a r a t i o n s of s - R N A c o n s i s t of p o p u l a t i o n s of s - R N A m o l e c u l e s e a c h s p e c i f i c f o r a p a r t i c u l a r a m i n o a c i d . In a d d i t i o n , a g i v e n a m i n o a c i d c a n h a v e m o r e t h a n one s - R N A . T h e r e m a y i n ; f a c t be as m a n y , as f i f t y d i f f e r e n t s - R N A s p e c i e s . A l l s - R N A s p e c i e s h a v e s i m i l a r m o l e c u l a r w e i g h t s r a n g i n g a r o u n d 25, 0 0 0 , a n d a r e of s i m i l a r c h a i n l e n g t h . S u c h a c o m p l e x m i x t u r e of c l o s e l y , r e l a t e d m o l e c u l e s p r e s e n t s a n u n u s u a l c h a l l e n g e to t h o s e a t t e m p t i n g to i s o l a t e a s i n g l e u n i q u e s - R N A . E a r l y a t t e m p t s , and u n t i l r e c e n t l y the o n l y s u c c e s s f u l a p p r o a c h , to p r e p a r e s - R N A s p e c i f i c f o r s i n g l e a m i n o a c i d s u t i l i z e d the d i f f e r e n c e s i n p a r t i t i o n c o e f f i c i e n t s of s - R N A m o l e c u l a r s p e c i e s i n t w o - p h a s e s o l v e n t s y s t e m s . - 6 6 -W a r n e r a n d V a i m b e r g (1958) f i r s t r e p o r t e d the e f f e c t i v e n e s s of the c o u n t e r c u r r e n t d i s t r i b u t i o n t e c h n i q u e f o r the f r a c t i o n a t i o n of r i b o -n u c l e i c a c i d s u s i n g a s o l v e n t s y s t e m c o m p o s e d of p h o s p h a t e b u f f e r ( p H 8) -• f o r m a m i d e • - i s o p r o p a n o l . H o l l e y a n d M e r r i l l (1959) a p p l i e d t h i s m e t h o d w i t h m i n o r m o d i f i c a t i o n s , to' the f r a c t i o n a t i o n of r a t l i v e r s - R N A . S u b s e q u e n t r e p o r t s b y H o l l e y and c o - w o r k e r s ( H o l l e y . et a l . , 1961; D o c t o r e t a l . ,. 1961; A p g a r . e t a l . , 1962) c l e a r l y d e m o n s t r a t e d the e f f e c t i v e n e s s of t h i s t e c h n i q u e f o r the p u r i f i c a t i o n of s e v e r a l s - R N A ' s f r o m y e a s t . T h e s e w o r k e r s a l s o s h o w e d that the l o s s of a m i n o ' a c i d a c c e p t o r a c t i v i t y , c o u l d be m i n i m i z e d b y l o w e r i n g the p_H of the p h o s p h a t e b u f f e r f r o m 8.to 6. T h e p h o s p h a t e - f o r m a m i d e - i s o p r o p a n o l s y s t e m h a s a l s o b e e n u s e d by. G o l d s t e i n et a l . , (1964) f o r the f r a c t i o n a t i o n of E . c o l i s - R N A ' s a n d by H o s k i n s o n and K h o r a n a (1965) f o r the p u r i f i c a t i o n of p h e n y l a l a n i n e - s p e c i f i c s - R N A f r o m y e a s t . T h e e l u c i d a t i o n , of the p r i m a r y s e q u e n c e s of y e a s t a l a n i n e ( H o l l e y et a l . , 1965) a n d p h e n y l a l a n i n e ( R a j B h a n d a r y et a l . , 1967) s - R N A ' s a t t e s t s to the e f f e c t i v e n e s s of t h i s m e t h o d f o r the i s o l a t i o n of c e r t a i n , s p e c i f i c s - R N A m o l e c u l e s . T h e two p h a s e s o l v e n t s y s t e m d e v i s e d b y K i r b y (1960a, 1960b) w a s e m p l o y e d , w i t h s o m e m o d i f i c a t i o n s , f o r the c o u n t e r c u r r e n t d i s t r i b u t i o n of y e a s t s - R N A ( D o c t o r a n d C o n n e l l y , 1961). T h e m o d i f i e d K i r b y s y s t e m a f f o r d e d a d i s t r i b u t i o n . p a t t e r n s i m i l a r to that o b t a i n e d by- A p g a r et a l . , (1962). - 6 7 -A n o t h e r t w o - p h a s e s o l v e n t s y s t e m c o n s i s t i n g of p h o s p h a t e b u f f e r -p o l y e t h y l e n e g l y c o l ( A l b e r t s s o n , 1961) w a s a d a p t e d by. W i e s m e y e r -et a l . , (1962) f o r the c o u n t e r c u r r e n t d i s t r i b u t i o n of E . c o l i s - R N A . S o m e s e p a r a t i o n . o f a c c e p t o r a c t i v i t i e s w a s o b s e r v e d but the o v e r a l l r e s u l t w a s not too e n c o u r a g i n g . Z a c h a u et a l . (1961), d e m o n s t r a t e d .the s e p a r a t i o n of the t r i - n -b u t y l a m m o n i u m s a l t s of a m i n o a c i d - s p e c i f i c s - R N A ' s i n a s o l v e n t s y s t e m c o m p o s e d of n - b u t a n o l - w a t e r - t r i - n - b u t y l a m i n e - a c e t i c a c i d - d i - n -b u t y l e t h e r . T h e m a j o r d r a w b a c k to the c o u n t e r c u r r e n t d i s t r i b u t i o n t e c h n i q u e l i e s i n the f a c t that the m a j o r i t y of s p e c i f i c a c c e p t o r R N A s p e c i e s a r e not c l e a r l y s e p a r a t e d f r o m one a n o t h e r w h e n the f r a c t i o n a t i o n i s c a r r i e d out i n . a 200 tube t r a n s f e r a p p a r a t u s . A t h o u s a n d p l a t e m a c h i n e gave e x t e n s i v e f r a c t i o n a t i o n of E . c o l i s - R N A a n d . r e v e a l e d 29 s - R N A s p e c i e s ( G o l d s t e i n . e t a l . , 1964),: but s u c h m a c h i n e s r e p r e s e n t a c o n s i d e r a b l e i n v e s t m e n t a n d a r e not w i d e l y u s e d . A n o t h e r d i s a d v a n t a g e of the c o u n t e r c u r r e n t d i s t r i b u t i o n m e t h o d l i e s i n the s e n s i t i v i t y of the s y s t e m to s m a l l v a r i a t i o n s i n . t e m p e r a t u r e . In a n a t t e m p t to c o m b i n e the r e s o l u t i o n a n d c a p a c i t y of c o u n t e r -c u r r e n t d i s t r i b u t i o n w i t h the o p e r a t i o n a l s i m p l i c i t y of c o l u m n c h r o m a t o g -r a p h y the t w o p h a s e s o l v e n t s y s t e m s u s e d i n the c o u n t e r c u r r e n t d i s t r i b u t i o n m e t h o d s have b e e n e m p l o y e d f o r c o l u m n p a r t i t i o n - 6 8 -c h r o m a t o g r a p h y of s - R N A . E v e r e t t et_ a l . , (I960) f r a c t i o n a t e d y e a s t s - R N A by p a r t i t i o n c h r o m a t o g r a p h y on s i l i c i c a c i d c o l u m n s u s i n g a m o d i f i e d p h o s p h a t e b u f f e r - f o r m a m i d e - i s o p r o p a n o l s o l v e n t s y s t e m . A l t h o u g h s o m e f r a c t i o n a t i o n ; o f a m i n o a c i d - s p e c i f i c s - R N A ' s w a s a c h i e v e d , o n l y 4 0 % of the a p p l i e d s - R N A w a s r e c o v e r e d . A s s u m i n g that t h i s d i s a d v a n t a g e w a s due to the s i l i c i c a c i d , T a n a k a et_ al_. , (1962) • u s e d S e p h a d e x G - 2 5 as the s u p p o r t i n g m e d i u m f o r the p a r t i t i o n c h r o m a t o g r a p h y of y e a s t s - R N A u s i n g the t w o p h a s e s y s t e m of Z a c h a u et a l . (1961). T h e s e p a r a t i o n of a m i n o a c i d - s p e c i f i c s - R N A ' s w a s c o m p a r a b l e to the 200 tube t r a n s f e r p r o c e d u r e of c o u n t e r c u r r e n t d i s t r i b u t i o n . B y s i m i l a r p r o c e d u r e s , B e r g q u i s t a n d S c o t t (1964), w e r e a b l e to f r a c t i o n a t e s - R N A f r o m y e a s t a n d f r o m E . c o l i . O n e d i s t i n c t i o n b e t w e e n c o l u m n p a r t i t i o n c h r o m a t o g r a p h y a n d c o u n t e r c u r r e n t d i s t r i b u t i o n i s the o p p o r t u n i t y i n the f o r m e r f o r g r a d i e n t e l u t i o n b y m e a n s of a n i n c r e a s i n g p a r t i t i o n c o e f f i c i e n t i n the m o b i l e p h a s e . . T h i s t e c h n i q u e w a s e x p l o i t e d by. M u e n c h . a n d B e r g : (1966b). T h e s e w o r k e r s f r a c t i o n a t e d . E . c o l i s - R N A on p a r t i t i o n c o l u m n s c o m p o s e d of G - 2 5 S e p h a d e x b e a d s a n d a b i p h a s i c s o l v e n t m i x t u r e of p h o s p h a t e b u f f e r ( p H 6. 88) - e t h o x y e t h a n o l -b u t o x y e t h a n o l - m e r c a p t o e t h a n o l - t r i e t h y l a m i n e . A n i n c r e a s e i n . t h e c o n c e n t r a t i o n of t r i e t h y l a m i n e w a s u s e d to e lute the s - R N A . One d i s t i n c t d i s a d v a n t a g e of the p a r t i t i o n c o l u m n s i s the t i m e r e q u i r e d f o r i s o l a t i n g and a s s a y i n g e a c h of the f r a c t i o n s f o r the d i f f e r e n t a c c e p t o r R N A ' s as r e l a t i v e l y l a r g e v o l u m e s of s o l v e n t a r e u s e d to d e v e l o p the c o l u m n . - 6 9 -A s i m p l e e x t r a c t i o n p r o c e s s e m p l o y i n g l i q u i d - l i q u i d i o n - e x c h a n g e ( K h y m , 1965) o f f e r e d a p o t e n t i a l l y , u s e f u l m o d i f i c a t i o n of the p a r t i t i o n p r o c e s s . In t h i s p r o c e s s , a l k y l a m m o n i u m s a l t s of s - R N A a r e p a r t i t i o n e d i n a b i p h a s i c s o l v e n t s y s t e m c o m p r i s e d of t r i c a p r y l y l m e t h y l -a m m o n i u m c h l o r i d e - t r i c h l o r o t r i f l u o r o e t h a n e - aqueous s o d i u m c h l o r i d e . T h i s t e c h n i q u e has b e e n e x p l o i t e d , w i t h s o m e m o d i f i c a t i o n s , f o r the r e v e r s e d p h a s e c o l u m n : . c h r o m a t o g r a p h i c f r a c t i o n a t i o n of E . c o l i s - R N A ( K e l m e r s et a l . , 1965). A l a r g e s a m p l e of h i g h l y p u r i f i e d p h e n y l a l a n i n e s - R N A w a s p r e p a r e d b y t h i s m e t h o d ( K e l m e r s , 1966). A n o t h e r a d s o r b a n t u s e d f o r the c o l u m n c h r o m a t o g r a p h i c f r a c t i o n a t i o n of s - R N A i s h y d r o x y a p a t i t e . T h e s e c o l u m n s a r e e l u t e d w i t h i n c r e a s i n g c o n c e n t r a t i o n s of p h o s p h a t e b u f f e r . T h i s m e t h o d w a s o r i g i n a l l y r e p o r t e d b y . H a r t m a n n , a n d C o y (1961), f o r the f r a c t i o n a t i o n of E . c o l i s - R N A . M u e n c h a n d B e r g . (1966a) have f u r t h e r r e f i n e d t h i s m e t h o d a n d c l a i m i t to be s u i t a b l e on b o t h a n a l y t i c a l a n d p r e p a r a t i v e s c a l e s . M e t h o d s f o r the f r a c t i o n a t i o n , o f s - R N A b y i o n e x c h a n g e c h r o m a t o g r a p h y have been, d e v e l o p e d b y a n u m b e r of i n v e s t i g a t o r s . N i s h i y a m a et aL (1961), r e p o r t e d the p a r t i a l r e s o l u t i o n of r a t l i v e r l e u c y l -s - R N A o n - D E A E c e l l u l o s e c o l u m n s . T h e s e w o r k e r s a l s o s t u d i e d the f r a c t i o n a t i o n of s - R N A o n D E A E - S e p h a d e x c o l u m n s a n d r e p o r t e d S e p h a d e x to be s o m e w h a t m o r e e f f e c t i v e t h a n D E A E - c e l l u l o s e ( K a w a d e - 7 0 -et a l . , 1963). M u c h m o r e i m p r e s s i v e f r a c t i o n a t i o n of s - R N A o n D E A E -c e l l u l o s e a n d D E A E - S e p h a d e x w a s r e p o r t e d by C h e r a y i l and B o c k (1964, 1965) w h o e m p l o y e d i n c r e a s i n g c o n c e n t r a t i o n s of s o d i u m c h l o r i d e , i n the p r e s e n c e of 7 M u r e a , to e l u t e the s - R N A . T h i s m e t h o d o f f e r e d the a d v a n t a g e s of good r e c o v e r y of a m i n o a c i d a c c e p t o r a c t i v i t y , good f l o w r a t e s a n d s i m p l e , i n e x p e n s i v e c h r o m a t o g r a p h i c e q u i p m e n t . . In t h i s m e t h o d i t w o u l d a p p e a r that u r e a opens the s - R N A s t r u c t u r e a l l o w i n g i n c r e a s e d i n t e r a c t i o n b e t w e e n . t h e c o n s t i t u e n t n u c l e o t i d e s a n d the e x c h a n g e r . A s i m i l a r o p e n i n g of the s - R N A s t r u c t u r e m a y be a c h i e v e d by e l e v a t i n g the t e m p e r a t u r e d u r i n g c h r o m a t o g r a p h y . T h i s i d e a has b e e n e x p l o i t e d r e c e n t l y , by B a g u l e y et a l . , (1965a, 1965b) f o r the f r a c t i o n a t i o n of y e a s t s - R N A . T h e s e w o r k e r s f r a c t i o n a t e d s - R N A on D E A E - c e l l u l o s e at 65° u n t i l i z i n g a n o n - l i n e a r s a l t g r a d i e n t to e lute the s - R N A . T h e y a l s o s h o w e d that s - R N A c o u l d be f r a c t i o n a t e d on. D E A E - S e p h a d e x b y e m p l o y i n g a d e c r e a s i n g ' t e m p e r a t u r e g r a d i e n t , at c o n s t a n t s a l t c o n c e n t r a t i o n , to e l u t e the s - R N A . A s the t e m p e r a t u r e i s l o w e r e d , the s - R N A m o l e c u l e s p r e s u m a b l y r e n a t u r e , thus e x p o s i n g f e w e r of the c o n -s t i t u e n t n u c l e o t i d e s to the i o n e x c h a n g e r . M o r e r e c e n t l y , S m i t h (1966) has r e p o r t e d the f r a c t i o n a t i o n of s - R N A f r o m S a l m o n e l l a t y p h i m u r i u m on D E A E - S e p h a d e x c o l u m n s by e l u t i o n w i t h .pH a n d s a l t g r a d i e n t s . T h e a u t h o r s u g g e s t s that r e s o l u t i o n - 7 1 -of the s - R N A d e p e n d s on a change i n the s - R N A f r o m a p a r t i a l l y d e n a t u r state at the b e g i n n i n g of e l u t i o n to the n a t i v e state at the e n d . In a d d i t i o n to the c o l u m n c h r o m a t o g r a p h i c t e c h n i q u e s a l r e a d y d i s c u s s e d , t h e r e i s the m e t h y l a t e d a l b u m i n c o l u m n of M a n d e l l a n d H e r s h e y (I960), w h i c h w a s u s e d b y Sue o k a a n d Y a m a n e (1962) f o r r e s o l v i n g , the a m i n o a c i d a c c e p t o r a c t i v i t i e s of E . c o l i s - R N A . M e l c h e r and Z a c h a u (1965) r e p o r t e d the f r a c t i o n a t i o n , of y e a s t s - R N A o n c o l u m n s of m e t h y l a t e d a l b u m i n - k i e s e l g u h r . T h e m a j o r d i s a d v a n t a g e of m e t h y l a t e d s e r u m a l b u m i n - k i e s e l g u h r c o l u m n s i s t h e i r s m a l l c a p a c i t y , a l t h o u g h t h e y a r e s t i l l v e r y u s e f u l i n . a n a l y t i c a l p r o c e d u r e s . O k a m o t o a n d K a w a d e (19 63), i m p r o v e d the c a p a c i t y , by u s i n g s i l i c i c a c i d as a s u p p o r t , w h i c h a d s o r b s m u c h m o r e m e t h y l a t e d s e r u m a l b u m i n t h a n does k i e s e l g u h r . A s u r v e y of e x i s t i n g m e t h o d s f o r p r e p a r i n g l a r g e a m o u n t s of s p e c i f i c s - R N A ' s f o r s t r u c t u r a l a n d b i o l o g i c a l s t u d i e s r e v e a l e d the n e c e s s i t y f o r a d d i t i o n a l f r a c t i o n a t i o n p r o c e d u r e s . A n u m b e r of a p p r o a c h e s w e r e t r i e d , m o s t of w h i c h m e t w i t h l i t t l e or no s u c c e s s . O f the f o u r m e t h o d s d e s c r i b e d i n t h i s p a r t of the t h e s i s , t w o i n v o l v e the i s o l a t i o n of f a m i l i e s of a m i n o • a c i d - s p e c i f i c s - R N A ' s , ( P a r t s I I - A a n d - D and two i n v o l v e the f r a c t i o n a t i o n of m i x e d s - R N A ( P a r t s I I - B a n d - C ) . P a r t I I - A d e s c r i b e s a new c h r o m a t o g r a p h i c m a t e r i a l c o n s i s t i n g of a b i f u n c t i o n a l m e r c u r i - d i o x a n e d e r i v a t i v e a t t a c h e d t h r o u g h a t h i o l g r o u p to the c e l l u l o s e m a t r i x . T h e u s e of m e r c u r i - c e l l u l o s e ( H g S -C e l l u l o s e ) f o r f r a c t i o n a t i n g n u c l e i c a c i d s and t h e i r d e r i v a t i v e s e v o l v e d f r o m the s t u d i e s of s e v e r a l g r o u p s on the i n t e r a c t i o n of v a r i o u s h e a v y m e t a l s ( i n c l u d i n g m e r c u r y ) w i t h D N A , R N A , e t c . ( T h o m a s , 1954; Y a m a n e a n d D a v i d s o n , 1961; D a v i d s o n _et a l . 1965) a n d a l s o f r o m a r e p o r t by E l d j a r n and J e l l u m (1963), i n w h i c h t h e y d e s c r i b e d the f r a c t i o n a t i o n of H S - p r o t e i n s o n i a b i f u n c t i o n a l m e r c u r y d e r i v a t i v e of a m i n o e t h y l c e l l u l o s e . In the p r e s e n t s t u d y , the u s e of H g S - c e l l u l o s e as a c h r o m a t o g r a p h i c m a t e r i a l has not b e e n f u l l y i n v e s t i g a t e d . H o w e v e r , p r e l i m i n a r y e x p e r i m e n t s s h o w e d that H g S - c e l l u l o s e c o u l d r e t a i n the 14 r a d i o a c t i v i t y a s s o c i a t e d w i t h C - c y s t e i n y l - s - R N A . T h e p r o c e d u r e s d e s c r i b e d .'in. P a r t s I I - B a n d - C f o r the f r a c t i o n -a t i o n of s - R N A i n v o l v e p a r t i t i o n c h r o m a t o g r a p h y o n a h i g h l y c r o s s -l i n k e d p o l y a c r y l a m i d e m a t r i x ( P a r t I I - B ) , and a d s o r p t i o n - i o n - e x c h a n g e c h r o m a t o g r a p h y on a n e w c h r o m a t o g r a p h i c m a t e r i a l , b e n z o y l a t e d -D E A E - c e l l u l o s e ( B D E A E - c e l l u l o s e ) ( P a r t I I - C ) . T h e l a t t e r p r o c e d u r e t u r n e d out to be a p a r t i c u l a r l y u s e f u l m e t h o d a n d may. be c o n s i d e r e d to be the m e t h o d of c h o i c e f o r the f r a c t i o n a t i o n of a c c e p t o r - R N A ' s . It m u s t be e m p h a s i z e d that the d e v e l o p m e n t of B D E A E - c e l l u l o s e w a s a c o o p e r a t i v e p r o j e c t i n v o l v i n g , s e v e r a l p e o p l e w o r k i n g i n the l a b o r a t o r y of D r . G . M . T e n e r . T h e s e w e r e , i n a d d i t i o n to D r . G . M . T e n e r , D r . I. C . G i l l a m , D r . E . W i m m e r , D r . I. M a x w e l l , M . v o n - 7 3 -T i g e r s t r o m , D . B l e w a n d the a u t h o r . T h e d e v e l o p m e n t of B D E A E -c e l l u l o s e w a s b a s e d o n a n a t t e m p t to d e t e r m i n e w h a t p r o p e r t i e s of m e t h y l a t e d s e r u m a l b u m i n m i g h t be r e s p o n s i b l e f o r the f r a c t i o n a t i o n of s - R N A ' s on, c o l u m n s of m e t h y l a t e d a l b u m i n - k i e s e l g u h r ( S u e o k a a n d Y a m a n e , 1962). W h e n the n e g a t i v e l y c h a r g e d c a r b o x y l g r o u p s of a l b u m i n . a r e m e t h y l a t e d , the a l b u m i n b e c o m e s a n a n i o n - e x c h a n g e r as a r e s u l t of the p o s i t i v e c h a r g e s on,the l y s i n e (12%) a n d a r g i n i n e (6%) r e s i d u e s . H o w e v e r , i n , a d d i t i o n to the b a s i c a m i n o a c i d s , . a l b u m i n , a l s o c o n t a i n s 13% a r o m a t i c a m i n o a c i d s a n d 3 0 % of o t h e r a m i n o a c i d s w i t h l i p o p h i l i c s i d e c h a i n s . One of the f u n c t i o n s of s e r u m a l b u m i n i s the t r a n s p o r t of l i p i d s . T h u s m e t h y l a t e d s e r u m a l b u m i n i s a l i p o p h i l i c a n i o n - e x c h a n g e r . W h e n D E A E - c e l l u l o s e i s b e n z o y l a t e d , i t s p r o p e r t i e s a r e c h a n g e d f r o m that of a h y d r o p h i l i c a n i o n - e x c h a n g e r to a l i p o p h i l i c a n i o n - e x c h a n g e r . P a r t I I - C d e s c r i b e s the c h r o m a t o g r a p h i c b e h a v i o u r of the g l y c i n e s - R N A ' s o n - B D E A E - c e l l u l o s e u n d e r d i f f e r e n t c o n d i t i o n s of e l u t i o n . N u m e r o u s r e p o r t s have a p p e a r e d d e s c r i b i n g c o m b i n e d c h e m i c a l a n d c h r o m a t o g r a p h i c t e c h n i q u e s f o r t h e i s o l a t i o n of f a m i l i e s of a m i n o a c i d - s p e c i f i c s - R N A ' s . M o s t i n v o l v e c h a r g i n g the m i x t u r e of s - R N A ' s w i t h a g i v e n a m i n o * a c i d f o l l o w e d by o x i d a t i o n of the uncharged s p e c i e s w i t h p e r i o d a t e to f o r m the R N A d i a l d e h y d e s . - 7 4 -U s i n g t h i s p r o c e d u r e , Z a m e c n i k et_ a l . , (I960) c o n v e r t e d the o x i d i z e d . R N A to a d y e - c o m p l e x a n d r e m o v e d the d y e - b o u n d R N A b y f r a c t i o n a l p r e c i p i t a t i o n . M o r e e f f i c i e n t m e t h o d s f o r r e m o v i n g the d i a l d e h y d e s have b e e n d e v e l o p e d by w a y of a c o u p l i n g r e a c t i o n b e t w e e n the d i a l d e h y d e s a n d s o l i d p o l y a c r y l i c a c i d h y d r a z i d e >(Zachau et a l . , 1961), p h e n y l h y d r a z i n e - c e l l u l o s e ( S a p o n a r a a n d B o c k , . 1961) o r a m i n o e t h y l -c e l l u l o s e Z u b a y , 1962). T h e s e m e t h o d s h o w e v e r , have the e c o n o m i c d i s a d v a n t a g e of d e g r a d i n g a l l s - R N A ' s e x c e p t that c h a r g e d w i t h the a m i n o a c i d . M o r e e c o n o m i c a l c h e m i c a l p r o c e d u r e s h a v e b e e n r e p o r t e d but do not a p p e a r to have b e e n v e r y p r o d u c t i v e . F o r e x a m p l e a m e t h o d d e v e l o p e d b y M e h l e r a n d B a n k (1963) i n v o l v e d a r e a c t i o n b e t w e e n s - R N A , p r e v i o u s l y c h a r g e d w i t h a n a m i n o a c i d , a n d £ . - t r i f l u o r o a c e t y H y s i n e N - c a r b o x y a n h y d r i d e . T h e L e u c h ' s a n h y d r i d e f o r m s a g r o w i n g p o l y -t r i f l u o r o a c e t y l l y s i n e ( p o l y T F A l y s i n e ) c h a i n on the f r e e OL-amino g r o u p of the b o u n d a m i n o a c i d of a m i n o a c y l - s - R N A . T h e g r o w i n g p o l y T F A l y s y l s i d e c h a i n e v e n t u a l l y e f fec ts p r e c i p i t a t i o n of the a m i n o a c y l - s - R N A . T h e r e c o v e r y of a m i n o a c i d a c c e p t o r a c t i v i t y a f t e r h y d r o l y s i s of the e s t e r l i n k a g e b e t w e e n the a m i n o a c i d a n d s - R N A w a s q u i t e l o w . A s i m i l a r m e t h o d w h i c h u t i l i z e d |*9-b e n z y l - N - c a r b o x y l - L - a s p a r t a t e a n h y d r i d e i n p l a c e of T F A l y s i n e N -c a r b o x y a n h y d r i d e w a s r e p o r t e d by S i m o n et a l . (1964). A m o r e r e c e n t - 7 5 -r e p o r t by the s a m e w o r k e r s u t i l i z e d the s a m e r e a c t i o n . f o r d e r i v a t i z i n g a n d p r e c i p i t a t i n g a m i n o a c y l - s - R N A ( K a t c h a l s k i et a l . ,. 1966). T h e y : r e m o v e d the p o l y a s p a r t y l m o i e t y , w i t h the a i d of the p r o t e o l y t i c - e n z y m e , P r o n a s e , thus, i m p r o v i n g o n the r e c o v e r y of b i o l o g i c a l a c t i v i t y . M e t h o d s u t i l i z i n g a m i n o a c y l - s - R N A s y n t h e s i s m u s t be c o m b i n e d w i t h a c h r o m a t o g r a p h i c : f r a c t i o n a t i o n . p r o c e d u r e b e c a u s e i t i s k n o w n ithat m a n y a m i n o a c i d - s p e c i f i c s - R N A ' s c o n s i s t of m o r e t h a n , o n e m o l e c u l a r c o m p o n e n t . In a d d i t i o n i t s h o u l d be p o i n t e d out that l a r g e a m o u n t s of p u r i f i e d a m i n o a c y l - s - R N A s y n t h e t a s e s a r e r e q u i r e d to p r e p a r e s u f f i c i e n t a m o u n t s of s - R N A f o r s t r u c t u r a l s t u d i e s . P a r t I I - D d e s c r i b e s a c o m b i n e d c h e m i c a l a n d c h r o m a t o g r a p h i c p r o c e d u r e f o r the i s o l a t i o n of the g l y c i n e a c c e p t o r - R N A . T h e m e t h o d d e s c r i b e d i n t h i s t h e s i s i s a n a d a p t a t i o n of a p r o c e d u r e d e v e l o p e d by D r s G . M . T e n e r a n d I. C . G i l l a m . It i n v o l v e s the c h e m i c a l a t t a c h m e n t of a l i p o p h i l i c . ' h a n d l e ' to a n a m i n o a c y l - s - R N A f o l l o w e d by s t e p w i s e e l u t i o n of the m i x t u r e f r o m B D E A E - c e l l u l o s e . I n t r a f a m i l y s - R N A s p e c i e s m a y t h e n be s e p a r a t e d by g r a d i e n t e l u t i o n c h r o m a t o g r a p h y on B D E A E - c e l l u l o s e . - 7 6 -E X P E R I M E N T A L A B B R E V I A T I O N S U S E D s - R N A : s o l u b l e r i b o n u c l e i c a c i d H T L : N - a c e t y l h o m o c y s t e i n e t h i o l a c t o n e I A A : i o d o a c e t i c a c i d T C A : t r i c h l o r o a c e t i c a c i d C i : c u r i e E D T A : e t h y l e n e d i a m i n e t e t r a a c e t i c a c i d : T o s y l : p - t o l u e n e s u l f o n i c a c i d D M F : N , N - d i m e t h y l f o r m a m i d e T L C : t h i n l a y e r c h r o m a t o g r a p h y D C C : d i - c y c l o h e x y l c a r b o d i i m i d e T H F : t e t r a h y d r o f u r a n A E - c e l l u l o s e: a m i n o e t h y l - c e l l u l o s e • H S A E - c e l l u l o s e : a m i n o e t h y l - c e l l u l o s e w h i c h has b e e n . t h i o l a t e d w i t h . H T L H g S A E - c e l l u l o s e : H S A E - c e l l u l o s e w h i c h has b e e n c o m p l e x e d w i t h b i s - 3, 6- ( a c e t a t o m e r c u r i m e t h y l ) - d i o x a n e • H S - c e l l u l o s e : 6 - m e r c a p t o - c e l l u l o s e ' H g S - c e l l u l o s e : H S - c e l l u l o s e c o m p l e x e d w i t h b i s - 3 , 6-( a c e t a t o m e r c u r i m e t h y l ) - d i o x a n e D E A E - c e l l u l o s e : d i e t h y l a m i n o e t h y l - c e l l u l o s e - 7 7 -B D E A E - c e l l u l o s e : b e n z o y l a t e d - D E A E - c e l l u l o s e p C , p U , p G a n d p A : the n u c l e o s i d e - . 5 ' p h o s p h a t e s of c y t i d i n e , u r i d i n e , g u a n o s i n e a n d a d e n o s i n e , r e s p e c t i v e l y , U p , G p , . a n d A p : the n u c l e o s i d e - 2 1 (3 ') p h o s p h a t e s of u r i d i n e , g u a n o s i n e a n d a d e n o s i n e , r e s p e c t i v e l y i P P O : 2, 5 - d i p h e n y l o x a z o l e d i m e t h y l P O P O P : . 1, 4 - b i s - 2 - ( 4 - m e t h y l - 5 - p h e n y l o x a z o l y l ) -b e n z e n e Tj^j-: m i d - p o i n t i n the t h e r m a l d e n a t u r a t i o n c u r v e d e f i n e d as the ' m e l t i n g ' t e m p e r a t u r e T h e s t a n d a r d a b b r e v i a t i o n s f o r the^ a m i n o a c i d s a r e u s e d . M A T E R I A L S s - R N A . B a k e r s ' y e a s t s - R N A w a s p u r c h a s e d f r o m . C a l b i o c h e m C o r p o r a t i o n , L o s A n g e l e s . B r e w e r s ' y e a s t s - R N A w a s p u r c h a s e d f r o m B o e h r i n g e r a n d S o e h n e , M a n n h e i m . 14 i C - a m i n o a c i d s w e r e o b t a i n e d f r o m N e w E n g l a n d N u c l e a r C o r p o r a t i o n , B o s t o n , M a s s . P P O ( S c i n t i l l a t i o n G r a d e ) , w a s o b t a i n e d f r o m K e n t C h e m i c a l s L t d . , V a n c o u v e r , C a n a d a . i d i m e t h y l P O P O P ( S c i n t i l l a t i o n G r a d e ) , w a s o b t a i n e d f r o m the P a c k a r d I n s t r u m e n t C o m p a n y I n c . , L a G r a n g e , 111. - 7 8 -O p t i c a l d e n s i t y m e a s u r e m e n t s w e r e m a d e u s i n g a C a r y M o d e l 11 s p e c t r o p h o t o m e t e r . O n e o p t i c a l d e n s i t y (O. D . ) u n i t i s d e f i n e d as that a m o u n t of m a t e r i a l p e r m l w h i c h , i n a 1 - c m l i g h t p a t h at 260 rryj., g i v e s a s p e c t r o p h o t o m e t r y r e a d i n g of one. T h e m g of p r o t e i n p e r m l of s o l u t i o n w e r e c a l c u l a t e d f r o m the r a t i o of a b s o r b a n c e s at 280 rryi- a n d 260 m / i - (280:260 r a t i o ) ( W a r b u r g and C h r i s t i a n , . 1941). N i t r o p r u s s i d e t e s t . W h e n .a few d r o p s of s o d i u m n i t r o p r u s s i d e s o l u t i o n w e r e a d d e d to the t e s t s o l u t i o n f'. a d e e p m a g e n t a c o l o u r f o r m e d i m m e d i a t e l y i n the p r e s e n c e of t h i o l s . T h e r e a c t i o n w i t h t h i o e s t e r s w a s m u c h s l o w e r . M E T H O D S P A R T I I - A . A T T E M P T S T O I S O L A T E C Y S T E I N Y L - s - R N A B Y  C H R O M A T O G R A P H Y O N M E R C U R I - C E L L U L O S E 1. T i t r a t i o n of A E - C e l l u l o s e w i t h A l k a l i A E - C e l l u l o s e (1. 00 g f w a s s u s p e n d e d . i n 20. 0 m l of 1 M N a C l f o r 2 h r at r o o m t e m p e r a t u r e , , a d j u s t e d to p H 1. 4 w i t h 2 N H C 1 , t h e n t i t r a t e d w i t h 0.100 N N a O H u s i n g a p H m e t e r e q u i p p e d w i t h ; a g l a s s e l e c t r o d e . A v a l u e of 0. 6 m e q p e r g r a m of dry. A E - c e l l u l o s e w a s o b t a i n e d . - 7 9 -2. T h i o l a t i o n of A E - C e l l u l o s e w i t h N - A c e t y l h o m o c y s t e i n e T h i o l a c t o n e  ( H T L ) A . T h i o l a t i o n of A E - C e l l u l o s e w i t h H T L i n D i o x a n e A t t e m p t s to t h i o l a t e A E - c e l l u l o s e w i t h H T L i n r e f l u x i n g d i o x a n e w e r e u n s u c c e s s f u l . W h e n c a t a l y t i c a m o u n t s of i m i d a z o l e w e r e i n c l u d e d i n the r e a c t i o n m i x t u r e d e s c r i b e d a b o v e , a p r o d u c t w a s o b t a i n e d w h i c h r e a c t e d w e a k l y , w i t h n i t r o p r u s s i d e . B . T h i o l a t i o n of A E - c e l l u l o s e w i t h H T L i n A q u e o u s M e d i u m i n the P r e s e n c e of S i l v e r N i t r a t e ( E l d j a r n . a n d , J e l l u m , 1963) W a s h e d a n d d r i e d A E - c e l l u l o s e (1; g, 0. 6 m e q ) w a s s u s p e n d e d i n 50 m l of d i s t i l l e d w a t e r , s t i r r e d f o r 1 m i n at l o w s p e e d i n a W a r i n g b l e n d o r a n d a l l o w e d to s w e l l i n w a t e r f o r 24 h r . T h e n 286 m g of H T L (1. 8 m e q ) d i s s o l v e d . i n a few m i s of d i s t i l l e d w a t e r , w a s a d d e d , f o l l o w e d by. 3. 0 m l of A g N O ^ s o l u t i o n (1. 8 m e q ) w h i c h w a s a d d e d i n i n c r e m e n t s of 0.10 m l p e r 2 m i n . D u r i n g a n d a f t e r the a d d i t i o n , t h e , p H w a s m a i n t a i n e d b e t w e e n 7. 5 a n d 8. 0 w i t h 1 N N a O H . T h e r e a c t i o n m i x t u r e w a s s t i r r e d s l o w l y . S a m p l e s of the c e l l u l o s e w e r e r e m o v e d at 1, 2 a n d 3 h r s ( s a m p l e s I, II , a n d III) a f t e r a l l the A g N O ^ h a d b e e n . a d d e d , a n d m i x e d i m m e d i a t e l y , w i t h a s o l u t i o n m a d e u p of e q u a l v o l u m e s of 1 N H N O ^ a n d s a t u r a t e d t h i o u r e a s o l u t i o n . ( T h e c o l o u r of the p r o d u c t c h a n g e s f r o m b r i g h t y e l l o w to v e r y p a l e y e l l o w ) . T h e s a m p l e s w e r e c e n t r i f u g e d a n d - 8 0 -the s u p e r n a t e s d e c a n t e d . T h e n i t r i c . a c i d - t h i o u r e a w a s h w a s r e p e a t e d ^ t h e n the p r o d u c t s w e r e w a s h e d r e p e a t e d l y , w i t h d i s t i l l e d w a t e r u n t i l n e u t r a l . A l l t h r e e s a m p l e s of t h i o l a t e d A E - c e l l u l o s e ( H S A E - c e l l u l o s e ) r e a c t e d s t r o n g l y / w i t h n i t r o p r u s s i d e . S a m p l e s I, II a n d III w e r e t i t r a t e d w i t h i o d o a c e t i c a c i d at _p_H 8 . 0 (see S e c t i o n 3), a n d c o n s u m e d 0. 225, 0. 226 a n d 0. 235 m e q of 0.100 N N a O H p e r d r y g r a m of H S A E -c e l l u l o s e r e s p e c t i v e l y , i n d i c a t i n g that the m a x i m u m d e g r e e of t h i o l a t i o n ( S a m p l e III) w a s 3 9 % of t h e o r e t i c a l b a s e d on a v a l u e of 0. 6 m e q p e r d r y g r a m of A E - c e l l u l o s e . 3. T i t r a t i o n of H S A E - C e l l u l o s e w i t h I o d o a c e t i c A c i d ( I A A ) T i t r a t i o n s w e r e c a r r i e d out u s i n g a R a d i o m e t e r T i t r i g r a p h ( C o p e n h a g e n ) . S a m p l e s of the H S A E - c e l l u l o s e (100 m g ) w e r e e a c h s u s p e n d e d i n . 2 . 0 m l of 0. 3 M K C 1 , 0. 005 M T r i s ( c h l o r i d e ) , p H 6. 8, f o r s e v e r a l h o u r s b e f o r e e a c h t i t r a t i o n . A s t o c k s o l u t i o n of I A A , c o n t a i n i n g 0. 27 m e q of I A A p e r m l , , w a s p r e p a r e d by d i s s o l v i n g 3. 0 g of I A A i n 50 m l d i s t i l l e d w a t e r , t i t r a t i n g to p H 6. 5 w i t h 1 N N a O H a n d e x t r a c t i n g the s o l u t i o n w i t h C H C l g u n t i l the o r g a n i c l a y e r w a s c o l o u r l e s s . S o l i d K C 1 ( 0 . 7 g) w a s a d d e d a n d the v o l u m e m a d e u p to 60 m l . A h y d r a t e d s a m p l e of H S A E - c e l l u l o s e w a s t r a n s f e r r e d q u a n t i t a t i v e l y to the T i t r i g r a p h r e a c t i o n v e s s e l at 2 5 ° , a n d the v o l u m e w a s a d j u s t e d to 4 . 0 m l w i t h 0. 3 M K C 1 , 0. 005 M T r i s -( c h l o r i d e ) , p H 6. 8. T h e p _ H w a s a d j u s t e d to the d e s i r e d v a l u e by the a d d i t i o n of 1 N N a O H a n d a 0. 37 m l a l i q u o t of the s t a n d a r d I A A s o l u t i o n w a s a d d e d to the r e a c t i o n v e s s e l w i t h the T i t r i g r a p h s t a t t e d at the d e s i r e d p H . T i t r a t i o n s w e r e c a r r i e d out at j>H 7. 0, 8 . 0 , 8 . 5 , 9 . 0 , 9 . 5 , 1 0 . 0 , 10. 5 a n d 11. 0. E a c h . t i t r a t i o n w a s f o l l o w e d f o r 50 m i n at 25^, T h e m e q of 0.100 N N a O H c o n s u m e d per d r y g r a m of H S A E - c e l l u l o s e i s s h o w n i n F i g u r e s 1A, B a n d C . A s t r a i g h t l i n e of s l o p e g r e a t e r t h a n 0 w a s o b t a i n e d a f t e r about 10 m i n r e a c t i o n t i m e (except f o r the p H 7. 0 t i t r a t i o n , F i g . 1-A), w h i c h , w h e n e x t r a p o l a t e d b a c k to z e r o t i m e , m a d e p o s s i b l e the c a l c u l a t i o n of a c o r r e c t i o n f a c t o r f o r the c a r b o x y m e t h y l a t i o n of a m i n o g r o u p s . T h e c o r r e c t e d t i t r a t i o n p l o t s a r e s h o w n i n F i g u r e s 2 - A a n d B . F r o m t h e s e p l o t s a r e o b t a i n e d the m e q of 0.100 N N a O H c o n s u m e d p e r g r a m of t h i o l a t e d c e l l u l o s e at v a r i o u s p H ' s w h i c h w h e n p l o t t e d a g a i n s t p H ( F i g . 2 - C ) , p e r m i t the c a l c u l a t i o n of the p K g - ^ of the t h i o l g r o u p by l o c a t i n g the m i d p o i n t i n the s l o p e of the c u r v e . A v a l u e of 9 . 7 5 w a s o b t a i n e d . T i t r a t i o n s of s a m p l e s w e r e r o u t i n e l y c a r r i e d out a t - p H 8. 0. 4. L a r g e S c a l e T h i o l a t i o n of A E - C e l l u l o s e w i t h N - A c e t y l h o m o c y s t e i n e T h i o l a c t o n e A E - C e l l u l o s e (100 g) w a s l e f t u n d e r w a t e r f o r about 12 h r a f t e r w h i c h the e x c e s s w a t e r w a s d e c a n t e d a n d 18. 5 g (116 m e q ) of H T L , d i s s o l v e d i n 1, 200 m l of d i s t i l l e d w a t e r , w a s a d d e d . A 1 N A g N O ^ s o l u t i o n w a s a d d e d s l o w l y , w i t h r a p i d s t i r r i n g a n d w i t h the s i m u l t a n e o u s -82-1 2 O x 10 8 cu If) o Z5 6 4 2 0 o £ 10 D L. cn cu U LU < 00 cu CL I O CO 8 6 4 2 O 0 O 510 o 8 cu £ 6 4 0 ( A ) / / (B) • pH 80 . pH 7.0 4 pH 9.5 * pH9.0 • pH 85 ( C ) pH 11.0 pH 10.5 pH 10.0 0 10 20 30 40 T I M E (min) F I G U R E 1. T i t r a t i o n c u r v e s f o r H S A E - c e l l u l o s e . 50 60 -83-8 6 o CD o u LU < Ul X / 0 6 4 2 o £ o CD 0 o / / ( A ) • pH70 o pH8.0 -+-!* —x--A- •A-+ -• A. -+ -•A-- + --A-- + • A- • A 8.5 ^ 9.0 9.5 10.0 10.5 11.0 (B) PH 0 10 20 30 40 50 TIME (min) 60 CD Q - 8 x O 03 7-6-g 5 r 3 s— o cr E 2 1 0 •0-( C ) P KSH 0 - i i i _ 7 8 11 12 9 10 PH F I G U R E 2. A , B - C o r r e c t e d t i t r a t i o n c u r v e s f o r H S A E - c e l l u l o s e . C - A p l o t of p H v s a l k a l i c o n s u m e d , f o r the t i t r a t i o n of H S A E - c e l l u l o s e w i t h I A A . - 8 4 -a d d i t i o n of 2 N N a O H , to m a i n t a i n the p_H at 7. 5 . A t o t a l of 116 m e q of A g + i o n w a s a d d e d . T h e r e a c t i o n m i x t u r e w a s t h e n s t i r r e d s l o w l y u n t i l the p H r e m a i n e d c o n s t a n t at 7. 5 f o r a p e r i o d of 15 m i n . T o t a l r e a c t i o n t i m e w a s . 4 . 5 ' h r . T h e b r o w n i s h - y e l l o w p r o d u c t w a s p o u r e d . i n t o a c h r o m a t o g r a p h i c , c o l u m n , a n d w a s h e d w i t h a s o l u t i o n of t h i o u r e a a n d H N O 3 ( s a t u r a t e d t h i o u r e a s o l u t i o n . : 1 N H N O 3 , 1:1 (v/v)) , u n t i l the ef f luent gave a n e g a t i v e n i t r o p r u s s i d e t e s t , a n d t h e n w a s h e d n e u t r a l w i t h d i s t i l l e d w a t e r . 5. S y n t h e s i s of b i s - 3 , 6 - ( A c e t a t o m e r c u r i m e t h y l ) - D i o x a n e ( C h a t t , 1951) M e r c u r i c n i t r a t e (16.5 g), . w a s d i s s o l v e d , i n , a m i n i m u m of d i l u t e H N O ^ a n d w a r m e d t o 5 0 ° . A l l y l a l c o h o l (3. 5 m l ) w a s a d d e d a n d the m i x t u r e a l l o w e d t o s t a n d at r o o m t e m p e r a t u r e f o r 1 h r . T h e r e s u l t i n g w h i t e p r e c i p i t a t e w a s c o l l e c t e d o n a B u c h n e r f u n n e l , w a s h e d w i t h i c e -c o l d w a t e r , r e d i s s o l v e d i n a m i n i m u m of 0. 5 N K O H a n d r e p r e c i p i t a t e d b y the c a r e f u l a d d i t i o n of d i l u t e H N O 3 . T h e s u p e r n a t e w a s d e c a n t e d a n d the w h i t e p r o d u c t p r e s s e d to d r y n e s s b e t w e e n f i l t e r p a p e r s a n d s t o r e d u n d e r v a c u u m i n a d e s i c c a t o r . T h e n i t r a t e f o r m of the m e r c u r i - d i o x a n e d e r i v a t i v e w a s c o n v e r t e d to a 1% s o l u t i o n of the a c e t a t e f o r m by d i s s o l v i n g . 6 . 2 g of the above p r o d u c t i n . a m i n i m u m of 0. 5 N N a O H , d i l u t i n g to 310 m l w i t h d i s t i l l e d w a t e r and p a s s i n g the s o l u t i o n t h r o u g h a D o w e x - 1 (acetate) c o l u m n . ( 3 8 x 1.4 c m d i a r m ). T h e e f f luent c o n t a i n i n g the o r g a n o m e r c u r y - 8 5 -c o m p o u n d w a s c o l l e c t e d i n 310 m l of i c e - c o l d 0. 02 M s o d i u m p h o s p h a t e b u f f e r , p H 7 . 4 . T h e c o n t a i n e r w a s p r o t e c t e d f r o m l i g h t by a n a l u m i n u m f o i l w r a p p e r a n d s t o r e d at 3 - 5 ° u n t i l r e a d y f o r u s e . 6. P r e p a r a t i o n of the M e r c u r i - C e l l u l o s e ( H g S A E - C e l l u l o s e ) C o l u m n H S A E - c e l l u l o s e ( c o n t a i n i n g about 0. 23 m e q of HS p e r d r y g r a m ) • w a s s u s p e n d e d i n 0.01 M s o d i u m p h o s p h a t e b u f f e r , . p H 7 . 4 , s t i r r e d f o r about 1 m i n at l o w s p e e d i n a W a r i n g b l e n d o r t h e n a l l o w e d to s w e l l o v e r n i g h t at 3 - 5 9 , A 1% s o l u t i o n of the b i s - m e r c u r i - d i o x a n e d e r i v a t i v e w a s a d d e d to the s u s p e n s i o n . A n e x c e s s of the o r g a n o m e r c u r y c o m p o u n d w a s m a i n t a i n e d ( N a l . t e s t ) d u r i n g 2 h r a n d t h e n . t h e s u s p e n s i o n w a s p o u r e d i n t o a c h r o m a t o g r a p h i c c o l u m n o f the d e s i r e d d i m e n s i o n s . A l t e r n a t i v e l y , the s u s p e n s i o n w a s p o u r e d d i r e c t l y . i n t o the c h r o m a t o g r a p h i c c o l u m n w h i c h w a s t h e n w a s h e d w i t h a .1% s o l u t i o n of the o r g a n o m e r c u r y d e r i v a t i v e u n t i l the l a t t e r w a s d e t e c t e d i n the e f f luent ( N a l t e s t ) . I n e i t h e r c a s e the c o l u m n w a s t h e n w a s h e d w i t h d i s t i l l e d w a t e r u n t i l a n e g a t i v e t e s t w i t h N a l w a s o b t a i n e d . A n a n a l y t i c a l ' p o r t i o n of the H g S A E - c e l l u l o s e w a s s u s p e n d e d i n . 0. 01 M N a O A c , p H 5. 2: a n d c e n t r i f u g e d . T h e s u p e r n a t e gave n e g a t i v e N a l a n d n i t r o p r u s s i d e t e s t s . The o r g a n o m e r c u r i - c e l l u l o s e a l s o g a v e . a n e g a t i v e n i t r o p r u s s i d e t e s t . H o w e v e r , w h e n i t w a s r e s u s p e n d e d i n very-d i l u t e m e r c a p t o e t h a n o l a n d w a s h e d r e p e a t e d l y w i t h d i s t i l l e d w a t e r , the r e s u l t i n g H S A E - c e l l u l o s e t h e n gave a s t r o n g p o s i t i v e r e a c t i o n to . - 8 6 -n i t r o p r u s s i d e . 7. B e h a v i o u r of s - R N A on H g S A E - C e l l u l o s e T o a s m a l l c o l u m n (7 x 1 c m d i a m . ) of H g S A E - c e l l u l o s e , p r e v i o u s l y e q u i l i b r a t e d w i t h 0. 01 M N a O A c , . p H 5. 2, w a s a d d e d 20 O . D . u n i t s of s - R N A d i s s o l v e d . i n 1. 0 m l of d i s t i l l e d w a t e r . T h e c o l u m n w a s w a s h e d s u c c e s s i v e l y w i t h 50 m l of 0. 01 M N a O A c , - p H 5. 2, 100 m l 0. 01 M N H 4 O A c , p H 5. 2, t h e n w i t h 150 m l 0. 01 M N a O A c , . p H 5. 2, 0.10 M N a C l . N o u l t r a v i o l e t a b s o r b i n g m a t e r i a l w a s e l u t e d . T h e s - R N A w a s f i n a l l y e l u t e d by w a s h i n g ; w i t h 0. 01 M N a O A c , p H 5. 2, 1. 0 M N a C l . T h e ef f luent gave a n e g a t i v e t e s t w i t h N a l i n d i c a t i n g . t h a t the H g h a d not b e e n e l u t e d . A f t e r w a s h i n g the c o l u m n f r e e of c h l o r i d e i o n s w i t h 0. 01 M N a O A c , p H 5. 2,. a n a d d i t i o n a l 10 O . D . u n i t s of s - R N A w e r e a p p l i e d a n d the c o l u m n d e v e l o p e d s u c c e s s i v e l y w i t h 50 m l of 0. 01 M N a O A c , , ; p H 5. 2, 75 m l of 0.10 M N a C l , 0. 01 M N a O A c , pH 5. 2, t h e n w i t h 0. 01 M N a O A c , p H 5. 2, 0. 01 M m e r c a p t o e t h a n o l . T h e O . D . o b s e r v e d at 260 m u . . w a s due to m e r c a p t o e t h a n o l a n d to the H g II i o n . F i n a l l y the c o l u m n w a s w a s h e d w i t h 1 M N a C l w h i c h e l u t e d the s - R N A o r i g i n a l l y a p p l i e d . T h a t the O . D . o b s e r v e d w a s due to s - R N A w a s c o n f i r m e d b y a d d i n g a s m a l l c r y s t a l of r i b o n u c l e a s e t o - a n a l i q u o t a n d o b s e r v i n g the h y p e r c h r o m i c i t y . T h e a l i q u o t s h o w e d 1 8 . 4 % h y p e r c h r o m i c i t y . i n 6 m i n . - 8 7 -8. P r e p a r a t i o n of A m i n o a c y l - s - R N A S y n t h e t a s e s f r o m Y e a s t T h e a m i n o a c y l - s - R N A s y n t h e t a s e s u s e d i n . t h i s p a r t of the e x p e r i m e n t a l w e r e p r e p a r e d f r o m f r e s h l y g r o w n b a k e r s ' y e a s t b y g r i n d i n g the c e l l s w i t h g l a s s b e a d s i n . a V i r t i s " 4 5 " h o m o g e n i z e r f o l l o w e d by c e n t r i f u g a t i o n a n d d i a l y s i s . T h e p r o c e d u r e i s d e s c r i b e d i n d e t a i l i n P a r t III S e c t i o n 1-A. 9. P r e p a r a t i o n of 1 4 C - C y s t e i n y l - s - R N A T h e r e a c t i o n m i x t u r e ( f i n a l v o l u m e 12. 0 m l ) c o n t a i n e d .3. 7 m m o l e s of T r i s ( c h l o r i d e ) , p H 7 . 4 , 1. 6 y x m o l e s of L - ^ 4 C - c y s t e i n e ( 3 . 7 4 jj^Ci), 130yu-moles of M g C ^ , 250^u.moles of K C l , 53^i_moles of A T P , 24 ^x-moles of C T P , 30 yuorioles of E D T A , 370 ^ o r i o l e s of m e r c a p t o e t h a n o l , 20 m g of s - R N A (about 40 m u m o l e s ) a n d a m i n o a c y l - s - R N A s y n t h e t a s e e n z y m e s ( c o n t a i n i n g about 10 m g of p r o t e i n ) . T h e r e a c t i o n m i x t u r e w a s i n c u b a t e d f o r 60 m i n at r o o m t e m p e r a t u r e t h e n c o o l e d i n - i c e . A l l s u b s e q u e n t o p e r a t i o n s w e r e c a r r i e d . o u t b e t w e e n 0 and 5 ° . T o the c o l d r e a c t i o n m i x t u r e w a s a d d e d .1. 0 m l of 5 M N a C l a n d 6. 0 m l of c o l d e t h a n o l w i t h t h o r o u g h m i x i n g . T h e m i x t u r e w a s l e f t i n i c e f o r 10 m i n b e f o r e c e n t r i f u g i n g . T h e s u p e r n a t e w a s d e c a n t e d a n d the p e l l e t w a s h e d by s u s p e n d i n g i t i n a n e t h a n o l : 1. 5 M N a C l (2:1). s o l u t i o n . (1 m l ) f o l l o w e d by c e n t r i f u g a t i o n . T h e w a s h i n g w a s r e p e a t e d t w i c e m o r e . T h e f i n a l p e l l e t w a s d i s s o l v e d i n c o l d 0. 5- M N a C l , 0. 01 M N a O A c , p H 5. 2. - 8 8 -A 20 y j l a l i q u o t w a s s p o t t e d on a p l a n c h e t , d r i e d a n d c o u n t e d i n a w i n d o w -l e s s g a s - f l o w c o u n t e r ( N u c l e a r C h i c a g o ) . A t o t a l of 500 c p m w e r e i n c o r p o r a t e d . 10. C h r o m a t o g r a p h y , of 1 4 C - C y s t e i n y l - s - R N A o n H g S A E - C e l l u l o s e T h e s o l u t i o n of ^ 4 C - c y s t e i n y l - s - R N A w a s a p p l i e d i m m e d i a t e l y to a c o l u m n of H g S A E - c e l l u l o s e ( p r e p a r e d as d e s c r i b e d i n S e c t i o n 6 a n d e q u i l i b r a t e d w i t h 0. 5 M N a C l , 0. 01 M N a O A c , p H 5. 2) a n d a l l o w e d to i n c u b a t e i n c o n t a c t w i t h the c o l u m n f o r 20 m i n b e f o r e e l u t i n g w i t h 3 c o l u m n v o l u m e s of 0. 5 M N a C l , . 0. 01 M N a O A c , p H 5. 2, f o l l o w e d by - 3 3 c o l u m n v o l u m e s of the s a m e b u f f e r c o n t a i n i n g 10 M c y s t e i n e . F r a c t i o n s of 1. 0 m l e a c h w e r e c o l l e c t e d . T h e a b s o r b a n c e at 260 rryj-w a s r e c o r d e d . a n d r a d i o a c t i v i t y w a s d e t e r m i n e d b y - a p p l y i n g 50 ^L . 1 f r o m e a c h f r a c t i o n . t o a p l a n c h e t , d r y i n g u n d e r a h e a t l a m p a n d c o u n t i n g as d e s c r i b e d p r e v i o u s l y . A l l the a p p l i e d r a d i o a c t i v i t y a n d n e a r l y , a l l the a p p l i e d u l t r a v i o l e t a b s o r b i n g m a t e r i a l a p p e a r e d at the e l u t i o n f r o n t ' of the c o l u m n . N o r a d i o a c t i v i t y w a s d e t e c t e d i n the c y s t e i n e w a s h . 11. A c e t y l a t i o n a n d D e a c e t y l a t i o n of C e l l u l o s e W h a t m a n c o a r s e g r a d e c e l l u l o s e p o w d e r (50 g) w a s m i x e d w i t h 1 l i t r e of g l a c i a l a c e t i c a c i d i n a W a r i n g , b l e n d o r at l o w s p e e d . T o the s u s p e n s i o n c o n t a i n e d i n a 2 l i t r e f lask w a s a d d e d 0. 6 m l of c o n e . H ^ S O ^ t h e n the m i x t u r e w a s l e f t 3 h r at r o o m t e m p e r a t u r e . T h e n 250 m l of - 8 9 -a c e t i c a n h y d r i d e w a s a d d e d a n d the m i x t u r e a g i t a t e d f o r 5 h r at r o o m t e m p e r a t u r e . T h e v i s c o u s l i q u i d w a s d i v i d e d a m o n g two 10 l i t r e v e s s e l s of c o l d t a p w a t e r i n the f o r m of a t h i n s t r e a m w i t h c o n s t a n t s t i r r i n g . T h e r e s u l t i n g f i l a m e n t o u s p r e c i p i t a t e w a s p o u r e d i n t o a l a r g e B u c h n e r f u n n e l l i n e d w i t h c h e e s e c l o t h a n d w a s h e d w i t h c o l d t a p w a t e r u n t i l the f i l t r a t e no l o n g e r f r o t h e d . E x c e s s w a t e r w a s r e m o v e d b y s u c t i o n a n d the c e l l u l o s e a c e t a t e s u s p e n d e d i n 2 l i t r e s of e t h a n o l , 0. 5 N N a O H . A f t e r 20 h r t h e s u s p e n s i o n w a s s t i r r e d i n a W a r i n g . b l e n d o r at l o w s p e e d f o r a few m i n t h e n l e f t a f u r t h e r 40 h r . T h e s u s p e n s i o n : w a s t h e n p o u r e d i n t o a c h r o m a t o g r a p h i c c o l u m n a n d w a s h e d w i t h 12 l i t r e s of d i s t i l l e d w a t e r . T h e c e l l u l o s e w a s e x t r u d e d , r e s u s p e n d e d i n , 1600 m l of d i s t i l l e d w a t e r , s t i r r e d . i n a W a r i n g b l e n d o r at l o w s p e e d t h e n p o u r e d b a c k . i n t o the c h r o m a t o g r a p h i c c o l u m n . T h e c o l u m n w a s w a s h e d w i t h a n ; a d d i t i o n a l 2 l i t r e s of d i s t i l l e d w a t e r . T h e e l u a t e n o w s h o w e d a n e u t r a l r e a c t i o n . t o p H p a p e r . T h e w a s h e d c e l l u l o s e w a s s p r e a d on a g l a s s p l a t e a n d d r i e d i n a v a c u u m o v e n at 60° i n the p r e s e n c e of s i l i c a g e l . T h e r e s u l t i n g w h i t e p o w d e r (43 g) w a s s t o r e d u n d e r v a c u u m u n t i l r e a d y f o r u s e . 12. P r e p a r a t i o n of T o s y l - C e l l u l o s e A . R e c r y s t a l l i z a t i o n of T o s y l c h l o r i d e T h e f o l l o w i n g p r o c e d u r e w a s s u g g e s t e d by D r . K . N . S l e s s o r . E q u a l v o l u m e s of w a t e r a n d t o s y l c h l o r i d e w e r e w a r m e d u n t i l the l a t t e r -90-f o r m e d a n o i l i n the b o t t o m of the f l a s k . U p o n c o o l i n g , the o i l s o l i d i f i e d and the w a t e r w a s d e c a n t e d . T h e s o l i d w a s d i s s o l v e d i n b o i l i n g p e t r o l e u m e t h e r (b. p . 3 0 - 6 0 ° ) , t r e a t e d w i t h c h a r c o a l a n d f i l t e r e d by-g r a v i t y . T h e w h i t e c r y s t a l s , w h i c h r e s u l t e d on c o o l i n g of the f i l t r a t e , w e r e c o l l e c t e d by d e c a n t i n g the s u p e r n a t e a n d s p r e a d i n g the c r y s t a l s on a f i l t e r p a p e r to d r y at r o o m t e m p e r a t u r e . B . T o s y l a t i o n of D e a c e t y l a t e d C e l l u l o s e A c e t a t e D e a c e t y l a t e d c e l l u l o s e a c e t a t e (16 g) w a s " a c t i v a t e d " by s h a k i n g o v e r n i g h t i n a f l a s k c o n t a i n i n g 100 m l of d i s t i l l e d w a t e r a n d 200 m l of p y r i d i n e ( M a l m et a l . , 1948). T h e c e l l u l o s e w a s t h e n s u c k e d d r y on a B u c h n e r f u n n e l w i t h the a i d of a r u b b e r sheet , w a s h e d 3 t i m e s w i t h a n h y d r o u s p y r i d i n e a n d t r a n s f e r r e d to 1 a f l a s k c o n t a i n i n g 38 g of r e c r y s t a l l i z e d t o s y l c h l o r i d e i n 250 m l of a n h y d r o u s p y r i d i n e . A f t e r s h a k i n g f o r 31 h r at r o o m t e m p e r a t u r e , the g e l w a s s t i r r e d w i t h 3 l i t r e s of m e t h a n o l . T h e g r a n u l a r , a l m o s t w h i t e , p r o d u c t w a s s u c t i o n - f i l t e r e d on a B u c h n e r f u n n e l , s t i r r e d i n . a W a r i n g b l e n d o r c o n t a i n i n g m e t h a n o l , f i l t e r e d a g a i n a n d w a s h e d f r e e of c h l o r i d e i o n s w i t h m e t h a n o l (AgNO-^ t e s t ) . T h e p r o d u c t w a s t h e n s p r e a d on g l a s s p l a t e s a n d d r i e d . i n a v a c u u m o v e n at 50° to y i e l d 33. 3 g. T h e o r e t i c a l y i e l d f o r 6 - O - t o s y l a t i o n ;was 31. 2 g. 13. C h a r a c t e r i z a t i o n of T o s y l - C e l l u l o s e S u l f u r a n a l y s i s w a s c a r r i e d out a c c o r d i n g to the method, of L y s y j and Z a r e m b o (1958). T o a 250 m l i o d i n e f l a s k w a s a d d e d 25 m l of a 6% s o l u t i o n of H^02> two d r o p s of m e t h y l r e d i n d i c a t o r a n d the contents t i t r a t e d to the end p o i n t w i t h 0. 02 N N a O H . A s a m p l e of the t o s y l a t e d c e l l u l o s e (about 5 m g ) w a s w e i g h e d on a n , a s h l e s s f i l t e r p a p e r ( W h a t m a n 41), 2 c m x 2 c m s q u a r e w i t h a 5 c m x 0 . 3 c m w i c k p r o j e c t i n g f r o m one c o r n e r . T h e p a p e r w a s f o l d e d t w i c e l e n g t h w a y s , r o l l e d i n t o a t i g h t c y l i n d e r a n d i n s e r t e d : i n t o the c e n t r e of a s p i r a l p l a t i n u m w i r e w i t h the w i c k p r o j e c t i n g u p w a r d s a n d the p l a t i n u m w i r e s u s p e n d e d f r o m the g l a s s hook b e l o w the s t o p p e r of the i o d i n e f l a s k . A f t e r f l u s h i n g the f l a s k w i t h o x y g e n f o r one m i n , the w i c k w a s l i g h t e d a n d the s t o p p e r h e l d d o w n f i r m l y u n t i l the c o m b u s t i o n w a s c o m p l e t e . T h e f l a s k w a s t h e n s h a k e n v i g o r o u s l y to a b s o r b , a l l the g a s e s , o p e n e d a n d the w a l l s r i n s e d w i t h 100 m l of d i s t i l l e d w a t e r . The v o l u m e w a s r e d u c e d to about 25 m l on a hot p l a t e , then, a n o t h e r 100 m l of d i s t i l l e d w a t e r w a s a d d e d . a n d the v o l u m e r e d u c e d a g a i n to about 25 m l . A f t e r c o o l i n g the f l a s k , 2 d r o p s of m e t h y l r e d i n d i c a t o r w e r e a d d e d and the c o n t e n t s of the f l a s k t i t r a t e d to t h end p o i n t w i t h . 0 . 02 N N a O H . T h e p e r c e n t S i n the s a m p l e w a s c a l c u l a t e d f r o m the f o r m u l a : ( m l ) ( N ) ( ! 6 . 03-)(100) /o O — ' w t . of s a m p l e w h e r e : N = n o r m a l i t y of N a O H u s e d i n the t i t r a t i o n m l = m l of N a O H u s e d i n the t i t r a t i o n . T h e a v e r a g e v a l u e f o r d u p l i c a t e a n a l y s e s w a s 1 0 . 9 % S. T h e o r e t i c a l f o r a D S - 1 w a s 10.14% S. 14. P r e p a r a t i o n of S - A c e t y l - C e l l u l o s e T h e p o t a s s i u m t h i o a c e t a t e w a s a g i f t f r o m D r . K . N . S l e s s o r a n d w a s p r e p a r e d a c c o r d i n g to B a n n i s t e r a n d K a g a n (I960). T o s y l - c e l l u l o s e (1. 0 g, D . S . = 1. 07) and 0. 8 g of p o t a s s i u m t h i o a c e t a t e ( K S A c ) w e r e d i s s o l v e d i n 20 m l of N , N - d i m e t h y l f o r m a m i d e ( D M F ) : a n d h e a t e d on a s t e a m b a t h f o r 2 h r w i t h o c c a s i o n a l s t i r r i n g . T h e s o l u t i o n b e c a m e v e r y v i s c o u s . Then.100 m l of m e t h a n o l w a s a d d e d s l o w l y , to e f fect the p r e c i p i t a t i o n of the c e l l u l o s e , f o l l o w e d by the c o n s e c u t i v e a d d i t i o n of 50 m l of 8 0 % a q u e o u s m e t h a n o l a n d 50 m l of d i s t i l l e d w a t e r . The . p r e c i p i t a t e w a s c o l l e c t e d b y c e n t r i f u g a t i o n a n d w a s h e d w i t h 8 0 % m e t h a n o l u n t i l the s u p e r n a t e gave a n e g a t i v e n i t r o -p r u s s i d e r e a c t i o n . T h e p r o d u c t w a s t h e n w a s h e d w i t h 25 m l of e t h a n o l f o l l o w e d by 25 m l of e t h y l e t h e r and d r i e d i n a v a c u u m o v e n at 4 5 ° . Y i e l d w a s 0. 63 g a n d a s l o w r e a c t i o n . w a s o b t a i n e d w i t h . t h e n i t r o p r u s s i d e r e a g e n t , as e x p e c t e d f o r t h i o l e s t e r s . - 9 3 -15. H y d r o l y s i s of S - A c e t y l - C e l l u l o s e A . H y d r o l y s i s i n 0. 02 N S o d i u m M e t h o x i d e T o the S - a c e t y l ^ c e l l u l o s e (0 . 60 g), s u s p e n d e d i n 8 m l of m e t h a n o l , w a s a d d e d 2. 0 m l of 0.1 N N a O C H 3 i n m e t h a n o l a n d the s o l u t i o n , s t i r r e d at r o o m t e m p e r a t u r e f o r 21 h r . A f t e r a c i d i f y i n g to p H 5. 0 w i t h a c e t i c a c i d , the p r o d u c t w a s f i l t e r e d , w a s h e d u n t i l n e u t r a l w i t h m e t h a n o l , t h e n s w a s h e d w i t h 8 0 % a q u e o u s m e t h a n o l a n d f i n a l l y w i t h m e t h a n o l . T h e p r o d u c t w a s t h e n d r i e d i n a v a c u u m o v e n . a t 4 0 ° to y i e l d 0. 4 8 6 g, d r y w e i g h t . It r e a c t e d s l o w l y w i t h .the n i t r o p r u s s i d e r e a g e n t but d i d not r e a c t w i t h i o d o a c e t i c a c i d u n d e r the c o n d i t i o n s d e s c r i b e d i n S e c t i o n 3. B. H y d r o l y s i s i n 0.10 N S o d i u m H y d r o x i d e 100 m g of S - a c e t y l - c e l l u l o s e w e r e s u s p e n d e d i n . 10 m l of 0.10 N N a O H and h e a t e d at 100° f o r 20 m i n . A g e l f o r m e d u p o n h e a t i n g a n d the i c e l l u l o s e c o u l d not be p r e c i p i t a t e d by c o o l i n g o r by d i l u t i o n w i t h a n e q u a l v o l u m e of w a t e r . P r e c i p i t a t i o n w a s e f f e c t e d by the a d d i t i o n of 1 v o l u m e of d i s t i l l e d w a t e r a n d 10 v o l u m e s of a c e t o n e u n d e r r a p i d s t i r r i n g . P r e c i p i t a t i o n j w a s c o m p l e t e d by the a d d i t i o n of e t h y l e t h e r to' the a c e t o n e -w a t e r m i x t u r e . T h e s u p e r n a t e w a s d e c a n t e d a n d the g r a n u l a r , l i g h t y e l l o w p r o d u c t c o l l e c t e d b y c e n t r i f u g a t i o n f r o m m e t h a n o l . It w a s w a s h e d . w i t h m e t h a n o l , t h e n w i t h e t h y l e t h e r and d r i e d i n v a c u u m o v e n o at 50 .. T h e p r o d u c t r e a c t e d r a p i d l y a n d s t r o n g l y w i t h n i t r o p r u s s i d e - 9 4 -a n d r e a c t e d w i t h I A A to the extent of 0. 5 m e q p e r d r y g r a m of p r o d u c t d u r i n g 80 m i n u n d e r the c o n d i t i o n s d e s c r i b e d i n S e c t i o n , 3. W h e n 1 g of S - a c e t y l - c e l l u l o s e w a s h y d r o l y z e d i n 3 5 - m l of 0.1 N N a O H , i . e . , , i n c r e a s e d r a t i o of S - a c e t y l - c e l l u l o s e : 0.1 N N a O H (w/v) the H S - c e l l u l o s e c o u l d be r e c o v e r e d b y n e u t r a l i z i n g the a l k a l i n e s o l u t i o n w i t h g l a c i a l a c e t i c a c i d , c e n t r i f u g a t i o n a n d w a s h i n g ; the p r o d u c t w i t h w a t e r . 16. L a r g e S c a l e P r e p a r a t i o n of H S - C e l l u l o s e T o 11. 4 g of t o s y l - c e l l u l o s e (36 m m o l e s of g l u c o s e ) a n d 8. 2 g of K S A c (7 2 m m o l e s ) , w a s a d d e d 100 m l of D M F a n d the m i x t u r e h e a t e d on a s t e a m b a t h f o r 2 h r . T h e t o s y l - c e l l u l o s e d i s s o l v e d i n the hot s o l v e n t . A f t e r 2 hr. on the s t e a m b a t h a p o r t i o n ( s a m p l e I) w a s r e m o v e d and a n a d d i t i o n a l 0. 3 g of K S A c w a s a d d e d to the r e m a i n i n g m i x t u r e a n d h e a t i n g c o n t i n u e d f o r a n o t h e r h o u r ( s a m p l e II) . A f t e r c o o l i n g , one v o l u m e of D M F i n 5 v o l u m e s of m e t h a n o l w a s a d d e d s l o w l y w i t h s t i r r i n g , to e a c h s a m p l e . T h e y e l l o w s u p e r n a t e w a s d e c a n t e d a n d 2 v o l u m e s of m e t h a n o l : D M F (9:1, v/v) w e r e a d d e d to the r e s i d u e . T h e s o l u t i o n w a s s t i r r e d f o r a few m i n u t e s , t h e n 4 v o l u m e s of m e t h a n o l w e r e a d d e d . a n d the p r o d u c t a l l o w e d t o s e t t l e . T h e p r o d u c t w a s c o l l e c t e d by c e n t r i f u g a t i o n , w a s h e d w i t h m e t h a n o l t h e n w i t h e t h y l e t h e r a n d d r i e d i n a v a c u u m o v e n .at 5 0 ° . T h e y i e l d w a s 1.7 g a n d 5.1 g, f o r s a m p l e s I and II r e s p e c t i v e l y . P o r t i o n s of s a m p l e s I a n d 11.(100 m g e a c h ) w e r e h y d r o l y z e d as d e s c r i b e d i n S e c t i o n 1 5 - B . B o t h r e a c t e d , r a p i d l y a n d s t r o n g l y , w i t h n i t r o p r u s s i d e a n d b o t h r e a c t e d w i t h I A A u n d e r the c o n d i t i o n s d e s c r i b e d i n S e c t i o n 3. T h e r e s u l t s of the t i t r a t i o n s s h o w e d 1. 2 m e q . a n d 1. 7 m e q p e r g r a m of d r y S a m p l e s I a n d II r e s p e c t i v e l y w h e n the t i t r a t i o n r e a c t i o n w a s f o l l o w e d f o r 3 h r . H o w e v e r , b o t h . s a m p l e s s t i l l r e a c t e d w i t h n i t r o p r u s s i d e i n d i c a t i n g that the c a r b o x y m e t h y l a t i o n of H S - g r o u p s w a s i n c o m p l e t e . W h e n the t i t r a t i o n of S a m p l e II w a s f o l l o w e d f o r 6 h r a v a l u e of 1. 9 m e q of H S - g r o u p s p e r g r a m of d r y H S - c e l l u l o s e w a s o b t a i n e d . A g a i n the r e a c t i o n w a s i n c o m p l e t e as s h o w n b y the t i t r a t i o n c u r v e a n d the n i t r o p r u s s i d e t e s t . T h a t the r e a c t i o n was. i n d e e d i n c o m p l e t e w a s s h o w n b y t i t r a t i n g t o s y l - c e l l u l o s e u n d e r the s a m e c o n d i t i o n s . N o a l k a l i w a s c o n s u m e d d u r i n g 5 h r . 17. - C h r o m a t o g r a p h y of s - R N A o n H S - C e l l u l o s e s - R N A (10 mg)iwas d i s s o l v e d i n a few m i s of 0.10 M N a O A c , ;_pH 5 . 2 a n d a p p l i e d to' a n H S - c e l l u l o s e c o l u m n (10- x 1 c m d i a m . ), p r e v i o u s l y e q u i l i b r a t e d w i t h 0.10 M N a O A c , p H 5. 2 a n d d e v e l o p e d w i t h a l i n e a r g r a d i e n t of N a C l (0 to 0. 3 M N a C l ) , t o t a l v o l u m e 600 m l , w i t h b o t h v e s s e l s b u f f e r ed at p H 5. 2 w i t h 0. 10 M N a O A c . A b o u t 75% of the O . D . w a s e l u t e d at the s o l v e n t f r o n t w i t h the r e m a i n d e r b e i n g e l u t e d at about 0. 03 M N a C l . B o t h . p e a k s h a d s i m i l a r u l t r a v i o l e t s p e c t r a a n d h y p e r c h r o m i c e f fec ts on d i g e s t i o n w i t h p a n c r e a t i c r i b o n u c l e a s e . - 9 6 -18. ; C o n v e r s i o n of HS - C e l l u l o s e to M e r c u r i - C e l l u l o s e ( H g S - C e l l u l o s e ) T h e H S - c e l l u l o s e w a s s u s p e n d e d i n w a t e r o v e r n i g h t then; p a c k e d i n a j a c k e t e d , w a t e r - c o o l e d (about 4 ° ) c o l u m n (14 x l c m d i a m . ); a n d w a s h e d w i t h d i s t i l l e d w a t e r u n t i l the o d o r of t h i o a c e t i c a c i d w a s no l o n g e r d e t e c t e d . i n ; the e f f luent . T h e c o l u m n , w a s then: w a s h e d w i t h a 1% s o l u t i o n of b i s - 3 , 6 - ( a c e t a t o m e r c u r i m e t h y l ) - d i o x a n e i n 0. 01 M s o d i u m p h o s p h a t e , . p H 7. 6 u n t i l a p o s i t i v e N a l t e s t (white p r e c i p i t a t e ) w a s o b t a i n e d , i n the e f f luent , t h e n w i t h d i s t i l l e d w a t e r u n t i l a n e g a t i v e N a l t e s t w a s o b t a i n e d . The c o l u m n w a s t h e n e q u i l i b r a t e d w i t h 0. 01 M N a O A c , p_H 5 . 2 . 14 19. C h r o m a t o g r a p h y of C - C y s t e i n y l - s - R N A o n H g S - C e l l u l o s e 14 C - C y s t e i n y l - s - R N A w a s p r e p a r e d as d e s c r i b e d i n S e c t i o n 9 a n d the p e l l e t d i s s o l v e d i n 6. 0 m l of 0. 01 M N a O A c , p H 5. 2. A 3. 5 m l a l i q u o t of the s o l u t i o n , c o n t a i n i n g p e r m l , 66 O . D . u n i t s a n d 1600 c p m , w a s a p p l i e d to the H g S - c e l l u l o s e c o l u m n (14 x 1 c m d i a m . ) a n d a l l o w e d to i n c u b a t e i n c o n t a c t w i t h the H g S - c e l l u l o s e f o r 10 m i n at about 4 ° . T h e c o l u m n w a s t h e n w a s h e d w i t h a 1 l i t r e l i n e a r g r a d i e n t f r o m 0 to 0. 6 M N a C l , b o t h v e s s e l s , b u f f e r e d a t . p H 5. 2 w i t h 0. 01 M N a O A c . T h e f l o w r a t e w a s 28 m l p e r h r a n d 2. 8 m l f r a c t i o n s w e r e c o l l e c t e d . T h e g r a d i e n t w a s t e r m i n a t e d . a t 0. 4 M N a C l a n d e l u t i o n c o n t i n u e d w i t h 0. 4 M N a C l , 0. 01 M N a O A c , p H 5. 2, 0. 01 M m e r c a p t o e t h a n o l . T h e a b s o r b a n c e at 260 rryx w a s r e c o r d e d a n d 0.10 m l a l i q u o t s of the f r a c t i o n s w e r e . a p p l i e d t o p l a n c h e t s , d r i e d a n d c o u n t e d i n a w i n d o w l e s s g a s - f l o w c o u n t e r . T h e r e s u l t s a r e s h o w n i n F i g u r e 3. W h e n the * 4 C - c y s t e i n y l - s - R N A w a s a p p l i e d to a H g S - c e l l u l o s e c o l u m n e q u i l i b r a t e d w i t h 0. 10 M N a C l , 0. 01 M N a O A c , p H 5. 2 the b u l k of the a p p l i e d m a t e r i a l a n d a l l the r a d i o a c t i v i t y w a s e l u t e d a f t e r about one c o l u m n . v o l u m e of the l i n e a r g r a d i e n t (0.1 to 0. 4 M N a C l ) h a d p a s s e d t h r o u g h the c o l u m n . N e i t h e r a p p l i e d m a t e r i a l n o r r a d i o a c t i v i t y w a s d e t e c t a b l e i n the m e r c a p t o e t h a n o l w a s h . P A R T I I - B . P A R T I T I O N C H R O M A T O G R A P H Y O F s - R N A O N B I O G E L 1. P a r t i t i o n C h r o m a t o g r a p h y of s - R N A on B i o g e l P - 1 0 u s i n g the S y s t e m of W a r n e r a n d V a i m b e r g (1958) A two p h a s e s y s t e m d e s c r i b e d by W a r n e r a n d V a i m b e r g (1958) w a s p r e p a r e d a c c o r d i n g to A p g a r _et a l . (1962). The b i p h a s i c s y s t e m c o n s i s t e d of 131 g of K 2 H P 0 4 ' 3 H z O , 156 g of N a H 2 P 0 4 ' H 2 0 , 13 60 m l of d i s t i l l e d w a t e r , 130 m l of f o r m a m i d e a n d 700 m l of i s o p r o p a n o l . The m i x t u r e w a s s h a k e n a n d a l l o w e d to e q u i l i b r a t e o v e r n i g h t . A t t e m p t s to c h r o m a t o g r a p h s - R N A i n t h i s s y s t e m u s i n g B i o g e l P - 3 0 0 , P - 1 5 0 , P - 6 0 a n d P - 3 0 w e r e t e c h n i c a l l y i m p r a c t i c a l due to the s l o w f l o w r a t e s . T h u s B i o g e l P-10 w a s u s e d as s u p p o r t i n g m a t e r i a l f o r the s t a t i o n a r y p h a s e . \ 1 120 140 1 6 0 1 8 0 F R A C T I O N No. ( 2 .8ml ) 2 4 0 2 6 0 0 . 5 T | Q 4 : i 0.3g 0 . 2 ^ z 0.1 0 F I G U R E 3. C h r o m a t o g r a p h y of 1 4 C - c y s t e i n y l - s - R N A on H g S - c e l l u l o s e . - 9 9 -A . < D e t e r m i n a t i o n of the D i s t r i b u t i o n C o e f f i c i e n t (K) A 1 m g s a m p l e of s - R N A w a s d i s s o l v e d i n 2 m l of the u p p e r p h a s e t h e n 2 m l of the l o w e r p h a s e w a s a d d e d a n d the m i x t u r e s h a k e n v i g o r o u s l y . A f t e r a l l o w i n g 4 h r f o r the s y s t e m to r e a c h e q u i l i b r i u m , the O . D . at 260 m>ju of the u p p e r a n d l o w e r p h a s e s w a s d e t e r m i n e d . T h e r a t i o of the O . D . . i n . t h e u p p e r p h a s e to the O . D . i n the l o w e r p h a s e ( K ) . w a s 0 . 9 3 . T h e . p r o c e d u r e d e s c r i b e d a b o v e w a s r e p e a t e d e x c e p t t h i s t i m e the s - R N A s a m p l e w a s d i s s o l v e d i n the l o w e r p h a s e , t h e n a n e q u a l v o l u m e of the u p p e r p h a s e w a s a d d e d . A v a l u e of K = 1 . 01 w a s o b t a i n e d i n d i c a t i n g that the s y s t e m h a d a l m o s t r e a c h e d e q u i l i b r i u m a f t e r 4 h r . B . P a r t i t i o n C h r o m a t o g r a p h y of s - R N A : E x p e r i m e n t 1 A 6 m g s a m p l e of s - R N A w a s d i s s o l v e d i n 5 m l of u p p e r p h a s e a n d . a p p l i e d to a B i o g e l P - 1 0 c o l u m n (22 x 1. 8 c m d i a m . ) w h i c h h a d b e e n p a c k e d a n d w a s h e d w i t h the l o w e r , p h a s e a n d e q u i l i b r a t e d w i t h s e v e r a l c o l u m n v o l u m e s of the u p p e r p h a s e . T h e c o l u m n w a s w a s h e d w i t h . t h e u p p e r p h a s e but no s - R N A w a s e l u t e d . W h e n the c o l u m n w a s w a s h e d w i t h a s o l u t i o n of u p p e r p h a s e : d i s t i l l e d w a t e r , i n a r a t i o of . 3:1 (v/v) the s - R N A w a s e l u t e d . C . P a r t i t i o n C h r o m a t o g r a p h y of s - R N A : E x p e r i m e n t 2 T h e P-10 B i o g e l c o l u m n (22 x 1. 8 c m d i a m . } w a s r e p a c k e d i n l o w e r p h a s e a n d e q u i l i b r a t e d w i t h s e v e r a l c o l u m n v o l u m e s of u p p e r - 1 0 0 -p h a s e . T o a 10 m g s a m p l e of s - R N A d i s s o l v e d i n 0. 5 m l of d i s t i l l e d w a t e r w a s a d d e d 0 . 4 5 m l of u p p e r p h a s e a n d a p p l i e d to the c o l u m n . T h e c o l u m n w a s d e v e l o p e d w i t h a l i n e a r g r a d i e n t m a d e u p of 200 m l of u p p e r p h a s e i n the m i x i n g v e s s e l a n d 200 m l of u p p e r p h a s e a n d d i s t i l l e d w a t e r , i n a r a t i o of 3:2 (v/v) , i n the r e s e r v o i r . T h e f l o w r a t e w a s 16 m l p e r h o u r a n d 4 . 0 m l f r a c t i o n s w e r e c o l l e c t e d . The s - R N A w a s e l u t e d b e t w e e n f r a c t i o n s 22 a n d 50. T h e O. D . p r o f i l e w a s d i v i d e d i n t o 3 s e c t i o n s and the s - R N A i n e a c h r e c o v e r e d by e x h a u s t i v e d i a l y s i s f o l l o w e d by l y o p h i l i z a t i o n . R e c o v e r y of the a p p l i e d s a m p l e w a s q u a n t i t a t i v e . F r a c t i o n s I a n d III w e r e c o m b i n e d a n d r e c h r o m a t o g r a p h e d u n d e r the c o n d i t i o n s d e s c r i b e d a b o v e . T h e s - R N A w a s e l u t e d as a b r o a d p e a k a n d no r e s o l u t i o n of the a p p l i e d s a m p l e s c o u l d be d e t e c t e d . 2. P a r t i t i o n C h r o m a t o g r a p h y of s - R N A o n B i o g e l P - 1 0 U s i n g the S y s t e m of Z a c h a u et a l . (1961) A . P r e p a r a t i o n of the T r i b u t y l a m m o n i u m S a l t of s - R N A M e t h o d 1: T h e s o d i u m s a l t of s - R N A w a s d i s s o l v e d i n w a t e r a n d p a s s e d t h r o u g h a D o w e x 50 ( t r i b u t y l a m m o n i u m ) c o l u m n . T h e e f f luent c o n t a i n i n g the s - R N A w a s c o l l e c t e d and l y o p h i l i z e d . M e t h o d 2: T h e s o d i u m s a l t of s - R N A w a s d i s s o l v e d i n d i s t i l l e d w a t e r , c o o l e d to 0° a n d 2. 5 v o l u m e s of c o l d 0. 2 N H C 1 w a s a d d e d . T h e m i x t u r e w a s l e f t 10 m i n i n i c e a n d t h e n c e n t r i f u g e d . T h e s u p e r n a t e w a s d e c a n t e d - 1 0 1 -and the p e l l e t w a s h e d t w i c e w i t h a s m a l l v o l u m e of 0. 05 N H C 1 , t h e n w i t h e t h y l e t h e r a n d d r i e d at r o o m t e m p e r a t u r e . T h e d r y p e l l e t , w h i c h w a s now i n the f o r m of the f r e e a c i d , w a s d i s s o l v e d i n the u p p e r p h a s e as the t r i b u t y l a m m o n i u m s a l t . B . P r e p a r a t i o n of the B i o g e l P - 1 0 f o r P a r t i t i o n C h r o m a t o g r a p h y T h e p a r t i t i o n s y s t e m u s e d w a s that d e s c r i b e d by Z a c h a u et a l . (1961) f o r the c o u n t e r c u r r e n t d i s t r i b u t i o n of s - R N A a n d c o n s i s t e d of n - b u t a n o l , H ^ O , t r i b u t y l a m i n e ( f r e s h l y d i s t i l l e d ) a c e t i c a c i d a n d b u t y l e t h e r , (100:130:10:2. 5:27). T h e B i o g e l P-10 w a s p a c k e d i n a c o l u m n u s i n g the l o w e r p h a s e , t h e n w a s h e d w i t h s e v e r a l c o l u m n v o l u m e s of l o w e r p h a s e a n d f i n a l l y e q u i l i b r a t e d w i t h s e v e r a l c o l u m n v o l u m e s of u p p e r p h a s e . C . P a r t i t i o n C h r o m a t o g r a p h y of s - R N A A 250 m g s a m p l e of s o d i u m s - R N A w a s c o n v e r t e d to the f r e e a c i d as d e s c r i b e d i n S e c t i o n 2 - A ( M e t h o d 2) a n d d i s s o l v e d i n 7. 0 m l of u p p e r p h a s e . T h i s w a s a p p l i e d to a B i o g e l P-10 c o l u m n (88 x 3. 0 c m d i a m . ) p r e p a r e d as d e s c r i b e d i n S e c t i o n 2 - B w i t h the t e m p e r a t u r e m a i n t a i n e d at 2 4 ° b y m e a n s of t h e r m o s t a t t e d w a t e r c i r c u l a t i n g t h r o u g h a g l a s s j a c k e t . T h e c o l u m n w a s w a s h e d w i t h u p p e r p h a s e . F r a c t i o n s of 8 m l e a c h w e r e c o l l e c t e d at a f l o w r a t e of 32 m l p e r h r . - 1 0 2 -T o e a c h f r a c t i o n w a s a d d e d 0. 60 m l of 2 M N a O A c , 0 . 0 0 1 M M g ( O A c ) 2 a n d 0. 60 m l of d i b u t y l e t h e r . E a c h f r a c t i o n w a s t h o r o u g h l y m i x e d on a V o r t e x m i x e r a n d t r a n s f e r r e d to the c o l d r o o m ( 3 - 5 ° ) o v e r n i g h t . T h e u p p e r p h a s e w a s r e m o v e d by s u c t i o n a n d to the l o w e r p h a s e w a s a d d e d 2 v o l u m e s of c o l d e t h a n o l . A f t e r a m i n i m u m of 20 m i n i n i c e , e a c h f r a c t i o n w a s c e n t r i f u g e d , and the p e l l e t w a s h e d w i t h 0. 50 m l of e t h a n o l : e t h y l e t h e r : 1 M N a O A c : 0. 5 m M M g ( O A c ) 2 , . (3:1:1:1). T o e a c h odd n u m b e r e d f r a c t i o n w a s a d d e d 1. 0 m l of d i s t i l l e d w a t e r . A 0.10 m l a l i q u o t w a s t h e n d i l u t e d to 2. 0 m l w i t h d i s t i l l e d w a t e r a n d the a b s o r b a n c e at 260 rrjM-was r e c o r d e d . T h e c o l u m n ef f luent , c o n t a i n i n g the s - R N A , w a s t h e n c o m b i n e d i n t o 8 f r a c t i o n s w h i c h w e r e d i a l y z e d a g a i n s t d i s t i l l e d w a t e r a n d a s s a y e d f o r a c c e p t o r a c t i v i t y f o r g l y c i n e a n d h i s t i d i n e . D . A s s a y of the S a m p l e s O b t a i n e d f r o m P a r t i t i o n C h r o m a t o g r a p h y E a c h a s s a y m i x t u r e ( f i n a l v o l u m e 0. 50 m l ) c o n t a i n e d 90 y u m o l e s of T r i s ( a c e t a t e ) , p H 7 . 4 , 1. 5 y O m o l e s of s o d i u m A T P , 7. 7 yu.moles of M g C l 2 , 15. 4 y o m o l e s of K C l , 0. 4 ^ . m o l e of E D T A , 0. 3 7 ^ . m o l e of 14 m e r c a p t o e t h a n o l , 4 O . D . u n i t s of s - R N A , 0 . 5 yU.Ci of C - L - a m i n o a c i d , 43 muaJtrioles of g l y c i n e o r 52 m^umoles of _ L - h i s t i d i n e , 0. 6 y u m o l e of the a m i n o a c i d s not b e i n g a s s a y e d a n d 0.12 m l of the a m i n o a c y l - s -R N A s y n t h e t a s e e n z y m e s , p r e p a r e d as d e s c r i b e d i n P a r t III , S e c t i o n 1. The m i x t u r e w a s i n c u b a t e d f o r 60 m i n at r o o m t e m p e r a t u r e t h e n 0. 15 m l of c o l d , 5 M N a C l a n d 1. 5 m l of c o l d e t h a n o l w e r e a d d e d w i t h m i x i n g . - 1 0 3 -A f t e r 20 m i n i n i c e the s o l u t i o n w a s c e n t r i f u g e d a n d the s u p e r n a t e d e c a n t e d . T h e p e l l e t w a s w a s h e d t h r e e t i m e s w i t h 0. 3 m l of a c o l d e t h a n o l - N a C l s o l u t i o n (2:1, e t h a n o l : 1. 5 M N a C l ) t h e n the p e l l e t w a s d i s s o l v e d i n 0. 15 m l of 1. 5 N N H ^ O H . A 25yo.1 a l i q u o t w a s a p p l i e d to a p l a n c h e t , d r i e d a n d c o u n t e d i n a w i n d o w l e s s g a s - f l o w c o u n t e r . P A R T I I - C . F R A C T I O N A T I O N O F s - R N A O N B E N Z O Y L A T E D - D E A E - C E L L U L O S E 1. P r e p a r a t i o n of B e n z o y l a t e d - D E A E - C e l l u l o s e 107 g of D E A E - c e l l u l o s e ( S c h l e i c h e r a n d S c h u e l l , c o n t a i n i n g 0.-91 m e q p e r g r a m and about 7% w a t e r (w/w) ) w e r e d r i e d at 80° i n a v a c u u m o v e n . f o r 20 h r . T h e d r y p o w d e r w a s s u s p e n d e d i n 2. 5 l i t r e s of p y r i d i n e , t h e n 200 m l of b e n z o y l c h l o r i d e (1. 74 m o l e s, i . e . , about 3,-; m o l e s of b e n z o y l c h l o r i d e p e r m o l e of g l u c o s e m o i e t y ) , w e r e a d d e d . T h e m i x t u r e w a s h e a t e d u n t i l r e f l u x i n g w a s o b s e r v e d a n d m a i n t a i n e d at r e f l u x t e m p e r a t u r e f o r 15 m i n , a f t e r w h i c h the m i x t u r e w a s a l l o w e d t o c o o l u n t i l r e f l u x i n g c e a s e d . A f u r t h e r 150 m l of b e n z o y l c h l o r i d e (1. 3 m o l e s ) w a s t h e n a d d e d w i t h t h o r o u g h m i x i n g . T h e m i x t u r e w a s a g a i n h e a t e d a n d r e f l u x i n g c o n t i n u e d f o r 30 m i n . T h e m i x t u r e w a s t h e n a l l o w e d to c o o l b e l o w r e f l u x t e m p e r a t u r e (but not b e l o w about 9 0 ° o r e l s e the p r o d u c t b e g a n to c o m e out of s o l u t i o n ) and p o u r e d , a s . a t h i n s t r e a m , i n t o 20 l i t r e s of t a p w a t e r , w i t h v i g o r o u s s t i r r i n g , to f o r m l o n g t u b u l a r - 1 0 4 -s t r a n d s . T h e c e n t r e s of the s t r a n d s h a d t r a p p e d u n r e a c t e d b e n z o y l c h l o r i d e and p y r i d i n e s o l v e n t . T h e w a t e r w a s d e c a n t e d a n d the s t r a n d s s u s p e n d e d o v e r n i g h t i n 40 l i t r e s of t a p w a t e r . A f t e r d e c a n t i n g the w a t e r , the s t r a n d s w e r e d i s r u p t e d i n a W a r i n g b l e n d o r , c o n t a i n i n g 75% aqueous e t h a n o l . T h e b l e n d o r w a s o p e r a t e d i n t e r m i t t e n t l y at h i g h s p e e d u n t i l a f i n e s l u r r y w a s f o r m e d . The s l u r r y w a s p o u r e d i n t o a l a r g e c h r o m a t o g r a p h i c c o l u m n , a n d w a s h e d w i t h 75% a q u e o u s e t h a n o l u n t i l the e f f luent c l e a r e d . T h e p r o d u c t w a s t r e a t e d a g a i n i n . t h e W a r i n g b l e n d o r t h e n g r a d e d b e t w e e n 50 and 200 m e s h s c r e e n s . T h e p a r t i c l e s w h i c h c o l l e c t i n , t h e 50 m e s h s c r e e n w e r e t r e a t e d a g a i n i n , t h e W a r i n g b l e n d o r as d e s c -r i b e d a b o v e . T h e 5 0 - 2 0 0 m e s h b e n z o y l a t e d - D E A E - c e l l u l o s e ( B D E A E -c e l l u l o s e ) w a s p a c k e d i n a c h r o m a t o g r a p h i c c o l u m n a n d w a s h e d w i t h 50% aqueous e t h a n o l , 1 M N a C l u n t i l the a b s o r b a n c e (260 nyx) of the ef f luent a p p r o a c h e d z e r o . T h e c o l u m n . w a s t h e n w a s h e d f r e e of c h l o r i d e i o n s w i t h d i s t i l l e d w a t e r a n d s t o r e d i n 10-15% aqueous e t h a n o l u n t i l r e q u i r e d . 2. P r e p a r a t i o n of the B D E A E - C e l l u l o s e C o l u m n B D E A E - c e l l u l o s e , p r e p a r e d as d e s c r i b e d a b o v e , w a s s u s p e n d e d i n 2 M N a C l a n d p a c k e d i n a c h r o m a t o g r a p h i c c o l u m n of the d e s i r e d d i m e n s i o n s . T h e c o l u m n w a s w a s h e d w i t h 2 M N a C l u n t i l the a b s o r b a n c e (at 260 mp-) of the ef f luent w a s z e r o t h e n t h o r o u g h l y e q u i l i b r a t e d w i t h the s t a r t i n g b u f f e r . T h e s - R N A , d i s s o l v e d i n s t a r t i n g b u f f e r , w a s a p p l i e d - 1 0 5 -to the c o l u m n , w a s h e d on w i t h a few m i s of the s t a r t i n g b u f f e r , t h e n an i n c r e a s i n g l i n e a r g r a d i e n t of s a l t w a s a p p l i e d . T h e f l o w r a t e w a s m a i n t a i n e d at about one m l p e r m i n w i t h the a i d of a n A c c u - F l o p u m p ( B e c k m a n I n s t r u m e n t s ) . 3. C h r o m a t o g r a p h y of M o n o n u c l e o t i d e s o n B D E A E - C e l l u l o s e 5 m g S a m p l e s of A p , G p , U p a n d p C w e r e d i s s o l v e d i n 20 m l of s t a r t i n g b u f f e r and the p H of the s o l u t i o n a d j u s t e d to 7. 4 w i t h 0. 2 N N a O H . T h e s o l u t i o n w a s t h e n a p p l i e d to a B D E A E - c e l l u l o s e c o l u m n (95 x 1. 5 c m diam-..) , . p r e v i o u s l y e q u i l i b r a t e d w i t h 1500 m l of s t a r t i n g b u f f e r . T h e c o l u m n w a s d e v e l o p e d w i t h a 3 l i t r e l i n e a r g r a d i e n t of N a C l , f r o m 0.01 to 0 . 0 5 M . B o t h v e s s e l s w e r e b u f f e r e d a t - p H 7 . 4 w i t h 0. 005 M s o d i u m p h o s p h a t e . T h e f l o w r a t e w a s 1 m l p e r m i n . a n d 10. 4 m l f r a c t i o n s w e r e c o l l e c t e d ( F i g . 4 ) . The p e a k s e l u t e d b y the s a l t g r a d i e n t w e r e i d e n t i f i e d by u l t r a v i o l e t s p e c t r a l a n a l y s i s at p H 1, 7 a n d 12. i P e a k s . 1 to 4 c o n s i s t e d of p C , U p , G p a n d A p , r e s p e c t i v e l y . P e a k 5 w a s m o s t l y A p . A l i q u o t s of 2 . 0 m l , f r o m t e s t tubes 30, 4 8 , 58, 78, 100, 115 a n d a 4 . 0 m l a l i q u o t f r o m t e s t tube 90 w e r e l y o p h i l i z e d , r e d i s s o l v e d i n a m i n i m u m of d i s t i l l e d w a t e r a n d c h r o m a t o g r a p h e d on; t h i n l a y e r c e l l -u l o s e p l a t e s i n , s a t u r a t e d a m m o n i u m s u l p h a t e , i s o p r o p a n o l , 0 . 1 0 M N a O A c , p H 6 . 0 , (80:2:18). T h e 2 ' - a n d 3 ' - i s o m e r s of U p were not c l e a r l y s e p a r a t e d . P e a k 3a (tube 78) w a s G - 3 ' p h o s p h a t e a n d p e a k 3b (tube 90) w a s G - 2 ' p h o s p h a t e . P e a k 4 a (tube 100) w a s A - 3 1 p h o s p h a t e and p e a k 4b o a, w o o 3 <-!-O TO P> o o 3 O i-l cr o 3 0 o 0) o . h-1 • CL fl> cn O 3 W a H !> i n re o 01 O P T I C A L D E N S I T Y (260mLj) O P r-1 ro ro ro w O ^ o o r o c D O ^ t D r o • 9 0 1 -- 1 0 7 -(tube #115) c o n t a i n e d A - 3 ' p h o s p h a t e a n d A - 2 ' p h o s p h a t e . T h e p u r i n e - 2 ' p h o s p h a t e i s o m e r s r a n a h e a d of the p u r i n e - 3 ' p h o s p h a t e i s o m e r s . W h e n 5 m g s a m p l e s of the 5' i s o m e r s of the f o u r c o m m o n r i b o m o n o n u c l e o t i d e s w e r e c h r o m a t o g r a p h e d u n d e r the c o n d i t i o n s d e s c r i b e d a b o v e ( e x c e p t the l i n e a r g r a d i e n t w a s 0. 01 to 0. 04 M N a C l ) the e l u t i o n ^ p r o f i l e s h o w n i n F i g u r e 5 w a s o b t a i n e d . 4. R e c o v e r y of s - R N A f r o m C o l u m n E l u a t e s M e t h o d 1: In the e a r l y s t a g e s of t h i s s t u d y , the d e s i r e d f r a c t i o n s f r o m the c o l u m n c h r o m a t o g r a p h y of m i x e d s - R N A w e r e c o n s o l i d a t e d , r e d u c e d i n v o l u m e by e v a p o r a t i o n at about 3 0 ° , u n d e r r e d u c e d p r e s s u r e , a n d p r e c i p i t a t e d i n the c o l d w i t h 2 to 3 v o l u m e s of e t h a n o l . F r a c t i o n s o b t a i n e d f r o m the u p p e r e n d of the g r a d i e n t ( i . e. , h i g h s a l t c o n c e n t r a t i o n s ) w e r e c o n c e n t r a t e d , d i a l y z e d a n d c o n c e n t r a t e d f u r t h e r b e f o r e p r e c i p i t a t i n g w i t h c o l d e t h a n o l . M a g n e s i u m c h l o r i d e w a s r o u t i n e l y a d d e d to a c o n c e n t r a t i o n of about 0. 01 M to t h o s e f r a c t i o n s o b t a i n e d f r o m c o l u m n s r u n i n the p r e s e n c e of E D T A . P r e c i p i t a t i o n s w e r e u s u a l l y c a r r i e d out at p H 5. T h e p r e c i p i t a t e s w e r e c o l l e c t e d by c e n t r i f u g a t i o n , r e d i s s o l v e d i n a s m a l l v o l u m e of 1 M N a C l , 0. 01 M M g C l 2 a n d p r e c i p i t a t e d a g a i n w i t h c o l d e t h a n o l . T h e r e s u l t i n g p e l l e t s w e r e w a s h e d w i t h c o l d e t h a n o l t h e n w i t h e t h y l e t h e r , d r i e d at r o o m t e m p e r a t u r e a n d s t o r e d i n the c o l d as a d r y p o w d e r . P o r t i o n s w e r e d i s s o l v e d i n d i s t i l l e d w a t e r f o r the p u r p o s e of a s s a y i n g a n d t h e s e w e r e s t o r e d i n the f r o z e n state at about - 1 8 ° . - 1 0 8 -NaCI(M)— 0.04 -0.03 -0.02 -0.01 0 40 60 80 100 FRACTION No. F I G U R E 5. C h r o m a t o g r a p h y of m o n o r i b o n u c l e o t i d e s o n B D E A E -c e l l u l o s e . -109-M e t h o d 2: F r a c t i o n s f r o m the c o l u m n c h r o m a t o g r a p h y of m i x e d s - R N A c o n t a i n i n g the s - R N A a c c e p t o r a c t i v i t y of i n t e r e s t , w e r e c o n s o l i d a t e d and c o n c e n t r a t e d to a s m a l l v o l u m e by u l t r a f i l t r a t i o n ( B l a t t et a l . , 1965) o n a n A m i c o r i D i a f l o a p p a r a t u s ( A m i c o n C o r p . , C a m b r i d g e , M a s s * ) e q u i p p e d w i t h a U M - 1 m e m b r a n e , at 0 - 5 ° . T h e c o l d c o n c e n t r a t e w a s p r e c i p i t a t e d as d e s c r i b e d a b o v e , w i t h a t t e n t i b n t o the p r e s e n c e of m a g n e s i u m and a s u f f i c i e n t l y h i g h i o n i c s t r e n g t h to e f fect q u a n t i t a t i v e p r e c i p i t a t i o n of the t - R N A ( R a z z e l l , 1963). T h e p r e c i p i t a t e w a s c o l l e c t e d b y c e n t r i f u g a t i o n , w a s h e d t w i c e w i t h c o l d e t h a n o l t h e n w i t h e t h y l , e t h e r a n d d r i e d at r o o m t e m p e r a t u r e . 5. P r e p a r a t i o n of Y e a s t A m i n o a c y l - s - R N A S y n t h e t a s e E n z y m e s M e t h o d 1: P r e p a r a t i o n of the a c t i v a t i n g e n z y m e s by g r i n d i n g the y e a s t c e l l s w i t h g l a s s b e a d s i n a V i r t i s " 4 5 " h o m o g e n i z e r , f o l l o w e d by c e n t r i f u g a t i o n a n d d i a l y s i s , i s d e s c r i b e d i n P a r t III , S e c t i o n 1. M e t h o d 2: P r e p a r a t i o n of the a m i n o a c y l - s - R N A s y n t h e t a s e e n z y m e s by t o l u e n e l y s i s at 3 7 ° u n d e r c o n d i t i o n s of c o n t r o l l e d p H , f o l l o w e d by c e n t r i f u g a t i o n a n d g e l f i l t r a t i o n i s d e s c r i b e d i n P a r t III , S e c t i o n 2. M e t h o d 3: T h i s p r e p a r a t i o n , i n v o l v i n g the l y s i s of y e a s t c e l l s at l o w t e m p e r a t u r e a n d p a r t i a l p u r i f i c a t i o n by c h r o m a t o g r a p h y on h y d r o x y -a p a t i t e i s d e s c r i b e d i n P a r t III , S e c t i o n . 3 . - 1 1 0 -6. A s s a y of C o l u m n F r a c t i o n s f o r S p e c i f i c A m i n o A c i d A c c e p t o r A c t i v i t y -M e t h o d 1: S t o c k s o l u t i o n s of the s - R N A f r a c t i o n s to be a s s a y e d w e r e p r e p a r e d as d e s c r i b e d i n S e c t i o n 4 ( M e t h o d 1) a n d . c o n t a i n e d b e t w e e n 50 a n d 100 O . D . u n i t s of s - R N A p e r m l . T h e i n c u b a t i o n m i x t u r e ( f i n a l v o l u m e 0. 5 m l ) c o n t a i n e d 120 yj jmoles of T r i s ( c h l o r i d e ) , p H 7 . 4 , 2 y u m o l e s of s o d i u m A T P , 1 ^umole of s o d i u m C T P , 7. 7^t_moles of M g C ^ , 15 y u m o l e s of K C l , 0 . 4 y u . m o l e of E D T A , 0. 37yu.mole ,of m e r c a p t o e t h a n o l , 14 0. 5 ^ t . C i a n d 4 0 - 5 0 mjuorioles of C - L - a m i n o a c i d ( u n l e s s o t h e r w i s e noted) , about 4 O . D . u n i t s of s - R N A a n d about 0. 10 m l of a m i n o a c y l -s - R N A s y n t h e t a s e e n z y m e s p r e p a r e d i n the V i r t i s " 4 5 " h o m o g e n i z e r (see S e c t i o n 5, M e t h o d 1). T h e b l a n k c o n t a i n e d d i s t i l l e d w a t e r i n p l a c e of s - R N A . T h e c o n t r o l s , a s s a y e d s i m u l t a n e o u s l y , c o n t a i n e d 4 a n d 2 O . D . u n i t s of m i x e d s - R N A . A f t e r i n c u b a t i n g at r o o m t e m p e r a t u r e f o r 60 m i n , t h e i n c u b a t i o n m i x t u r e w a s p l a c e d i n i c e a n d t r e a t e d a c c o r d i n g to one of the t w o f o l l o w i n g p r o c e d u r e s : a) E t h a n o l p r e c i p i t a t i o n - to the i n c u b a t i o n m i x t u r e w a s a d d e d 0.15 m l of c o l d 5 M N a C l , 0. 05 m l of c a r r i e r - R N A (30 m g / m l ) , and 1. 5 m l of c o l d e t h a n o l w i t h t h o r o u g h m i x i n g . A f t e r 10 m i n i n i c e , the m i x t u r e w a s c e n t r i f u g e d , the s u p e r n a t e d e c a n t e d a n d the p e l l e t w a s h e d t h r e e t i m e s w i t h 0. 3 m l of c o l d e t h a n o l - N a C l s o l u t i o n (2:1, e t h a n o l : 1. 5 M N a C l ) . F i n a l l y the p e l l e t w a s d i s s o l v e d i n 0. 20 m l of 1. 5 N N H 4 O H a n d a 25 ^ol a l i q u o t a p p l i e d to a p l a n c h e t , d r i e d a n d c o u n t e d i n a w i n d o w l e s s g a s -f l o w c o u n t e r . -111-b) T C A p r e c i p i t a t i o n - to the i n c u b a t i o n m i x t u r e w a s a d d e d 0.15 m l of c o l d 5 M N a C l and 1. 5 m l of c o l d e t h a n o l . A f t e r l e a v i n g i n i c e at l e a s t 10 m i n , the m i x t u r e w a s p o u r e d i n t o a M i l l i p o r e a p p a r a t u s c o n t a i n i n g a few m i s of c o l d 10% T C A a n d e q u i p p e d w i t h a 2. 5 c m ( d i a m . ) "Whatman 1 f i l t e r d i s k . T h e i n c u b a t i o n v e s s e l w a s r i n s e d t w i c e w i t h c o l d , 10% T C A and the r i n s e s a d d e d to the M i l l i p o r e a p p a r a t u s . S u c t i o n w a s a p p l i e d , and the f i l t e r d i s k w a s h e d t w i c e w i t h 10% T C A . T o t a l w a s h i n g v o l u m e w a s 50 to 60 m l of c o l d , 10% T C A . T h e f i l t e r d i s k w a s r e m o v e d , g l u e d to a p l a n c h e t , d r i e d a n d c o u n t e d as i n (a) a b o v e . M e t h o d 2: T h e f o l l o w i n g m e t h o d w a s b a s e d on the a s s a y p r o c e d u r e deve|oped b y C h e r a y i l a n d B o c k (1965). T h e ! a m i n o a c i d a c c e p t o r a s s a y s w e r e p e r f o r m e d o n W h a t m a n 3 M M , 2. 3 c m d i a m . p a p e r d i s k s . A l i q u o t s of 100 JJA e a c h f r o m the c o l u m n f r a c t i o n s w e r e a d d e d t o the n u m b e r e d p a p e r d i s k s . T h e d i s k s w e r e a l l o w e d to d r y u n t i l the w e t s h e e n w a s gone a n d t h e n t r a n s f e r r e d to a v e s s e l c o n t a i n i n g c o l d 75% e t h a n o l , 0. 03 M K C l (about 10 m l of w a s h s o l v e n t p e r p a p e r d i s k ) 1 f o r about 10 m i n w i t h o c c a s i o n a l s t i r r i n g . T h e d i s k s w e r e w a s h e d a s e c o n d t i m e i n . t h e s a m e s o l v e n t t h e n the e x c e s s s o l v e n t w a s r e m o v e d by s u c t i o n f i l t r a t i o n a n d the d i s k s d r i e d at r o o m t e m p e r a t u r e . T h e d r y d i s k s , w e r e s u s p e n d e d o n s t a i n l e s s s t e e l p i n s a n c h o r e d i n a b l o c k of s t y r o f o a m . A 100yjJ. a l i q u o t of a n i n c u b a t i o n m i x t u r e , d e s c r i b e d b e l o w , w a s a d d e d to the s u s p e n d e d d i s k s . T h e - 1 1 2 -s t y r o f o a m b l o c k w a s t h e n p l a c e d i n a p o l y e t h y l e n e b o x l i n e d w i t h w e t p a p e r t o w e l s o r i n a g l a s s b o x c o n t a i n i n g w a t e r a n d the b o x s e a l e d to m a i n t a i n a h u m i d a t m o s p h e r e d u r i n g the i n c u b a t i o n p e r i o d . T h e i n c u b a t i o n m i x t u r e c o n t a i n e d p e r 100^/-l, 50 yul of the a p p r o p r i a t e a c t i v a t i n g e n z y m e s o l u t i o n a n d 50 yul of a s o l u t i o n c o n t a i n i n g 3. 7yu_moles of M g C ^ , 6. 7 y o m o l e s of K C l , 27yU.moles of N a c a c o d y l a t e , p H 7. 3, 0. 5 yumole of s o d i u m A T P , 0. l y u m o l e of s o d i u m C T P , 0.12yu_mole of m e r c a p t o e t h a n o l , 0.1yLt.mole of E D T A , 75 rtyjurnoles of g l y c i n e a n d 2. 68 jjJOi p e r yucM of ^ C - g l y c i n e . T h e s o l u t i o n of a c t i v a t i n g e n z y m e s w h i c h w a s s t o r e d at about - 1 8 ° i n 4 0 % g l y c e r o l , w a s d i l u t e d 1:1 w i t h d i s t i l l e d w a t e r b e f o r e a d d i n g i t to the i n c u b a t i o n m i x t u r e . A f t e r i n c u b a t i n g f o r 30 m i n the d i s k s w e r e r e m o v e d a n d w a s h e d at 0° f o r a m i n i m u m of. 10 m i n , w i t h o c c a s i o n a l s t i r r i n g , i n e a c h of the f o l l o w i n g b a t h s , t w o c o n s e c u t i v e b a t h s of 10% T C A , one e t h a n o l b a t h a n d f i n a l l y a n e t h y l e t h e r b a t h . E a c h b a t h c o n t a i n e d 10 m l of s o l v e n t p e r p a p e r d i s k e x c e p t the e t h e r bath w h i c h c o n t a i n e d 3 to 5 m l of s o l v e n t p e r d i s k . T h e d i s k s w e r e t h e n s p r e a d o n . p a p e r t o w e l s a n d d r i e d w i t h the a i d of a heat l a m p . T h e r a d i o a c t i v i t y w a s d e t e r m i n e d i n a s c i n t i l l a t i o n s p e c t r o m e t e r by s u s p e n d i n g . t h e d i s k s i n 5 m l of s c i n t i l l a t i o n f l u i d c o n t a i n i n g 3 g of P P O a n d 0. 3 g of d i m e t h y l P O P O P p e r l i t r e of r e a g e n t t o l u e n e . - 1 1 3 -7. C h r o m a t o g r a p h y of Y e a s t s - R N A on B D E A E - C e l l u l o s e u n d e r C o n d i t i o n s of V a r y i n g N a C l a n d / o r M g C ^ A . . N a C l G r a d i e n t i n . t h e P r e s e n c e of 0.10 M M g C l 2 , p H 5. 0 T h e B D E A E - c e l l u l o s e c o l u m n (75. 5 x 2 . 5 c m d i a m . ) w a s e q u i l i b r a t e d w i t h 2 l i t r e s of 0.10 M M g C l 2 , 0. 005 M N a O A c , p H 5. 0, t h e n 600 m g of s - R N A ( C a l b i o c h e m . ) d i s s o l v e d i n 35 m l of the s t a r t i n g b u f f e r w a s a d d e d . T h e c o l u m n w a s d e v e l o p e d w i t h a n 8 l i t r e l i n e a r g r a d i e n t of N a C l f r o m 0 to 0 . 4 5 M , b o t h v e s s e l s c o n t a i n i n g 0.10 M M g C l 2 a n d 0. 005 M N a O A c , p_H 5. 0. T h e f l o w r a t e w a s about 1 m l p e r m i n and 15. 2 m l f r a c t i o n s w e r e c o l l e c t e d . T h e e l u t i o n p r o f i l e w a s d i v i d e d i n t o f r a c t i o n s (I - X I ) , as s h o w n i n F i g u r e 6, a n d a s s a y e d f o r a c c e p t o r a c t i v i t y t o w a r d g l y c i n e a n d h i s t i d i n e a c c o r d i n g to M e t h o d 1-a ( S e c t i o n 6). T h e r a d i o a c t i v i t y d a t a a r e e x p r e s s e d i n F i g u r e 6 as c p m p e r O . D . u n i t of s - R N A . F r a c t i o n s I to V w e r e 1. 4 , 1.8, 3 . 9 , 2 . 7 , a n d 1. 7 f o l d e n r i c h e d , r e s p e c t i v e l y , f o r g l y c i n e a c c e p t o r a c t i v i t y w h i l e F r a c t i o n s V I I I to X w e r e 3. 3, 2. 2 a n d 1. 3 f o l d e n r i c h e d r e s p e c t i v e l y , f o r h i s t i d i n e a c c e p t o r a c t i v i t y . B . N a C l G r a d i e n t i n the P r e s e n c e of 0. 02 M M g C l 2 , p H 5. 0 T h e B D E A E - c e l l u l o s e c o l u m n (91 x 3 . 5 c m d i a m . ) w a s e q u i l i b -r a t e d w i t h s e v e r a l c o l u m n v o l u m e s of 0.10 M N a C l , 0. 02 M MgCl2» 0. 005 M N a O A c , p H 5 . 0 , t h e n 1. 00 g of s - R N A ( C a l b i o c h e m . ), d i s s o l v e d o d H o 3" ^ ~ o -o o *-t 3 fD 51) cn <-r 0 ) 3 n ft) o H i '0 H i ro <=> cn Crq cn 1-d X U M > H i o o c n ro C c n »—* • 3 Crq P n i—> OTP fO 3 OPTICAL DENSITY(260mu) and cpm per O.D. unit x 10"3 ^ 0 0 0 0 - ^ . - » - - ^ - > - » rj)0 ro ^ 0) CX) Q_ w 01 CD ro O < < < < < x X X O P ro O P O r" 00 O • t i l - 1 1 5 -i n 150 m l of s t a r t i n g b u f f e r , w a s a d d e d . T h e c o l u m n w a s d e v e l o p e d w i t h a n 8 l i t r e l i n e a r g r a d i e n t of N a C l b e t w e e n 0. 10 a n d 1.0 M , b o t h v e s s e l s c o n t a i n i n g 0. 02 M M g C l 2 a n d 0. 005 M N a O A c , p_H 5. 0. T h e f l o w r a t e w a s about 1 m l p e r m i n and 18. 5 m l f r a c t i o n s w e r e c o l l e c t e d . T h e e l u t i o n p r o f i l e w a s d i v i d e d i n t o F r a c t i o n s I to X V , as s h o w n i n F i g u r e 7, a n d the f r a c t i o n s a s s a y e d foy g l y c i n e , h i s t i d i n e a n d a l a n i n e a c c e p t o r a c t i v i t y a c c o r d i n g to M e t h o d 1-a. T h e r a d i o a c t i v i t y d a t a a r e e x p r e s s e d i n F i g u r e 7 as c p m p e r O . D . u n i t of s - R N A . T h e s p e c i f i c a c t i v i t y of the ^ C - a m i n o a c i d s i n the a s s a y s w e r e : a l a n i n e a n d g l y c i n e , 1.1 ^i_Ci p e r p^M; h i s t i d i n e , 10 pjCi p e r fjJs/L. F r a c t i o n s I, II , III, V a n d V I w e r e 9.1 , 11, 1.7, 3. 3 a n d 1. 6 f o l d e n r i c h e d r e s p e c t i v e l y i n g l y c i n e a c c e p t o r a c t i v i t y . F r a c t i o n s X , X I , X I I , and X I I I w e r e 1. 2, 4 . 6, 4 . 0 a n d 1. 9 f o l d e n r i c h e d r e s p e c t i v e l y i n h i s t i d i n e a c c e p t o r a c t i v i t y . F r a c t i o n s III, I V , V , V I a n d V I I w e r e 4 . 3, 2. 6, 6. 2, 2. 2 a n d 1. 3 f o l d e n r i c h e d r e s p e c t i v e l y i n a l a n i n e a c c e p t o r a c t i v i t y . C . N a C l G r a d i e n t i n the P r e s e n c e of 0. 005 M M g C l 2 , p H 5. 0 T h e B D E A E - c e l l u l o s e c o l u m n (89. 2 x 3 . 5 c m d i a m . ) w a s e q u i l i b r a t e d w i t h s e v e r a l c o l u m n v o l u m e s of 0 . 1 0 ' M N a C l , 0 . 0 0 5 M M g C l 2 , 0. 005 M N a O A c , p H 5. 0, t h e n 1. 00 g of s - R N A ( C a l b i o c h e m . , 14, 250 O . D . u n i t s ) d i s s o l v e d i n 150 m l of s t a r t i n g b u f f e r w a s a d d e d . T h e c o l u m n w a s d e v e l o p e d w i t h a n 8 l i t r e l i n e a r g r a d i e n t of N a C l b e t w e e n 0.10 and:.1. 0 M , b o t h v e s s e l s c o n t a i n i n g 0. 005 M M g C l 2 , a n d 0 . 0 0 5 M r—I o a 5' * J S * " O tn 2 O fD O ° O H (N» tn >^< re & P> CTQ tn O ~ ^ ? X > Ul 0 © 3 • td . D W > o tn n> C tn )-•• TO OPTICAL DENSITY (260mu) cpm per O. D. unit x1CT2 [V) w 4^  cn co 2 CTQ H &> rt> 3 6 6 o o 6 p p ^ M LO J> Dl (J) CCQ-— P Q P o M CO CD ° ho - 9 1 1 -- 1 1 7 -N a O A c , p_H 5. 0. T h e f l o w r a t e w a s 8 8 m l p e r h r a n d f r a c t i o n s of 18. 6 m l e a c h w e r e c o l l e c t e d . T h e e l u t i o n p r o f i l e w a s d i v i d e d i n t o F r a c t i o n s I to X V I , as s h o w n i n F i g u r e 8. F r a c t i o n s I to X V I w e r e a s s a y e d f o r a c c e p t o r a c t i v i t y t o w a r d g l y c i n e , v a l i n e a n d p h e n y l a l a n i n e . G l y c i n e a n d p h e n y l a l a n i n e a c c e p t o r a c t i v i t i e s w e r e a s s a y e d by M e t h o d 1-a a n d v a l i n e a c c e p t o r a c t i v i t y w a s a s s a y e d by M e t h o d 1 - b . T h e s p e c i f i c a c t i v i t i e s a n d c o n c e n t r a t i o n s of the ^ 4 C - a m i n o a c i d s i n the a s s a y s w e r e : g l y c i n e , 8. 2 5 ^j_Ci p e r j^Js/L a n d 7 7 m / u m o l e s ; p h e n y l a l a n i n e , 11.1 yJZi p e r jjJs/L a n d 4 7 . 4 m / u m o l e s ; v a l i n e , 5 . 5 6 ^uCi p e r ^JLM , a n d 4 8 r r j u m o l e s . F r a c t i o n s I, II , III, I V a n d V w e r e e n r i c h e d i n g l y c i n e a c c e p t o r a c t i v i t y 11. 2, 3.3, 3.3, 4.1, a n d 2. 2 f o l d r e s p e c t i v e l y . F r a c t i o n s II , III, I V , V a n d V I w e r e e n r i c h e d i n v a l i n e a c c e p t o r a c t i v i t y 3. 6, 5.4, 4.1, 1.7 a n d 2. 0 f o l d r e s p e c t i v e l y . The o n l y f r a c t i o n e x h i b i t i n g s i g n i f i c a n t a c c e p t o r a c t i v i t y t o w a r d p h e n y l a l a n i n e w a s f r a c t i o n X I I ( 0 . 8 f o l d ) . F r a c t i o n s I to X V I r e p r e s e n t e d 8 3 % of the a p p l i e d O . D . -3 D . N a C l G r a d i e n t i n the P r e s e n c e of 10 M E D T A , p H 5. 0 T h e B D E A E - c e l l u l o s e c o l u m n ( 9 0 x 3. 5 c m d i a m . ) w a s e q u i l i b -r a t e d w i t h s e v e r a l c o l u m n v o l u m e s of 0. 3 M N a C l , 1 0 " 3 M E D T A , 0 . 0 0 5 M N a O A c , p H 5. 0, t h e n 1. 0 0 g of s - R N A ( C a l b i o c h e m . , 16, 5 0 0 O . D . u n i t s ) , d i s s o l v e d i n 150 m l of s t a r t i n g b u f f e r , w a s a d d e d . T h e c o l u m n w a s d e v e l o p e d w i t h a n 8 l i t r e l i n e a r g r a d i e n t of N a C l f r o m 0. 3 to 1.1 M , -3 b o t h v e s s e l s c o n t a i n i n g 10 M E D T A a n d 0. 0 0 5 M N a O A c , £ H 5. 0. - 1 1 9 -The flow r a t e w a s about 1 m l p e r m i n , a n d f r a c t i o n s of 18. 6 m l e a c h w e r e c o l l e c t e d . T h e e l u t i o n p r o f i l e w a s d i v i d e d i n t o F r a c t i o n s I t o ' X I V , as s h o w n i n F i g u r e 9, a n d a s s a y e d f o r a c c e p t o r a c t i v i t y t o w a r d g l y c i n e , a c c o r d i n g to M e t h o d 1-a. T h e s p e c i f i c a c t i v i t y of the ^ 4 C - g l y c i n e i n e a c h a s s a y w a s 1. l ^ u C i p e r y j . M . F r a c t i o n s I, II , III a n d I V w e r e e n r i c h e d i n g l y c i n e a c c e p t o r a c t i v i t y 5. 5, 2. 6, 2. 9 a n d 2. 6 f o l d , r e s p e c t i v e l y . F r a c t i o n s I t o X I V r e p r e s e n t e d 7 2% of the a p p l i e d m a t e r i a l . E . M g C l 2 G r a d i e n t i n the P r e s e n c e of 0.10 M N a C l , ; 5 . 0 T h e B D E A E - c e l l u l o s e c o l u m n (90 x 2 c m d i a m . ) w a s e q u i l i b r a t e d w i t h 2 l i t r e s of 0. 02 M M g C l 2 , 0.10 M N a C l , 0. 005 M N a O A c , p H 5. 0, t h e n 300 m g of s - R N A ( C a l b i o c h e m . ), d i s s o l v e d i n 50 m l of s t a r t i n g b u f f e r , w a s a d d e d . T h e c o l u m n w a s d e v e l o p e d w i t h a 6 l i t r e l i n e a r g r a d i e n t of M g C l 2 b e t w e e n 0. 02 a n d 0. 45 M , b o t h v e s s e l s c o n t a i n i n g 0 . 1 0 M N a C l a n d 0. 005 M N a O A c , p H 5 . 0 . T h e f l o w r a t e w a s 1 m l p e r m i n a n d f r a c t i o n s of 19 m l e a c h w e r e c o l l e c t e d . T h e e l u t i o n p r o f i l e w a s d i v i d e d i n t o F r a c t i o n s I to X I I , as s h o w n i n F i g u r e 10, a n d a s s a y e d f o r a c c e p t o r a c t i v i t y t o w a r d g l y c i n e a n d h i s t i d i n e a c c o r d i n g to M e t h o d 1-a. F r a c t i o n s I t o V I I w e r e e n r i c h e d i n g l y c i n e a c c e p t o r a c t i v i t y 1. 2* 1.5, 1.4,, 2 . 6 , 2 . 7 , 2. 9 a n d 2. 3 f o l d , r e s p e c t i v e l y . F r a c t i o n s X to X I I w e r e e n r i c h e d i n h i s t i d i n e a c c e p t o r a c t i v i t y 1. 2, 4 . 8 a n d 3. 0 f o l d , r e s p e c t i v e l y . I—I o c M 2, ^  °> ^ O g a -I T ) P ro o -' ^ Q P cn > o W a > w 1 n Q O cn 3' TO 2 n TO rH &. a> 3 OPTICAL DENSITY (260mu) and cpm per O D unit x 1 0 i gly ) (Jl 0) M 05 (0 cn n 4 1—1 o a JO 3 *J r+ CD " ? n 0 ) rt-*d 0 - i 3 CD CD cn rr OPTICAL DENSITY (260mu) • and cpm per O.D. unit x 1 0 - 3 o o o o P o o O ^ W W 0 1 * 0 ) \1 o P e 00 CD O CD n CD 2, C f P *•* cn 2 CD O S. cn c n > O O • 3 Cd a > H i n CD o cn CD c n 0 Q O ru (TO - f f" CD 3 < < < <~ X X ^ O O P P P P ^ r o ^ o~) co O - T Z I -- 1 2 2 -F . P u l s e G r a d i e n t of N a C l i n the P r e s e n c e of 0. 005 M M g C l 2 , p H 5. 0 (i) C h r o m a t o g r a p h i c M e t h o d T h e a p p a r a t u s u s e d to s u p p l y p u l s e s of i n c r e a s i n g c o n c e n t r a t i o n s of N a C l i s s h o w n i n F i g u r e 11. T h e r e s e r v o i r a n d m i x i n g v e s s e l w e r e a r r a n g e d i n the u s u a l w a y , f o r a l i n e a r g r a d i e n t of i n c r e a s i n g N a C l c o n c e n t r a t i o n . T h e s o l u t i o n f r o m the m i x i n g ; v e s s e l p a s s e d t h r o u g h a n A c c u - F l o p u m p ( B e c k m a n I n s t r u m e n t s ) , w h i c h c o n t r o l l e d the f l o w r a t e , i n t o a v e s s e l e q u i p p e d w i t h a r e m o v a b l e s y p h o n ( S y p h o n v e s s e l ) . T h e s y p h o n u s e d i n the f o l l o w i n g e x p e r i m e n t d e l i v e r e d 200 m l v o l u m e s to the c o l u m n r e s e r v o i r v e s s e l . T h e a i r s p a c e s i n the s y p h o n v e s s e l a n d c o l u m n r e s e r v o i r v e s s e l w e r e j o i n e d b y m e a n s of T y g o n t u b i n g . T h e c l o s e d s y s t e m w a s n e c e s s a r y so the p u m p c o u l d c o n t r o l the f l o w r a t e of the c o l u m n . P r i o r to a p p l y i n g the p u l s e g r a d i e n t to the c o l u m n , the s y p h o n v e s s e l w a s a l l o w e d to f i l l a n d f l o w i n t o the c o l u m n r e s e r v o i r , t h e n v a l v e ' A ' w a s c l o s e d a n d the s y s t e m c o n n e c t e d to the c h r o m a t o g r a p h i c c o l u m n . T h e m a i n r e s e r v o i r c o n t a i n e d 4 l i t r e s of 1. 0 M N a C l a n d the m i x e r c o n t a i n e d 4 l i t r e s of 0. 10 M N a C l . B o t h v e s s e l s c o n t a i n e d 0 . 0 0 5 • M M g C l 2 a n d 0. 005 M N a O A c , p H 5. 0. T o the B D E A E - c e l l u l o s e c o l u m n (114 x 3 . 0 c m d i a m . ), p r e v i o u s l y e q u i l i b r a t e d w i t h 0.10 M N a C l , 0 . 005 M M g C l 2 , 0. 005 M N a O A c , p H 5 . 0 , w a s a d d e d 1. 00 g of s - R N A ( C a l b i o c h e m . , 14, 300 O . D . u n i t s ) , d i s s o l v e d - 1 2 3 -kESERVOIR MIXER PUMP 2 0 0 m l — H SYPHON VESSEL COLUMN RESERVOIR VALVE A COLUMN F I G U R E 11. A p p a r a t u s f o r s u p p l y i n g p u l s e g r a d i e n t s d u r i n g c o l u m n c h r o m a t o g r a p h y . - 1 2 4 -i n 150 m l of s t a r t i n g b u f f e r . A f t e r w a s h i n g the c o l u m n w i t h 50 m l of s t a r t i n g ; b u f f e r , the p u l s e g r a d i e n t w a s a p p l i e d . T h e f l o w r a t e w a s about 1 m l p e r m i n a n d 14. 3 m l f r a c t i o n s w e r e c o l l e c t e d , e x c e p t f o r the m e t h o x y e t h a n o l e l u a t e (see F i g . 12) w h i c h w a s c o l l e c t e d as 11. 2 m l f r a c t i o n s . T h e e l u t i o n p r o f i l e w a s d i v i d e d i n t o F r a c t i o n s I to X V I as s h o w n i n F i g u r e 12 a n d a s s a y e d f o r a m i n o a c i d a c c e p t o r a c t i v i t y t o w a r d g l y c i n e and p h e n y l a l a n i n e by M e t h o d 1-a. S p e c i f i c a c t i v i t i e s a n d c o n c e n t r a t i o n s 14 of the C - a m i n o a c i d s i n the a s s a y s w e r e : g l y c i n e , 4 . 45 ^ x C i p e r ^ L M and 120 n y j j n o l e s ; p h e n y l a l a n i n e , 11.1 jjjCi p e r y u M a n d 4 7 . 4 m ^ m o l e s . F r a c t i o n s I, II , III , I V a n d V w e r e e n r i c h e d i n g l y c i n e a c c e p t o r a c t i v i t y , 9 . 3 , 9 . 0 , 4 . 1 , 2. 7 a n d 1. 3 f o l d r e s p e c t i v e l y . F r a c t i o n s X V and X V I w e r e s i m i l a r l y e n r i c h e d 2. 8 a n d 4 . 3 f o l d , r e s p e c t i v e l y i n - p h e n y l a l a n i n e a c c e p t o r a c t i v i t y . O v e r 9 5 % of the a p p l i e d m a t e r i a l w a s r e c o v e r e d , w h i l e about 12% of the r e c o v e r e d m a t e r i a l w a s e l u t e d i n the m e t h o x y e t h a n o l F r a c t i o n s X V a n d X V I . ( i i ) D e t e r m i n a t i o n of the M e l t i n g C u r v e s T h e m e l t i n g p r o f i l e s ( T j ^ ' s ) w e r e d e t e r m i n e d on F r a c t i o n s I, V , I X , X I I I , X V I a n d o n m i x e d s - R N A i n a G i l f o r d , M o d e l 2000 s p e c t r o -p h o t o m e t e r . A c u v e t t e c o n t a i n i n g 3. 0 m l of 0. 005 M T r i s ( c h l o r i d e ) , p H 7 . 4 , 0 . 0 0 5 M E D T A , a n d 0. 006 M M g C l 2 w a s d e g a s s e d u n d e r v a c u u m , t h e n about 1 O . D . u n i t of s - R N A w a s a d d e d a n d the c u v e t t e p l a c e d i n the |I|O|III|IV|V|VI|VI^ /III| ix I x| xi I XII I xiff I xTv fxv] xv i I NaCI (M)— (Integrated) 15%MeOEtOH J — 1 . 0 M NaCl 320 3 60 4 0 0 440 4 80 5 2 0 5 6 0 6 0 0 640 FRACTION No. F I G U R E 12 P u l s e g r a d i e n t e l u t i o n of b a k e r s ' y e a s t s - R N A f r o m B D E A E - c e l l u l o s e . i n the p r e s e n c e of 0 . 005 M M g C b , _p_H 5. 0. N a C l g r a d i e n t - 1 2 6 -h e a t i n g c h a m b e r of the G i l f o r d s p e c t r o p h o t o m e t e r . T h e t e m p e r a t u r e w a s i n c r e a s e d at the r a t e of 1 ° p e r m i n . T h e T w c u r v e s of F r a c t i o n s I, V , I X M and X I I I s h o w e d c l e a r i n f l e c t i o n s w i t h m e l t i n g o c c u r r i n g b e t w e e n 55 a n d 8 5 ° . F r a c t i o n X V I a n d the m i x e d s - R N A s a m p l e s h o w e d e a r l y m e l t i n g s t a r t i n g at about 35 - 4 0 ° . T h e T j ^ ' s of a l l the s a m p l e s w e r e 68 - 7 0 ° . ( i i i ) G-100 S e p h a d e x C h r o m a t o g r a p h y of F r a c t i o n s X V a n d X V I A G-100 S e p h a d e x c o l u m n (40 x 2. 5 c m d i a m , ) w a s e q u i l i b r a t e d w i t h 1. O M N a C l . F r a c t i o n s X V and X V I w e r e e a c h d i s s o l v e d i n 1. 0 m l of 1. 0 M N a C l a n d c h r o m a t o g r a p h e d , s e p a r a t e l y , on the c o l u m n . T h e f l o w r a t e w a s 0. 35 m l p e r m i n . T h e e l u t i o n p r o f i l e s a r e s h o w n i n F i g u r e 1 3 - A a n d B ( F r a c t i o n s X V a n d X V I r e s p e c t i v e l y ) . T h e e l u t i o n p r o f i l e of F r a c t i o n X V s h o w e d that 34% of the O . D . ( test t u b e s 19 to 36) w a s not s - R N A , w h i l e that of F r a c t i o n X V I s h o w e d that 27% of the O . D . ( test tubes 18 to 34) w a s not s - R N A . T h e p h e n y l a l a n i n e a c c e p t o r a c t i v i t y of. the m a j o r p e a k f r o m F r a c t i o n s X V and X V I (peak a i n F i g u r e s 1 3 - A a n d B ) w a s d o u b l e d . P e a k b f r o m F r a c t i o n X V I s h o w e d no a c c e p t o r a c t i v i t y t o w a r d p h e n y l a l a n i n e . G . P r e p a r a t i v e C o l u m n C h r o m a t o g r a p h y of s - R N A o n B D E A E - C e l l u l o s e C h r o m a t o g r a p h y of 5 g of b r e w e r s ' y e a s t s - R N A , as d e s c r i b e d b e l o w , w a s c a r r i e d out by D r . I . C . G i l l a m . *1 o 0 a 1 re i — • i—> o ^ P 0 3 o O O o o OPTICAL GO DENSITY (260mjj) CO piBlue D extra n GO 4^  3 p n o 5 0 0 I > P a h - t H OQ i — ' O J l td o re p a re ro O -LZl-- 1 2 8 -T h e B D E A E - c e l l u l o s e c o l u m n (110 x 3 . 2 c m d i a m . ) w a s e q u i l i b r a t e d w i t h 0. 45 M N a C l , 0 .01 M M g S 0 4 , t h e n 5. 0 g of s - R N A (70, 000 O . D . u n i t s ) d i s s o l v e d i n 200 m l of 0. 45 M N a C l , 0 . 01 M M g S 0 4 w a s a d d e d . T h e c o l u m n w a s d e v e l o p e d w i t h a 10 l i t r e l i n e a r g r a d i e n t of N a C l f r o m 0 . 4 5 to 1. 0 M , b o t h v e s s e l s c o n t a i n i n g 0 . 0 1 M M g S 0 4 . W h e n the l i n e a r N a C l g r a d i e n t h a d f i n i s h e d , the c o l u m n w a s w a s h e d w i t h 1 M N a C l , 0 . 01 M M g S 0 4 10% m e t h o x y e t h a n o l ( v / v ) . T h e f l o w r a t e w a s m a i n t a i n e d at 80 m l p e r h o u r w i t h the a i d of a n A c c u - F l o p u m p and f r a c t i o n s of about 20 m l e a c h w e r e c o l l e c t e d . T h e e l u t i o n p r o f i l e i s s h o w n i n F i g u r e 14. T h e c o l u m n f r a c t i o n s ' w e r e a s s a y e d f o r a c c e p t o r a c t i v i t y t o w a r d 20 a m i n o a c i d s , as d e s c r i b e d by G i l l a m et a l . , (1967). The p o s i t i o n of e l u t i o n of the g l y c i n e a c c e p t o r a c t i v i t y i s a l s o s h o w n i n F i g u r e 14. H . R e c h r o m a t o g r a p h y of the G l y c i n e - ! F r a c t i o n (i) C h r o m a t o g r a p h y at _gH 3. 5 i n the P r e s e n c e of 0. 05 M M g C l 2 T h e B D E A E - c e l l u l o s e c o l u m n ( 1 1 9 x 1. 2 c m d i a m . ) w a s p r e p a r e d a n d e q u i l i b r a t e d as u s u a l , t h e n the g l y c i n e - I f r a c t i o n f r o m the 5 g p r e p a r a t i v e c o l u m n . w a s d i s s o l v e d i n 40 m l of s t a r t i n g b u f f e r (2880 O . D . u n i t s ) and a d d e d to the c o l u m n . A 2 l i t r e l i n e a r g r a d i e n t of N a C l f r o m 0 . 4 0 to 1. 0 M w a s a p p l i e d . B o t h v e s s e l s c o n t a i n e d 0. 05 M M g C l 2 a n d 0. 005 M s o d i u m f o r m a t e , p H 3. 5. F r a c t i o n s of 11. 4 m l e a c h w e r e -6ZI " - 1 3 0 -c o l l e c t e d at a f l o w r a t e of 1.1 m l p e r m i n ( F i g . 15). T h e c o l u m n f r a c t i o n s w e r e a s s a y e d f o r g l y c i n e a c c e p t o r a c t i v i t y a c c o r d i n g to M e t h o d 2 ( S e c t i o n 6) a n d the r e s u l t s a r e s h o w n i n F i g u r e 15. F r a c t i o n s 28 to 60, (378 m l , 1930 O . D . u n i t s ) w e r e c o m b i n e d ( F r a c t i o n , I , F i g . 15), a n d the s - R N A i s o l a t e d by M e t h o d 2 ( S e c t i o n 4 ) . ( i i ) C h r o m a t o g r a p h y at _p_H 4 . 0 i n the P r e s e n c e of 1 0 " 3 ' M E D T A F r a c t i o n I ( F i g . 15) w a s d i s s o l v e d i n 10. 6 m l of s t a r t i n g b u f f e r (1880 O . D . u n i t s ) a n d a p p l i e d to a B D E A E - c e l l u l o s e c o l u m n , (119 x l . ? c m d i a m . ), p r e v i o u s l y e q u i l i b r a t e d w i t h 0. 45 M N a C l , 10" M E D T A a n d 0. 005 M s o d i u m f o r m a t e , p_H 4. 0. A 2 l i t r e l i n e a r g r a d i e n t of N a C l f r o m 0 . 4 5 to 1. 2 M w a s a p p l i e d . B o t h v e s s e l s c o n t a i n e d 1 0 " 3 M E D T A a n d 0. 005 M s o d i u m f o r m a t e p H 4. 0. T h e f l o w r a t e w a s 1. 1 m l p e r m i n and 11 m l f r a c t i o n s w e r e c o l l e c t e d ( F i g . 16). T h e c o l u m n f r a c t i o n s w e r e a s s a y e d f o r g l y c i n e a n d m e t h i o n i n e a c c e p t o r a c t i v i t y a c c o r d i n g to M e t h o d 2 ( S e c t i o n 6) a n d the r e s u l t s a r e s h o w n i n F i g u r e 16. F r a c t i o n s 86 to 98 w e r e 95 t o 100% g l y c i n e - I s - R N A . F r a c t i o n s 78 to 100 w e r e c o m b i n e d (253 m l , 970 O . D . u n i t s ) a n d the s - R N A w a s i s o l a t e d by M e t h o d 2 ( S e c t i o n 4) to y i e l d 4 5 . 7 m g of w h i t e p o w d e r . J . R e c h r o m a t o g r a p h y of the G l y c i n e - I I F r a c t i o n (i) C h r o m a t o g r a p h y at _pH 5 i n the P r e s e n c e of 0. 01 M M g C l ? T h e B D E A E - c e l l u l o s e c o l u m n (119 x 1. 2 c m d i a m . ) w a s p r e p a r e d 2 OPTICAL DENSITY ( 2 6 0 m j j ) o c o ro -N CD CO JO O H 1 >—! 1 r I — i — I i ( m e t « • = • » , gly — ) -zei -- 1 3 3 -and e q u i l i b r a t e d as u s u a l , t h e n the g l y c i n e - I I f r a c t i o n f r o m the 5 g p r e p a r a t i v e c o l u m n w a s d i s s o l v e d i n 30 m l of s t a r t i n g b u f f e r (6870 O . D . u n i t s ) a n d a d d e d to the c o l u m n . A 2 l i t r e l i n e a r g r a d i e n t of N a C l , f r o m 0. 40 to 1. 0 M , w i t h b o t h v e s s e l s c o n t a i n i n g 0. 01 M M g C l 2 a n d 0. 005 M N a O A c , p H 5 . 0 , w a s a p p l i e d . T h e f l o w r a t e w a s 1 m l p e r m i n a n d f r a c t i o n s of 10 m l e a c h w e r e c o l l e c t e d a n d a s s a y e d f o r g l y c i n e a c c e p t o r a c t i v i t y b y M e t h o d 2 ( S e c t i o n 6). T h e r e s u l t s a r e s h o w n i n F i g u r e 17. F r a c t i o n s 55 to 90 w e r e c o m b i n e d ( F r a c t i o n I) a n d the s - R N A i s o l a t e d by M e t h o d 2 ( S e c t i o n 4) f o r r e c h r o m a t o g r a p h y at j > H 3 . 5 . ( i i ) C h r o m a t o g r a p h y at J D H 3. 5 i n the P r e s e n c e of 10" 3 M E D T A T h e s - R N A f r o m F r a c t i o n I ( F i g . 17) w a s d i s s o l v e d i n 12 m l of s t a r t i n g b u f f e r (4800 O . D . u n i t s ) a n d a p p l i e d to a B D E A E - c e l l u l o s e c o l u m n (119 x 1. 2 c m d i a m . ), p r e v i o u s l y e q u i l i b r a t e d w i t h 0. 45 M N a C l , 10" 3 M E D T A , 0. 005 M s o d i u m f o r m a t e , p H 3. 5. T h e c o l u m n w a s d e v e l o p e d w i t h a 2 l i t r e l i n e a r g r a d i e n t of N a C l , f r o m 0 . 4 5 to 1. 2 M , w i t h b o t h v e s s e l s c o n t a i n i n g 10"-' M E D T A a n d 0. 005 M s o d i u m f o r m a t e , p H 3. 5. F r a c t i o n s of 10. 5 m l e a c h w e r e c o l l e c t e d at a f l o w r a t e of 1 m l p e r m i n . T h e c o l u m n f r a c t i o n s w e r e a s s a y e d f o r a c c e p t o r a c t i v i t y t o w a r d s g l y c i n e a n d a l a n i n e a c c o r d i n g to M e t h o d 2 ( S e c t i o n 6). T h e r e s u l t s a r e s h o w n i n F i g u r e 18. C o l u m n f r a c t i o n s 125 to 205 w e r e c o m -b i n e d (855 m l , 1639 O . D . u n i t s ) and the s - R N A i s o l a t e d by M e t h o d 2 ( S e c t i o n 4) t o y i e l d 9 3 . 3 m g of w h i t e p o w d e r (Glycine-11-/3 f r a c t i o n ) . 4 t—I 0 o cn cn o ro OPTICAL DENSITY (260mjj) &cpm per 0.10ml of Eluate x 10 - 2 , _ A _ ^ _ ^ _ A _ > r o r o r o 4^  CD oo o ro CD GO O ro ^ O O O O O O ro w ^ cn NaCI(M)—-O o H H o c o O r o O P T I C A L O CO Co - I r — o r o D E N S I T Y ( 2 6 0 m u ) CT) CO O ro O O ^ 4^ oo r o CD NaCl (M) cScji o o mz b r+CU O O o - V -o o ^ CO ^ NaCl (M) -CO o r o w 4^ 0 1 O cpm x 1 0 _ l ( g l y ) r o 4^  c (J) CO pm x 10 " 3( 9a) r o - 1 3 6 -P A R T I I - D . A C O M B I N E D C H E M I C A L A N D C H R O M A T O G R A P H I C  M E T H O D F O R T H E I S O L A T I O N O F A M I N O A C I D - S P E C I F I C s - R N A ' S 1. P r e p a r a t i o n of the A c y l a t i n g A g e n t T h e N - h y d r o x y s u c c i n i m i d e e s t e r of p h e n o x y a c e t i c a c i d w a s p r e p a r e d by D r . G . M . T e n e r a c c o r d i n g to a g e n e r a l p r o c e d u r e f o r the p r e p a r a t i o n of the N - h y d r o x y s u c c i n i m i d e e s t e r s of a c y l a m i n o a c i d s d e s c r i b e d by A n d e r s o n ^ t a l . (1964). 2. E x p e r i m e n t I A . C o n d i t i o n s f o r M a x i m u m C h a r g i n g of s - R N A w i t h G l y c i n e S e v e r a l a s s a y s w e r e s e t u p , e a c h c o n t a i n i n g the s a m e c o n c e n t -r a t i o n of c o ^ f a c t o r s a n d s y n t h e t a s e e n z y m e s but v a r y i n g a m o u n t s of s-R N A . In t h i s w a y the c o n d i t i o n s f o r m a x i m a l c h a r g i n g of the s - R N A w e r e e s t a b l i s h e d . T h u s , the a m i n o a c y l - s - R N A u s e d i n the f o l l o w i n g s e c t i o n s w a s p r e p a r e d u n d e r c o n d i t i o n s i n w h i c h a l i n e a r r e l a t i o n s h i p e x i s t e d b e t w e e n the s - R N A c o n c e n t r a t i o n a n d the a m o u n t of a m i n o a c i d i n c o r p o r a t e d . B . P r e p a r a t i o n of 1 4 C - G l y c y l - s - R N A A 50 m g s a m p l e of s - R N A , e n r i c h e d 6 f o l d i n g l y c i n e a c c e p t o r a c t i v i t y , w a s o b t a i n e d f r o m the g l y c i n e - I p e a k f o l l o w i n g c h r o m a t o g r a p h y - 1 3 7 -on B D E A E - c e l l u l o s e as d e s c r i b e d i n P a r t II , S e c t i o n 7 - G . T h e r e a c t i o n m i x t u r e ( f i n a l v o l u m e 6. 5 m l ) , c o n t a i n e d p e r m l of f i n a l v o l u m e , 4 3 . 5 ^j-moles M g C l 2 , 87 / OLmoles K C l , I80^j_moles s o d i u m c a c o d y l a t e , p H 7. 3, 2. 03 yLLmoles s o d i u m A T P , 0. 615 ^ t m o l e s o d i u m C T P , 1 . 1 5^jLmoles m e r c a p t o e t h a n o l , 0. 615^t-mole E D T A , 3. 8 5 y u j n o l e s s o d i u m p h o s p h a t e , p H 7. 5, 11% g l y c e r o l ( v / v ) , 128 O . D . u n i t s of s - R N A , 14 6 .16^j .moles g l y c i n e , 1. 23 ^ i . C i C - g l y c i n e a n d 3. 6 m g p r o t e i n c o n t a i n i n g a m i n o a c y l - s - R N A s y n t h e t a s e e n z y m e s p r e p a r e d as d e s c r i b e d i n P a r t III, S e c t i o n 2 - D . (The e n z y m e s h a d b e e n s t o r e d i n 4 0 % g l y c e r o l ( v / v ) , 2 3 10 M s o d i u m p h o s p h a t e a n d 10 M m e r c a p t o e t h a n o l ) . T h e m i x t u r e w a s i n c u b a t e d at 2 5 ° f o r 50 m i n . T h e r e a c t i o n w a s f o l l o w e d b y r e m o v i n g 20 a l i q u o t s at i n t e r v a l s , a p p l y i n g t h e m to W h a t m a n 3 M M f i l t e r d i s k s a n d w a s h i n g the d i s k s t w i c e i n c o l d , 10% T C A , o n c e i n c o l d e t h a n o l a n d f i n a l l y i n e t h y l e t h e r . T h e d i s k s w e r e d r i e d a n d c o u n t e d i n a s c i n t i l l a t i o n s p e c t r o m e t e r . T h e r e a c t i o n w a s s t o p p e d a f t e r the 50 m i n i n c u b a t i o n p e r i o d b y p l a c i n g i n i c e a n d a d d i n g 1. 0 m l 0. 5 N s o d i u m f o r m a t e , p H 3 . 0 , 2 . 0 m l 5 M N a C l a n d 25 m l e t h a n o l , a l l s o l u t i o n s p r e - c o o l e d i n i c e . T h e p e l l e t , o b t a i n e d by c e n t r i f u g a t i o n , w a s w a s h e d t h r e e t i m e s w i t h 2 m l of c o l d e t h a n o l : s a l t s o l u t i o n (2:1, e t h a n o l : 1. 5 M N a C l ) . T h e p e l l e t w a s d i s s o l v e d i n 15 m l of 0 . 3 M N a C l , 0. 01 M M g C l 2 , 0. 005 M N a O A c , p_H 4. 5, at 0 ° , a n d the s o l u t i o n e x t r a c t e d w i t h 7 m l of w a t e r - s a t u r a t e d p h e n o l . T h e a q u e o u s l a y e r w a s r e m o v e d a f t e r c e n t r i f u g a t i o n a n d the p h e n o l l a y e r w a s e x t r a c t e d t w i c e w i t h c o l d b u f f e r . T h e w a s h i n g s w e r e -138-added to the aqueous layer which was then extracted with ethyl ether. The s-RNA in the aqueous layer was precipitated by the addition of two volumes of cold ethanol. The precipitate was collected by centrifugation.and the resulting pellet washed three times with ethanol : salt solution. 14 C. Derivatization of O G l y c y l - s - R N A 14 A portion of the previously-prepared C'-glycyl-s-RNA (400 O. D. units and 114, 700 cpm) was dissolved in 5. 0 ml of cold, 0.10 M triethanol-amine hydrochloride, 0. 02 M M g C ^ . pH 4. 0. The derivatization was carried out in an ice bath by adding 0. 20 ml of tetrahydrofuran, containing 10 mg of the acylating agent, to the cold solution and adjusting the j>H to 8. 0 with 1 N NaOH. The reaction was allowed to proceed for 10 min, then the jj>H of the solution was adjusted to 4. 5 with cold 2 N acetic acid. The s-RNA was precipitated by the addition of 3 volumes of cold ethanol. The white precipitate, which appeared when the acylating agent was added, dissolved in the cold, aqueous ethanol, solution. After 20 min in ice the solution was centrifuged and the resulting pellet washed first with an ethanol - salt solution (2:1, ethanol : 1. 5 M NaCl) then with ethanol. The pellet was drained free of excess ethanol, then dissolved in 5 ml of 0. 3 M NaCl, 0. 01 M M g C l 2 , 0. 005 M NaOAc, pH 4. 5. . The radio-activity in. a 0.10 ml aliquot was determined as described in the next section. The recovery of radioactivity was 68%. - 1 3 9 -D . C h r o m a t o g r a p h y of the N - ( P h e n o x y a c e t y l ) - C - g l y c y l - s - R N A  on B D E A E - C e l l u l o s e T h e d e r i v a t i z e d glycylr s - R N A , d i s s o l v e d i n 5. 0 m l of 0. 3 M N a C l , 0. 01 M M g C l 2 , 0. 005 M N a O A c , p H 4 . 5, w a s a p p l i e d to a B D E A E - c e l l -u l o s e c o l u m n (16 x 2. 2 c m d i a m . ), w h i c h h a d b e e n p r e v i o u s l y e q u i l i b r a t e d w i t h the s a m e b u f f e r . T h e c o l u m n w a s w a s h e d c o n s e c u t i v e l y w i t h the f o l l o w i n g s o l u t i o n s : 50 m l of 0 . 3 M N a C l , 200 m l 0. 8 M N a C l t h e n w i t h 1. 5 M N a C l , 10% e t h a n o l . A l l the s o l u t i o n s w e r e b u f f e r e d at p_H 4 . 5 w i t h 0. 005 M N a O A c a n d c o n t a i n e d 0. 01 M M g C l 2 . F r a c t i o n v o l u m e s of 4 . 5 m l w e r e c o l l e c t e d e x c e p t f o r the f r a c t i o n s f r o m the 10% e t h a n o l w a s h w h i c h w e r e 2. 8 m l e a c h . A 0.10 m l a l i q u o t f r o m e a c h f r a c t i o n w a s a d d e d to 10 m l of m o d i f i e d B r a y ' s s o l u t i o n a n d the r a d i o a c t i v i t y d e t e r m i n e d i n a s c i n t i l l a t i o n s p e c t r o m e t e r . T h e m o d i f i e d B r a y ' s s o l u t i o n c o n s i s t e d of 60 g n a p h t h a l e n e , 4 g P P O a n d 0. 2 g d i m e t h y l P O P O P i n 1 l i t r e of d i o x a n e . T h e O . D . of e a c h f r a c t i o n w a s a l s o r e c o r d e d . B o t h d a t a a r e s h o w n i n F i g u r e 19. F r a c t i o n s I a n d II ( F i g . 19) c o n s i s t e d of 6 3 . 5 % a n d 3 3 . 8 % r e s p e c t i v e l y , of the O . D . a p p l i e d t o the c o l u m n . F r a c t i o n I (64 m l ) a n d F r a c t i o n II (50 m l ) w e r e ; a d j u s t e d to p H 9 w i t h 1. 5 N N H 4 O H a n d l e f t 2 h r at r o o m t e m p e r a t u r e . B o t h f r a c t i o n s w e r e t h e n c o n c e n t r a t e d to about 7 m l e a c h i n a D i a f l o a p p a r a t u s e q u i p p e d w i t h a U M - 1 m e m b r a n e . T h e s - R N A w a s p r e c i p i t a t e d i n the c o l d w i t h e t h a n o l a n d c o l l e c t e d by OPTICAL DENSITY ( 2 6 0 r n u ) O O co co o ro O w O o "n > O oi —I o O o o o CO O CD O O O C — i O 1=1 ro w 8 8 8 cpm per 0.1ml of E lua te -OH --141-c e n t r i f u g a t i o n . T h e p e l l e t s w e r e w a s h e d t w i c e w i t h a n e t h a n o l - s a l t s o l u t i o n (2:1, e t h a n o l : 1. 5 M N a C l ) t h e n d i s s o l v e d i n 0. 5 m l of d i s t i l l e d w a t e r a n d s t o r e d at about - 1 8 ° . F r a c t i o n s I a n d II w e r e a s s a y e d f o r a c c e p t o r a c t i v i t y t o w a r d g l y c i n e . U n f r a c t i o n a t e d s - R N A w a s u s e d as c o n t r o l . T h e i n c u b a t i o n m i x t u r e ( f i n a l v o l u m e , 150^j_l) c o n t a i n e d 3 . 7 ^ i . m o l e s M g C ^ , 6. 7 ^ j m o l e s . . K C l , 2 7 ^ u n o l e s s o d i u m c a c o d y l a t e , p_H 7. 3, 0. 33 ^ Lmole s o d i u m A T P , O . l O ^ x m o l e s o d i u m C T P , 0.10yu.mole E D T A , 0 . 1 7 ^ . m o l e m e r c a p t o e t h a n o l , 75 mu . m o l e s g l y c i n e a n d 0. 2yU.Ci 1 4 C - g l y c i n e (2 . 67JULCI p e r y x M ) , 4 6 . 3 O . D . u n i t s of m i x e d s - R N A o r 12. 7 O . D . u n i t s of F r a c t i o n I o r 1. 64 O . D . u n i t s of F r a c t i o n II, a n d 5OyLLl of the s a m e c r u d e s y n t h e t a s e e n z y m e p r e p a r a t i o n u s e d i n S e c t i o n 2 - B . T h e m i x t u r e w a s i n c u b a t e d at 2 5 ° a n d 15ju.1 a l i q u o t s w e r e r e m o v e d at 4 m i n i n t e r v a l s . A l l t h r e e a s s a y s s h o w e d m a x i m u m i n c o r p o r a t i o n of ^ C - g l y c i n e i n l e s s t h a n 8 m i n . F r a c t i o n II w a s 9 . 4 f o l d e n r i c h e d a n d F r a c t i o n I w a s s t i l l 2. 6 f o l d e n r i c h e d i n g l y c i n e a c c e p t o r a c t i v i t y . F r a c t i o n II w a s r e c h r o m a t o g r a p h e d o n B D E A E - c e l l u l o s e i n the s t e p w i s e m a n n e r , d e s c r i b e d a b o v e . T h e 0. 8 M N a C l e l u t e d 9 3 . 6% of the a p p l i e d m a t e r i a l w h i l e the r e m a i n i n g 6. 4 % w a s e l u t e d by the 1. 5 M N a C l 15% e t h a n o l ( v / v ) . T h e s - R N A e l u t e d b y the 0. 8 M N a C l w a s h w a s r e c o v e r e d a n d a s s a y e d f o r a c c e p t o r a c t i v i t y t o w a r d g l y c i n e as d e s c r i b e d a b o v e . F r a c t i o n II w a s 10. 0 f o l d e n r i c h e d i n g l y c i n e a c c e p t o r a c t i v i t y . - 1 4 2 -T h u s the a c c e p t o r a c t i v i t y w a s not s i g n i f i c a n t l y e n r i c h e d b y r e c h r o m a t o g -r a p h y . 3. E x p e r i m e n t II A . C o n d i t i o n s f o r M a x i m u m C h a r g i n g of s - R N A w i t h G l y c i n e u s i n g  a P a r t i a l l y P u r i f i e d G l y c y l - s - R N A S y n t h e t a s e F r a c t i o n 198 ( f r o m P a r t III , S e c t i o n 6 - B , F i g . 4 ) , c o n t a i n i n g . the g l y c y l - s - R N A s y n t h e t a s e a c t i v i t y w a s p a s s e d t h r o u g h a G - 2 5 S e p h a d e x ? 3 c o l u m n e q u i l i b r a t e d w i t h 10 M s o d i u m p h o s p h a t e , 10" M m e r c a p t o -e t h a n o l , 10"^ M E D T A , 4 0 % g l y c e r o l ( v / v ) , p _ H 7. 5, to r e m o v e the e x c e s s a m m o n i u m s u l f a t e . T h e e l u a t e f r o m the S e p h a d e x c o l u m n c o n t a i n i n g the g l y c y l - s - R N A s y n t h e t a s e a c t i v i t y c o n t a i n e d 0. 38 m g of p r o t e i n p e r m l a n d w a s s t o r e d at - 1 8 ° . E x p e r i m e n t s w e r e c a r r i e d out u s i n g c o n s t a n t a m o u n t s of e n z y m e and c o f a c t o r s w h i l e v a r y i n g the a m o u n t of m i x e d s - R N A i n the i n c u b a t i o n m i x t u r e . T h e i n c u b a t i o n s w e r e c a r r i e d out i n 1 m l g l a s s t u b e s ( f i n a l volume 300 ^ j_l) c o n t a i n i n g 3. 3^jLmoles M g C ^ , 6. 6^j_moles K C l , 27 yUmoles s o d i u m c a c o d y l a t e , p H 7. 3, 0. 33yj_mole s o d i u m A T P , 0.12 ^janole m e r c a p t o e t h a n o l , 0. lOyU-mole E D T A , 75 m ^ u n o l e s g l y c i n e ( 2 . 6 8 ^U-Ci peryU-M), 19 / j L g p r o t e i n (as d e s c r i b e d a b o v e ) , a n d s - R N A f r o m 2. 8 mg to 11. 2 m g . T h e b l a n k s c o n t a i n e d d i s t i l l e d w a t e r i n p l a c e of the s - R N A . - 1 4 3 -T h e i n c u b a t i o n s w e r e c a r r i e d out at 2 5 ° a n d 50^j_l a l i q u o t s w e r e r e m o v e d at i n t e r v a l s a n d a p p l i e d to W h a t m a n 3 M M p a p e r d i s k s . T h e d i s k s w e r e w a s h e d a n d c o u n t e d as d e s c r i b e d i n S e c t i o n 2 - B . T h e s y n t h e s i s of g l y c y l - s - R N A w a s not c o m p l e t e d u r i n g the 30 m i n i n c u b a t i o n p e r i o d u n d e r any of the c o n d i t i o n s s t u d i e d . In a d d i t i o n , i t a p p e a r e d that the i n i t i a l r a t e s of g l y c y l - s - R N A s y n t h e s i s d e c r e a s e d w i t h i n c r e a s i n g c o n c e n t r a t i o n s of s - R N A . T h i s o b s e r v a t i o n l e d to a n i n v e s t i g a t i o n of the e f fect of m a g n e s i u m i o n c o n c e n t r a t i o n on the r e a c t i o n v e l o c i t y . T h u s i n c u b a t i o n s w e r e c a r r i e d out as d e s c r i b e d a b o v e e x c e p t t h e y a l l c o n t a i n e d 5. 6 m g m i x e d s - R N A a n d the c o n c e n t r a t i o n of m a g n e s i u m i o n s v a r i e d f r o m 3. 2 j U m o l e s to 103 y o m o l e s . A l i q u o t s of 50 yuJ e a c h w e r e r e m o v e d at 10, 20, 30 a n d 40 m i n , a n d a p p l i e d to W h a t m a n 3 M M p a p e r d i s k s . T h e d i s k s w e r e w a s h e d a n d c o u n t e d as d e s c r i b e d p r e v i o u s l y . The r e s u l t s a r e s h o w n i n F i g u r e 20. A n o t h e r s e r i e s of i n c u b a t i o n s w e r e c a r r i e d out to s t u d y the ef fect of m a g n e s i u m i n the r a n g e 0 t o 28^j_moles p e r a s s a y . T h e i n c u b a t i o n s w e r e c a r r i e d out as d e s c r i b e d above e x c e p t that E D T A w a s a d d e d to o b t a i n m a g n e s i u m i o n c o n c e n t r a t i o n s b e l o w 3. 3y_»_moles p e r a s s a y . T h e r e s u l t s a r e s h o w n i n , F i g u r e 21, i n w h i c h the c p m p e r 50yu_l a l i q u o t i s p l o t t e d a g a i n s t e f f e c t i v e m a g n e s i u m i o n c o n c e n t r a t i o n f o r i n c u b a t i o n t i m e s of 10, 20 a n d 30 m i n . T h e e f f e c t i v e c o n c e n t r a t i o n of m a g n e s i u m i o n w a s c a l c u l a t e d b y s u b t r a c t i n g the c o n c e n t r a t i o n of E D T A f r o m the - 144-40 i O X 32 24 3 . O ^ 16 £_ <D o. £ 8 CL U 0 10 min 0 • < 20 40 60 80 [Mg] (umoles) 100 F I G U R E 20. E f f e c t of m a g n e s i u m i o n s on the s y n t h e s i s of 1 4 C - g l y c y l - s - R N A ( R a n g e , 3. 2 to 103 ^ m o l e s ~ M g C l 2 ) . . 9 [Mg] - [EDTA] (j j m o l e s ) F I G U R E 21. E f f e c t of m a g n e s i u m i o n s on the s y n t h e s i s of 1 4 C - g l y c y l - s - R N A ( R a n g e , 0 to 28 ^ m o l e s M g C ^ ) -- 1 4 6 -c o n c e n t r a t i o n of m a g n e s i u m i o n s a d d e d to e a c h i n c u b a t i o n m i x t u r e . T h e r e s u l t s ( F i g . 21) s u g g e s t t h a t 0. 5 to 0. 7 ^ . m o l e of m a g n e s i u m i s r e q u i r e d p e r m g of s - R N A f o r m a x i m u m r a t e u n d e r the c o n d i t i o n s s t u d i e d . H a v i n g e s t a b l i s h e d a n o p t i m a l c o n c e n t r a t i o n of m a g n e s i u m i o n , the m i n i m a l a m o u n t of s y n t h e t a s e e n z y m e r e q u i r e d to f u l l y c h a r g e the m i x e d s - R N A w i t h g l y c i n e w a s t h e n d e t e r m i n e d as d e s c r i b e d i n S e c t i o n 2^A. T h e c o n d i t i o n s w e r e s u c h that m a x i m u m g l y c y l - s - R N A s y n t h e s i s w a s o b t a i n e d i n a 10 to 20 m i n ; i n c u b a t i o n p e r i o d . B . I s o l a t i o n of G l y c i n e - S p e c i f i c s - R N A S t e p 1. R e m o v a l of P h e n y l a l a n i n e A c c e p t o r s - R N A f r o m  m i x e d s - R N A T h e s - R N A ( c a . 5 g) w a s d i s s o l v e d i n O . 3 M N a C l , . 0. 005 M M g S 0 4 , . 0 . 0 0 5 - M N a O A c , - p H 4 . 5 a n d a p p l i e d o n B D E A E - c e l l u l o s e c o l u m n p r e v i o u s l y e q u i l i b r a t e d w i t h the s a m e b u f f e r . T h e c o l u m n w a s t h e n w a s h e d , f i r s t w i t h about one c o l u m n v o l u m e of the s a m e b u f f e r , t h e n w i t h 0. 9 M N a C l , 0 . 01 M M g S 0 4 , 0. 005 M N a O A c , p H 4 . 5, a n d f i n a l l y w i t h 1. 5 M N a C l , 0. 005 M M g S 0 4 , 0. 005 M N a O A c , p H 4 . 5, 15% e t h a n o l ( v / v ) . T h e s - R N A r e m o v e d b y the 0. 9 M N a C l w a s h w a s r e c o v e r e d b y e t h a n o l p r e c i p i t a t i o n . i n the u s u a l w a y a n d u s e d i n S t e p 2. T h e m a t e r i a l e l u t e d by the e t h a n o l - s a l t w a s h c o n t a i n s a l l the p h e n y l -a l a n i n e a c c e p t o r a c t i v i t y . - 1 4 7 -S t e p 2. " S h a m " P h e n o x y a c e t y l a t i o n T h e p e l l e t ( c a . 4 . 5 g ) o b t a i n e d f r o m S t e p 1 ( e l u t e d b y 0. 9 M N a C l ) w a s d i s s o l v e d i n 125 m l of d i s t i l l e d w a t e r ( i n c l u d i n g w a s h i n g s ) a n d a d d e d to 200 m l of 0.10 M t r i e t h a n o l a m i n e * H C 1 , p H 4. 5. T h e s o l u t i o n w a s c o o l e d to 0 ° , t h e n 1 g of p h e n o x y a c e t y l a t i n g agent, d i s s o l v e d i n 25 m l of T H F w a s a d d e d . T h e p H of the s o l u t i o n w a s a d j u s t e d to 8. 0 w i t h 1 N N a O H a n d the m i x t u r e s t i r r e d f o r 20 m i n at 0 ° . T h e p H w a s t h e n a d j u s t e d to 4 . 5 w i t h a c e t i c a c i d a n d the s - R N A c o l l e c t e d by p r e c i p i t a t i o n w i t h 3 v o l u m e s of c o l d e t h a n o l f o l l o w e d by c e n t r i f u g a t i o n . T h e r e s u l t i n g p e l l e t w a s d i s s o l v e d i n 0. 3 M N a C l , 0. 005 M M g S 0 4 , 0. 005 M N a O A c , p H 4 . 5 a n d c h r o m a t o g r a p h e d on B D E A E - c e l l u l o s e as d e s c r i b e d i n S t e p 1. T h e s - R N A e l u t e d by the 0. 9 M N a C l (about 94% of the a p p l i e d m a t e r i a l ) w a s r e c o v e r e d i n the u s u a l w a y a n d u s e d i n S t e p 3. T h e e t h a n o l - s a l t w a s h r e m o v e s the m a t e r i a l w h i c h w a s r e a d i l y p h e n o x y a c e t y l a t e d u n d e r the c o n d i t i o n s d e s c r i b e d . S t e p 3. P r e p a r a t i o n of 1 4 C - G l y c y l - s - R N A T h e r e a c t i o n m i x t u r e ( f i n a l v o l u m e 21 m l ) c o n t a i n e d 0. 6 m m o l e s M g C ^ , 1. 2 m m o l e s K C l , 4 . 8 " m m o l e s s o d i u m c a c o d y l a t e , 60yLt.moles s o d i u m A T P , 18yu_moles E D T A , 22yu_moles m e r c a p t o e t h a n o l , 2 7 . 7 ^ j L m o l e s g l y c i n e (1. 27 y u C i p e r y u - M ) , 1. 00 g of s - R N A p r e v i o u s l y t r e a t e d as d e s c r i b e d i n S t e p s 1 a n d 2 a n d 2 . 7 m g of p r o t e i n p r e p a r e d as d e s c r i b e d - 1 4 8 -i n S e c t i o n 3 - A . T h e i n c u b a t i o n w a s c a r r i e d out i n a 150 m l C o r e x c e n t r i f u g e b o t t l e at 3 0 ° . A l i q u o t s of 50^i_l e a c h w e r e r e m o v e d at i n t e r v a l s d u r i n g the i n c u b a t i o n a n d a p p l i e d t o W h a t m a n 3 M M f i l t e r d i s k s . T h e d i s k s w e r e w a s h e d a n d c o u n t e d as d e s c r i b e d i n S e c t i o n 2 - B . A f t e r 40 m i n , the i n c u b a t i o n m i x t u r e w a s p l a c e d i n i c e a n d the jpH of the s o l u t i o n a d j u s t e d to 4 . 5 w i t h c o l d , 2 N a c e t i c a c i d . T h e s o l u t i o n w a s t h e n e x t r a c t e d w i t h 20 m l of w a t e r - s a t u r a t e d p h e n o l . F o l l o w i n g c e n t r i f u g a t i o n , the p h e n o l l a y e r w a s r e m o v e d a n d w a s h e d t w i c e w i t h a few m i s of 0. 3 M N a C l , 0. 01 M M g C ^ . T h e w a s h i n g s w e r e a d d e d to the a q u e o u s l a y e r w h i c h w a s s u b s e q u e n t l y e x t r a c t e d w i t h i c e - c o l d e t h y l e t h e r . 14 S t e p 4 . P h e n o x y a c e t y l a t i o n of C - G l y c y l - s - R N A T o the c o l d a q u e o u s l a y e r o b t a i n e d i n S t e p 3 w a s a d d e d 5 m l of T H F c o n t a i n i n g 1 g of p h e n o x y a c e t y l a t i n g agent . T h e p H of the s o l u t i o n w a s a d j u s t e d to 8. 0 by the c a r e f u l a d d i t i o n of 1 N N a O H a n d the m i x t u r e s t i r r e d f o r 10 m i n at 0 ° . T h e p H w a s t h e n a d j u s t e d to 4 . 5 w i t h c o l d , 2 N a c e t i c a c i d , a n d , a f t e r a d d i n g 2 m l of 5 M N a C l to the s o l u t i o n , the s - R N A w a s p r e c i p i t a t e d by the a d d i t i o n of 2. 5 v o l u m e s of c o l d e t h a n o l . T h e m i x t u r e w a s l e f t at 0° f o r 20 m i n t h e n c e n t r i f u g e d . T h e r e s u l t i n g p e l l e t w a s w a s h e d , f i r s t w i t h 15 m l of c o l d e t h a n o l : s a l t s o l u t i o n (2:1, - 1 4 9 -e t h a n o l : 1. 5 M N a C l ) , t h e n w i t h 30 m l of i c e - c o l d e t h a n o l . S t e p 5. I s o l a t i o n of the N - ( P h e n o x y a c e t y l ) — C - g r y c y l - s - R N A  f r o m the M i x t u r e T h e p e l l e t , o b t a i n e d f r o m S t e p 4, w a s d i s s o l v e d i n 50 m l of 0. 3 M N a C l , 0. 01 M M g C l 2 0. 005 M N a O A c , p H 4 . 0 a n d the s o l u t i o n c e n t r i f u g e d to r e m o v e the u n r e a c t e d , s p a r i n g l y s o l u b l e p h e n o x y a c e t y l a t i n g agent . T h e s u p e r n a t e w a s a d d e d to a B D E A E - c e l l u l o s e c o l u m n (24 x 3 . 5 c m d i a r m ), p r e v i o u s l y e q u i l i b r a t e d w i t h the s a m e b u f f e r . T h e c o l u m n w a s w a s h e d , f i r s t w i t h about 150 m l of 0. 3 M N a C l , 0 . 01 M M g C l 2 , 0. 005 M N a O A c , p H 4 . 0, t h e n w i t h 1200 m l of 0 . 8 M N a C l , 0. 01 M M g C l 2 , 0. 005 M N a O A c , p H 4. 0, a n d f i n a l l y w i t h 1. 5 M N a C l , 0. 01 M M g C l 2 , 0. 005 M N a O A c , p H 4. 0, 15% e t h a n o l (v/v) u n t i l the a b s o r b a n c e of the e luate w a s l e s s t h a n 0. 2 (about 300 m l ) . T h e f l o w r a t e w a s about 4 m l p e r m i n a n d 19 m l f r a c t i o n s w e r e c o l l e c t e d . T h e a b s o r b a n c e at 260 m j x w a s r e c o r d e d a n d the r a d i o a c t i v i t y i n the c o l u m n f r a c t i o n s w a s d e t e r m i n e d by a d d i n g a 0. 10 m l a l i q u o t t o 10 m l of a m o d i f i e d B r a y ' s s o l u t i o n a n d c o u n t i n g i n a s c i n t i l l a t i o n s p e c t r o m e t e r . T h e f i r s t t w o f r a c t i o n s of the O . D . p e a k e l u t e d by the 0. 8 M 14 N a C l w a s h c o n t a i n e d s o m e u n r e a c t e d C - g l y c y l - s - R N A . T h e t w o f r a c t i o n s w e r e t h e r e f o r e c o m b i n e d (38 m l ) a n d c o o l e d to 0 ° . T h e n 1 m l of 0.10 M t r i e t h a n o l a m i n e - H C 1 , p H 4. 5 w a s a d d e d (to f a c i l i t a t e a d j u s t m e n t of the p H to 8) a l o n g w i t h 1 m l of T H F c o n t a i n i n g 50 m g of -150-p h e n o x y a c e t y l a t i n g agent . T h e s o l u t i o n w a s a d j u s t e d to p H 8 w i t h 1 N N a O H , s t i r r e d f o r 10 m i n at 0 ° , t h e n the j>H w a s r e a d j u s t e d to 4 . 5 w i t h 2 N a c e t i c a c i d . T h e s o l u t i o n w a s t h e n c o m b i n e d w i t h the f r a c t i o n s c o n t a i n i n g the r a d i o a c t i v i t y e l u t e d b y the e t h a n o l - 1. 5 M N a C l w a s h . T h e s - R N A w a s i s o l a t e d f r o m the c o m b i n e d s o l u t i o n s by p r e c i p i t a t i o n w i t h c o l d e t h a n o l f o l l o w e d b y c e n t r i f u g a t i o n . T h e r e s u l t i n g p e l l e t w a s r e c h r o m a t o g r a p h e d on B D E A E - c e l l u l o s e as d e s c r i b e d a b o v e e x c e p t that 12. 5 m l f r a c t i o n s w e r e c o l l e c t e d at a f l o w r a t e of about 3 m l p e r m i n . T h e e l u t i o n p r o f i l e a n d r a d i o a c t i v i t y d a t a a r e s h o w n i n F i g u r e 22. T h e n o n - p h e n o x y a c e t y l a t e d s - R N A ( e l u t e d by the 0. 8 M N a C l w a s h ) w a s r e c o v e r e d (8, 190 O . D . u n i t s ) i n a m a n n e r a n a l o g o u s to that d e s c r i b e d b e l o w . F r a c t i o n s 83 to 96 i n c l u s i v e w e r e c o m b i n e d (175 m l , 1275 O . D . u n i t s , 1,113, 000 c p m ) a n d the p H of the s o l u t i o n w a s a d j u s t e d to 9 w i t h 1.5 N N H ^ O H . A f t e r 2 h r s at r o o m t e m p e r a t u r e the p H w a s a d j u s t e d to 5 w i t h a c e t i c a c i d a n d the s - R N A s o l u t i o n c o n c e n t r a t e d to about 45 m l i n a D i a f l o a p p a r a t u s e q u i p p e d w i t h a U M - 1 m e m b r a n e . T h e c o n c e n t r a t e w a s c o o l e d to 0° a n d the s - R N A i s o l a t e d by p r e c i p i t a t i o n w i t h c o l d e t h a n o l f o l l o w e d b y c e n t r i f u g a t i o n . A f t e r w a s h i n g once w i t h c o l d e t h a n o l , the p e l l e t w a s d i s s o l v e d i n 15 m l of 0 . 4 0 M N a C l , 0 . 01 M M g C l ^ . L e s s t h a n . 0 . 1 % of the r a d i o a c t i v i t y w a s r e t a i n e d . T h e s o l u t i o n w a s t h e n c h r o m a t o g r a p h e d on B D E A E - c e l l u l o s e as d e s c r i b e d i n S t e p 6. - 1 5 1 -£ ° 4 0 CO ^ C\J >-t:30 00 z LU Q 20 _ J < (J Ix 10 O 0 I 0 F I G U R E 22. 20 40 6 0 80 100 FRACTION No. (12.5 ml) 60 to 50'O x 40 30 o 13 o E 20 o •HO 0 •O Q. U C h r o m a t o g r a p h y of N - ( p h e n o x y a c e t y l ) o n B D E A E - c e l l u l o s e . 14 C - g l y c y l - s - R N A - 1 5 2 -S t e p 6. C h r o m a t o g r a p h y of G l y c i n e - s - R N A on B D E A E - C e l l u l o s e T h e 15 m l s o l u t i o n of g l y c i n e - s - R N A o b t a i n e d f r o m S t e p 5 w a s a p p l i e d to a B D E A E - c e l l u l o s e c o l u m n (103 x 1. 2 c m d i a m . ), p r e v i o u s l y e q u i l i b r a t e d w i t h 0 . 4 0 M N a C l , 0. 01 M M g C l 2 . A f t e r w a s h i n g the c o l u m n w i t h a few m i s of the s a m e b u f f e r , a 2 l i t r e l i n e a r g r a d i e n t of N a C l f r o m 0. 4 0 to 0. 80 M w a s a p p l i e d . B o t h v e s s e l s c o n t a i n e d 0. 01 M M g C l ^ . W h e n the l i n e a r g r a d i e n t h a d f i n i s h e d , the c o l u m n w a s w a s h e d w i t h 1. 5 M N a C l , 0. 01 M M g C l 2 , 15% e t h a n o l ( v / v ) . T h e f l o w r a t e w a s 1 m l p e r m i n a n d 16 m l f r a c t i o n s w e r e c o l l e c t e d . T h e c o l u m n f r a c t i o n s w e r e a s s a y e d f o r a c c e p t o r a c t i v i t y t o w a r d g l y c i n e as d e s c r i b e d i n P a r t I I - C , S e c t i o n 6, M e t h o d 2. T h e e l u t i o n p r o f i l e a n d a s s a y d a t a a r e s h o w n i n F i g u r e 23. T h e s - R N A f r o m P e a k I i n F i g u r e 23 w a s r e c o v e r e d a n d r e c h r o m a t o g r a p h e d a t j a H 4 . 0 i n the p r e s e n c e of 0. 001 M E D T A as d e s c r i b e d i n P a r t I I - C , S e c t i o n 7 H . T h e s - R N A w a s e l u t e d as a s i n g l e p e a k i n the e l u t i o n p r o f i l e . A s s a y of the c o l u m n f r a c t i o n s , as d e s c r i b e d a b o v e , gave a s i n g l e p e a k of g l y c i n e a c c e p t o r a c t i v i t y w h i c h c o i n c i d e d w i t h the O . D . p r o f i l e . - 1 5 3 -.E o CD C\J >-h-1 4 r 12 l a -s ' 6 4 ^ 2 4 UJ Q _ J < y i s O 12 0.G 11 30 50 - . 7 0 90 FRACTION No. F I G U R E 23 . C h r o m a t o g r a p h y of g l y c i n e a c c e p t o r R N A on B D E A E -c e l l u l o s e . - 1 5 4 -D I S C U S S I O N T h e p a s t s i x y e a r s o r so have w i t n e s s e d a n e v e r i n c r e a s i n g e f f o r t by m a n y i n v e s t i g a t o r s t o f r a c t i o n a t e the c o m p l e x m i x t u r e of c l o s e l y r e l a t e d m o l e c u l e s c a l l e d s o l u b l e - R N A . T h e d i f f i c u l t i e s p r e s e n t e d by t h i s f a m i l y of R N A ' s h a v e b e e n d i s c u s s e d . W e w i s h e d to i s o l a t e s p e c i e s of s - R N A o n w h i c h no o t h e r g r o u p w a s w o r k i n g . T h i s m e a n t the d e v e l o p m e n t of u n i q u e m e t h o d s f o r the f r a c t i o n a t i o n of s - R N A . A n u m b e r of a p p r o a c h e s w e r e t r i e d , m o s t of w h i c h w e r e u n s u c c e s s f u l . T h e m e t h o d d e s c r i b e d i n P a r t I I - A i n v o l v e s the i m m o b i l i z a t i o n of m e r c u r y o n a c e l l u l o s e s u p p o r t . A r e p o r t by E l d j a r n a n d J e l l u m (1963) d e s c r i b e d the p r e p a r a t i o n of a n o r g a n o m e r c u r i a l - p o l y s a c c h a r i d e w h i c h w a s u s e d as a c h r o m a t o g r a p h i c m a t e r i a l f o r the s e p a r a t i o n a n d i s o l a t i o n of H S - p r o t e i n s . T h e s e p a r a t i o n d e p e n d e d o n the p r e s e n c e of a r e a c t i v e s u l f h y d r y l g r o u p i n the p r o t e i n , w h i c h c o u l d i n t e r a c t w i t h the i m m o b i l i z e d o r g a n o m e r c u r i a l , f i r m l y a n c h o r e d to c r o s s l i n k e d d e x t r a n . I n i t h e p r e s e n t w o r k , a m i n o e t h y l - c e l l u l o s e w a s t h i o l a t e d w i t h N - a c e t y l h o m o c y s t e i n e t h i o l a c t o n e ( H T L ) . T h e u s e of the l a t t e r c o m p o u n d as a t h i o l a t i n g agent w a s f i r s t i n t r o d u c e d by B e n e s c h a n d B e n e s c h (1958). A t t e m p t s to t h i o l a t e a m i n o e t h y l - c e l l u l o s e w i t h H T L i n r e f l u x i n g d i o x a n e w e r e u n s u c c e s s f u l . W h e n a c a t a l y t i c a m o u n t of -155-i m i d a z o l e w a s i n c l u d e d i n the r e a c t i o n m i x t u r e , a p r o d u c t w a s o b t a i n e d w h i c h r e a c t e d o n l y w e a k l y w i t h n i t r o p r u s s i d e . T h i o l a t i o n of p r o t e i n s w i t h t h i o l a c t o n e s i s k n o w n to o c c u r m u c h m o r e r e a d i l y i n the p r e s e n c e t h a n i n the a b s e n c e of i m i d a z o l e ( K l o t z a n d E l f b a u m , 1964). W h e n the t h i o l a t i o n of a m i n o e t h y l - c e l l u l o s e w i t h H T L w a s c a r r i e d out at r o o m t e m p e r a t u r e , a n d i n the p r e s e n c e of s i l v e r n i t r a t e ( S c h w y z e r a n d H u e r l i m a n , 1954; B e n e s c h a n d B e n e s c h , 1958) a p r o d u c t w a s o b t a i n e d w h i c h r e a c t e d s t r o n g l y w i t h n i t r o p r u s s i d e . T h e s i l v e r n i t r a t e w a s a d d e d i n i n c r e m e n t s , w i t h the s i m u l t a n e o u s a d d i t i o n of s o d i u m h y d r o x i d e . T h e s i l v e r n i t r a t e r e a c t s w i t h H T L to f o r m a s p a r i n g l y s o l u b l e c o m p l e x w i t h the c o n c o m i t a n t r e l e a s e of a p r o t o n . It i s the s i l v e r c o m p l e x w h i c h r e a c t s w i t h the a m i n o g r o u p s on the c e l l u l o s e ( B e n e s c h a n d B e n e s c h , 1958). T h e a l k a l i ( u s e d f o r n e u t r a l i z i n g the r e l e a s e d p r o t o n s ) m u s t be a d d e d c a u t i o u s l y s o t h a t the _p_H of the s o l u t i o n does not e x c e e d 9. O t h e r -w i s e the s i l v e r i o n s w i l l be r e d u c e d by the r e d u c i n g e n d g r o u p s of the c e l l u l o s e . T h e s i l v e r i o n s w e r e r e a d i l y r e m o v e d f r o m the t h i o l a t e d p r o d u c t by w a s h i n g i t w i t h a s o l u t i o n c o n s i s t i n g of n i t r i c a c i d a n d c o n c e n t r a t e d t h i o u r e a . T h e n u m b e r of H S - g r o u p s i n the t h i o l a t e d a m i n o e t h y l - c e l l u l o s e w a s d e t e r m i n e d b y t i t r a t i n g s a m p l e s of the p r o d u c t w i t h i o d o a c e t i c a c i d ( P a r t I I ^ A , S e c t i o n 3) . I o d o a c e t i c a c i d o r i o d o a c e t a m i d e has b e e n u s e d by m a n y i n v e s t i g a t o r s f o r d e t e r m i n i n g the n u m b e r of H S - g r o u p s i n p r o t e i n . T h e m e t h o d d e p e n d s on.the f o r m a t i o n of c a r b o x y m e t h y l t h i o e t h e r - 1 5 6 -w h i c h i s a c c o m p a n i e d by the r e l e a s e of a p r o t o n f r o m the H S - g r o u p . T h u s b y c a r r y i n g out the t i t r a t i o n i n a p H stat , the n u m b e r of e q u i v a l e n t s of a l k a l i r e q u i r e d to n e u t r a l i z e the p r o t o n s r e l e a s e d c o r r e s p o n d to the n u m b e r of e q u i v a l e n t s of H S - g r o u p s r e a c t i n g w i t h the i o d o a c e t i c a c i d . O b v i o u s l y t h e n , one m u s t k n o w the a p p r o x i m a t e p K of the t h i o l g r o u p . A n ; a p p r o x i m a t e p K g p j w a s o b t a i n e d i n the p r e s e n t s t u d y b y c a r r y i n g out a s e r i e s of t i t r a t i o n s o v e r a w i d e r a n g e of p H ' s ( P a r t I I - A , S e c t i o n 3 a n d F i g u r e s 1-A, B , C and 2 - A , B ) . W h e n a m i n o e t h y l - c e l l u l o s e w a s t h i o l a t e d w i t h H T L i n the p r e s e n c e of s i l v e r n i t r a t e , the m a x i m u m d e g r e e of t h i o l a t i o n o b t a i n e d w a s about 0. 23 m e q of H S - g r o u p s p e r g r a m of d r y p r o d u c t . A c o l u m n of the t h i o l a t e d a m i n o e t h y l - c e l l u l o s e w a s r e a d i l y c o n v e r t e d to the o r g a n o m e r c u r i a l d e r i v a t i v e b y pas s i n g a s o l u t i o n of b i s - 3 , 6 - ( a c e t a t o m e r c u r i m e t h y l ) - d i o x a n e t h r o u g h the c o l u m n . P r e l i m i n a r y e x p e r i m e n t s , d e s i g n e d to t e s t the b e h a v i o u r of s - R N A on the o r g a n o -m e r c u r i a l - c e l l u l o s e , s h o w e d that the a p p l i e d s - R N A w a s b e i n g r e t a r d e d as a r e s u l t of the i o n - e x c h a n g e p r o p e r t i e s of the d e r i v a t i z e d a m i n o e t h y l -c e l l u l o s e . W h e n ^ C - c y s t e i n y l - s - R N A w a s c h r o m a t o g r a p h e d on t h i s o r g a n o m e r c u r i a l - c e l l u l o s e i n the p r e s e n c e of 0. 5 M s o d i u m c h l o r i d e , the r a d i o a c t i v i t y w a s e l u t e d at the s o l v e n t f r o n t a l o n g w i t h the r e s t of the s - R N A . T h e e a s i e s t e x p l a n a t i o n f o r t h i s i s that the c o n c e n t r a t i o n of s o d i u m c h l o r i d e r e q u i r e d to o v e r c o m e the i o n i c i n t e r a c t i o n s w a s h i g h - 1 5 7 -e n o u g h to c o m p e t e e f f e c t i v e l y f o r the m e r c u r y . T o get a r o u n d the s a l t r e q u i r e m e n t i t w a s d e c i d e d to p r e p a r e a t h i o l a t e d c e l l u l o s e w h i c h d i d not have i o n - e x c h a n g e p r o p e r t i e s . T h i s w a s a c h i e v e d by the p r o c e d u r e s d e s c r i b e d i n P a r t I I - A , S e c t i o n s 11 to 16 i n c l u s i v e . T h e p r i n c i p l e w a s to p r e p a r e the 6-O - t o s y l -c e l l u l o s e w h i c h c o u l d be c o n v e r t e d to 6 - S - a c e t y l - c e l l u l o s e b y a n u c l e o -p h i l i c d i s p l a c e m e n t r e a c t i o n w i t h p o t a s s i u m t h i o l a c e t a t e . A l k a l i n e h y d r o l y s i s of the 6 - S - a c e t y l - c e l l u l o s e w o u l d t h e n g i v e the d e s i r e d 6-d e o x y - 6 - m e r c a p t o - c e l l u l o s e . T h e m a x i m u m d e g r e e of s u b s t i t u t i o n f o r the p r e p a r a t i o n of 6 -O - t o s y l - c e l l u l o s e w a s o b t a i n e d b y a c t i v a t i n g the d e a c e t y l a t e d c e l l u l o s e a c e t a t e i n a q u e o u s p y r i d i n e ( M a l m et a l . , 1948; W h i s t l e r a n d S h a s h a , 1964) p r i o r to t o s y l a t i o n . S u l f u r a n a l y s i s by the m e t h o d of L y s y j a n d Z a r e m b o , (1958), i n d i c a t e d a D . S. of 1. 07. T h e t o s y l - c e l l u l o s e w a s r e a d i l y c o n v e r t e d to 6 - S - a c e t y l - c e l l u l o s e by r e a c t i o n w i t h p o t a s s i u m t h i o a c e t a t e . T h e 6 - S - a c e t y l - c e l l u l o s e r e a c t e d w e a k l y w i t h n i t r o p r u s s i d e , as e x p e c t e d f o r t h i o e s t e r s a n d w a s c o n v e r t e d to 6 - d e o x y - 6 - m e r c a p t o - c e l l u l o s e by a l k a l i n e h y d r o l y s i s . T h e n u m b e r of H S - g r o u p s i n the m e r c a p t o - c e l l u l o s e c o u l d not be a c c u r a t e l y d e t e r m i n e d by t i t r a t i o n w i t h i o d o a c e t i c a c i d . T h u s , e v e n a f t e r 6 h r the c a r b o x y m e t h -y l a t e d m e r c a p t o - c e l l u l o s e s t i l l r e a c t e d s t r o n g l y w i t h n i t r o p r u s s i d e . W h e n s - R N A w a s c h r o m a t o g r a p h e d on a c o l u m n of m e r c a p t o -c e l l u l o s e i n 0.10 M s o d i u m a c e t a t e , m o s t of the a p p l i e d m a t e r i a l (75%) -158-w a s e l u t e d at the s o l v e n t f r o n t w h i l e the r e m a i n d e r w a s e l u t e d by about 0. 03 M s o d i u m c h l o r i d e . B o t h p e a k s s h o w e d s i m i l a r h y p e r c h r o m i c e f fects w h e n d i g e s t e d w i t h p a n c r e a t i c r i b o n u c l e a s e a n d b o t h h a d s i m i l a r u l t r a v i o l e t s p e c t r a . T h e m e r c a p t o - c e l l u l o s e t h e r e f o r e , a p p e a r e d to have the d e s i r e d c h a r a c t e r i s t i c s i n that a l l the a p p l i e d s - R N A c o u l d be r e c o v e r e d . a t v e r y l o w s a l t c o n c e n t r a t i o n s . N o f u r t h e r s t u d i e s h a v e b e e n c a r r i e d out on the m e r c a p t o - c e l l u l o s e p r e p a r a t i o n s e x c e p t as the o r g a n o m e r c u r i a l - c e l l u l o s e d e r i v a t i v e . T h e m e r c a p t o - c e l l u l o s e w a s c o n v e r t e d to o r g a n o m e r c u r i a l -c e l l u l o s e , as d e s c r i b e d p r e v i o u s l y . W h e n ^C-cysteinyl-s-RNA w a s c h r o m a t o g r a p h e d on the o r g a n o m e r c u r i a l - c e l l u l o s e , u s i n g a n i n c r e a s i n g g r a d i e n t of s o d i u m c h l o r i d e , the r e s u l t s w e r e e n c o u r a g i n g ( F i g . 3). T h e f i r s t c o m p o n e n t to be e l u t e d h a d s p e c t r a l p r o p e r t i e s s i m i l a r to c y t i d i n e . T h e s e c o n d , a n d s l i g h t l y l a r g e r p e a k , h a d the s p e c t r a l p r o p e r t i e s of a d e n o s i n e . T h e r a t i o s of the f i r s t t w o p e a k s a r e w h a t w o u l d be e x p e c t e d f o r the c y t i d i n e - a n d a d e n o s i n e - 5 ' t r i p h o s p h a t e s u s e d i n the p r e p a r a t i o n 14 of the C - c y s t e i n y l - s - R N A . T h i s o b s e r v a t i o n s u g g e s t s that a d e n o s i n e i n t e r a c t s w i t h m e r c u r y m o r e s t r o n g l y t h a n does c y t i d i n e , a n d i s i n a g r e e m e n t w i t h the r e s u l t s of o t h e r i n v e s t i g a t i o n s on the i n t e r a c t i o n of m e r c u r y , w i t h n u c l e i c a c i d s (see D a v i d s o n et a l . , 1965, a n d l a t e r d i s c u s s i o n ) . T h e s - R N A w a s e l u t e d as a s h a r p p e a k at about 0. 3 M s o d i u m c h l o r i d e ( P e a k I, F i g u r e 3). T h e n a t u r e of the m a t e r i a l p r e c e d i n g p e a k I h a s not b e e n i n v e s t i g a t e d a l t h o u g h the m i x e d s - R N A u s e d i n t h i s - 1 5 9 -e x p e r i m e n t i s k n o w n to be c o n t a m i n a t e d w i t h n o n - s - R N A m a t e r i a l . N o r a d i o a c t i v i t y c o u l d be d e t e c t e d u n t i l the c o l u m n w a s w a s h e d w i t h 0 . 4 M s o d i u m c h l o r i d e c o n t a i n i n g 0. 01 M 2 - m e r c a p t o e t h a n o l ( P e a k II) . I n s u f f i c i e n t m a t e r i a l w a s r e c o v e r e d i n P e a k II f o r f u r t h e r i n v e s t i g a t i o n . T h e u s e of the o r g a n o m e r c u r i a l - c e l l u l o s e d e s c r i b e d h e r e , f o r the f r a c t i o n a t i o n of n u c l e i c a c i d s as w e l l as s u l p h y d r y l c o n t a i n i n g p r o t e i n s , has not b e e n i n v e s t i g a t e d f u r t h e r . P l a n s w e r e to u s e the m e t h o d f o r i s o l a t i n g c y s t e i n e - s p e c i f i c s - R N A i n l a r g e q u a n t i t i e s s u i t a b l e f o r s t r u c t u r a l w o r k . In a d d i t i o n a t t e m p t s w e r e to be m a d e to m a k e the m e t h o d m o r e g e n e r a l by i n v e s t i g a t i n g p r o c e d u r e s f o r t h i o l a t i n g a m i n o a c y l - s - R N A s. S t u d i e s on the i n t e r a c t i o n b e t w e e n n u c l e i c a c i d s a n d m e t a l c a t i o n s have b e e n r e p o r t e d b y a n u m b e r of i n v e s t i g a t o r s . E a r l y s t u d i e s b y T h o m a s (1954) and by Y a m a n e a n d D a v i d s o n (1961) s h o w e d that D N A c o m p l e x e d w i t h m e r c u r y . It w a s c o n c l u d e d f r o m the s p e c t r a l p r o p e r t i e s that the m e t a l i n t e r a c t e d w i t h the b a s e s i n D N A . S i n g e r (1964), s t u d i e d the c o m p l e x e s f o r m e d b e t w e e n t o b a c c o m o s a i c v i r u s a n d s e v e r a l m u l t i v a l e n t c a t i o n s ( i n c l u d i n g m e r c u r y ) . T h i s i n v e s t i g a t o r found that s l i g h t l y l e s s t h a n one m o l e of e i t h e r s i l v e r o r m e r c u r y w e r e b o u n d i n d e p e n d e n t l y , a n d c o m p e t i t i o n e x p e r i m e n t s s u g g e s t e d that 0. 5 m o l e of e a c h w a s b o u n d on s p e c i f i c i n d e p e n d e n t s i t e s . D a v i d s o n et a l . (19 65) d e m o n s t r a t e d that the a m o u n t of b i n d i n g at a g i v e n c o n c e n t -r a t i o n of m e r c u r y v a r i e d w i t h the b a s e c o m p o s i t i o n of D N A , b a s e d o n - 1 6 0 -C s ^ S O ^ d e n s i t y g r a d i e n t c e n t r i f u g a t i o n . T h e s e w o r k e r s u t i l i z e d c e s i u m s u l f a t e d e n s i t y g r a d i e n t c e n t r i f u g a t i o n i n the p r e s e n c e of m e r c u r y f o r the s e p a r a t i o n of c r a b d A T p o l y m e r f r o m the m a i n D N A c o m p o n e n t . T h u s i t w o u l d a p p e a r that o r g a n o m e r c u r i a l - c e l l u l o s e m i g h t be u s e f u l f o r f r a c t i o n a t i o n of n u c l e i c a c i d s by e x p l o i t i n g the i n t e r a c t i o n s b e t w e e n m e r c u r y a n d the c o n s t i t u e n t n u c l e o t i d e s . H a v i n g a n o r g a n o -m e r c u r i a l - c e l l u l o s e d e v o i d of i o n - e x c h a n g e p r o p e r t i e s m a k e s s u c h a s tudy f e a s i b l e . F u r t h e r i n v e s t i g a t i o n s on the u s e of o r g a n o m e r c u r i a l - c e l l u l o s e f o r f r a c t i o n a t i n g n u c l e i c a c i d s w a s d i s c o n t i n u e d w h e n the p o t e n t i a l of b e n z o y l a t e d d i e t h y l a m i n o e t h y l - c e l l u l o s e f o r the f r a c t i o n a t i o n of s - R N A w a s r e a l i z e d (see P a r t I I - C ) . T h e e x p e r i m e n t s r e p o r t e d i n P a r t I I - B u t i l i z e the d i f f e r e n c e s i n p a r t i t i o n c o - e f f i c i e n t s of s - R N A m o l e c u l a r s p e c i e s i n t w o - p h a s e s o l v e n t s y s t e m s . T h e a d v a n t a g e s and d i s a d v a n t a g e s of c o l u m n p a r t i t i o n c h r o m -a t r o g r a p h y , c o m p a r e d to c o u n t e r c u r r e n t d i s t r i b u t i o n m e t h o d s , have b e e n m e n t i o n e d i n the I n t r o d u c t i o n to P a r t I I . C o m m e n t s p e r t a i n i n g to the e x p e r i m e n t s d e s c r i b e d i n P a r t I I - B c a n be s u m m a r i z e d by s t a t i n g that , i n g e n e r a l , the r e s o l u t i o n of a c c e p t o r a c t i v i t i e s as w e l l as the r e c o v e r y of b i o l o g i c a l a c t i v i t y of s - R N A w a s p o o r . I n f a i r n e s s h o w e v e r , i t s h o u l d be p o i n t e d out that - 1 6 1 -o t h e r i n v e s t i g a t o r s h a v e h a d c o n s i d e r a b l y g r e a t e r s u c c e s s i n f r a c t i o n a t i n g s - R N A by c o l u m n p a r t i t i o n c h r o m a t o g r a p h y ( T a n a k a et a l . , 1962; B e r g q u i s t a n d S c o t t , 1964; M u e n c h a n d B e r g , 1966a). T h e r e a s o n s f o r the p o o r r e s u l t s d e s c r i b e d i n P a r t I I - B a r e by no m e a n s c l e a r but p r o b a b l y r e f l e c t to a l a r g e extent , the u s e of B i o g e l as a s u p p o r t f o r the l o w e r p h a s e . O t h e r w o r k e r s , e . g . T a n a k a et a l . (1962), and M u e n c h a n d B e r g , (1966a) u s e d G - 2 5 S e p h a d e x as a s u p p o r t i n g m e d i u m f o r the s t a t i o n a r y p h a s e . O f the m a n y p r o c e d u r e s r e p o r t e d f o r the f r a c t i o n a t i o n of s - R N A (see I n t r o d u c t i o n ) M A K ( m e t h y l a t e d a l b u m i n - k i e s e l g u h r ) c o l u m n s p r o v i d e r e a s o n a b l y good r e s o l u t i o n of a m i n o a c i d a c c e p t o r a c t i v i t i e s (Sueoka a n d Y a m a n e , 1962). T h e l o w c a p a c i t y of t h e s e c o l u m n s i s t h e i r m a j o r d i s a d v a n t a g e f o r f r a c t i o n a t i n g s - R N A on. a p r e p a r a t i v e s c a l e . A s m e n t i o n e d i n the i n t r o d u c t i o n to t h i s p a r t of the t h e s i s , i t w a s c o n c l u d e d that the f o r c e s r e s p o n s i b l e f o r the f r a c t i o n a t i o n of s - R N A on c o l u m n s of M A K w e r e p r o b a b l y of a l i p o i d a l n a t u r e r e s u l t i n g f r o m the a r o m a t i c a m i n o a c i d c o n t e n t of the a l b u m i n . It s e e m e d r e a s o n a b l e to t e s t the v a l i d i t y of t h i s c o n c l u s i o n b y c o n v e r t i n g D E A E - c e l l u l o s e f r o m a h y d r o p h i l i c a n i o n - e x c h a n g e r to a l i p o p h i l i c a n i o n - e x c h a n g e r . T h i s w a s e a s i l y a c h i e v e d by r e a c t i n g the h y d r o x y l g r o u p s of the c e l l u l o s e w i t h b e n z o y l c h l o r i d e ( S e c t i o n 1) to y i e l d a p r o d u c t r e f e r r e d to h e r e as b e n z o y l a t e d - D E A E - c e l l u l o s e ( B D E A E - c e l l u l o s e ) . T h e p r o d u c t h a d a - 1 6 2 -s a p o n i f i c a t i o n e q u i v a l e n t of 159 ( G i l l a m et a l . , 1967) ( t r i - O - b e n z o y l -c e l l u l o s e h a s a c a l c u l a t e d s a p o n i f i c a t i o n n u m b e r of 158). B D E A E -c e l l u l o s e , w h e n p r e p a r e d as d e s c r i b e d i n Section' . 1, c a n be p a c k e d i n c h r o m a t o g r a p h i c c o l u m n s w i t h o u t the a i d of a i r p r e s s u r e a n d h a s e x c e l l e n t f l o w c h a r a c t e r i s t i c s . C o l u m n c h r o m a t o g r a p h y of s - R N A on B D E A E - c e l l u l o s e i s o p e r a t i o n a l l y s i m p l e . T h e s - R N A i s d i s s o l v e d i n a s m a l l v o l u m e of s t a r t i n g b u f f e r t h e n a p p l i e d to the t o p of the c o l u m n p a c k i n g p r e v i o u s l y e q u i l i b r a t e d w i t h the s a m e b u f f e r . A f t e r w a s h i n g the c o l u m n w i t h a few m i s of s t a r t i n g b u f f e r a l i n e a r g r a d i e n t of i n c r e a s i n g s a l t c o n c e n t r a t i o n i s a p p l i e d . A n u m b e r of e x p e r i m e n t s w e r e c a r r i e d out on the f r a c t i o n a t i o n of s - R N A u s i n g l i n e a r g r a d i e n t s of i n c r e a s i n g c o n c e n t r a t i o n s of s o d i u m c h l o r i d e a n d c o n s t a n t c o n c e n t r a t i o n s of m a g n e s i u m c h l o r i d e o r E D T A . It s o o n b e c a m e a p p a r e n t that the p r e s e n c e of m a g n e s i u m i o n h a d a p r o f o u n d af fect on,the e l u t i o n p r o f i l e . W h e n s - R N A w a s c h r o m a t o g r a p h e d on D E A E - c e l l u l o s e u s i n g a n i n c r e a s i n g l i n e a r g r a d i e n t of s a l t (see P a r t I , F i g . 1), about 9 0 % of the O. D . w a s e l u t e d b e t w e e n 0. 55 a n d 0. 7 M c h l o r i d e i o n c o n c e n t r a t i o n . W h e n s - R N A w a s c h r o m a t o g r a p h e d on B D E A E - c e l l u l o s e i n the p r e s e n c e of E D T A (to c h e l a t e any m u l t i v a l e n t m e t a l i o n s p r e s e n t ) , the l e a d i n g edge of the e l u t i o n p r o f i l e a p p e a r e d at about 0. 68 M c h l o r i d e i o n - 1 6 3 -c o n c e n t r a t i o n a n d o n l y 7 0 % of the a p p l i e d m a t e r i a l w a s r e c o v e r e d u p to 1 . 1 M c h l o r i d e i o n c o n c e n t r a t i o n ( F i g . 9). W h e n m a g n e s i u m i o n s a r e i n c l u d e d i n the e l u t i n g s o l v e n t at c o n c e n t r a t i o n s of 0. 02 M a n d 0. 005 M ( F i g s 7 a n d 8, r e s p e c t i v e l y ) the l e a d i n g edge of the e l u t i o n p r o f i l e a p p e a r s at a s i m i l a r c o n c e n t r a t i o n of c h l o r i d e i o n (0. 64 a n d 0. 65 M r e s p e c t i v e l y ) . H o w e v e r w h e n the m a g n e s i u m i o n c o n c e n t r a t i o n i n the e l u t i n g s o l v e n t i s i n c r e a s e d to 0.10 M ( F i g . 6), o r w h e n the c o l u m n i s d e v e l o p e d w i t h a n i n c r e a s i n g g r a d i e n t of m a g n e s i u m c h l o r i d e ( F i g . 10), the l e a d i n g edge of the e l u t i o n p r o f i l e a p p e a r s b e t w e e n 0. 3 a n d 0 . 4 M c h l o r i d e i o n c o n c e n t r a t i o n . T o d e t e r m i n e i f the s p r e a d i n g of the O . D . w a s a g e n e r a l p h e n o m e n o n o r i f i t t r u l y r e f l e c t e d the f r a c t i o n a t i o n of i n d i v i d u a l s p e c i e s of s - R N A , the e l u t i o n p r o f i l e w a s d i v i d e d i n t o f r a c t i o n s , t h e n the s - R N A w a s r e c o v e r e d f r o m the f r a c t i o n s a n d a s s a y e d f o r a c c e p t o r a c t i v i t y t o w a r d s e v e r a l a m i n o a c i d s . T h e r e s u l t s of t h e s e a s s a y s a r e s h o w n i n F i g u r e s 6 t o 10. T h e s e p a r a t i o n of i n d i v i d u a l a c c e p t o r a c t i v i t i e s i s c l e a r l y e v i d e n t . It s h o u l d be p o i n t e d out h e r e that the s h a r p n e s s of the a c c e p t o r p r o f i l e s s h o w n i n F i g u r e s 6 to 10 d e p e n d i n p a r t on how one g r o u p s the c o l u m n f r a c t i o n s f o r a s s a y p u r p o s e s . I n l a t e r s t u d i e s , the c o l u m n f r a c t i o n s w e r e a s s a y e d i n d i v i d u a l l y a c c o r d i n g to the r a p i d m e t h o d r e p o r t e d by C h e r a y i l a n d B o c k (1965). - 1 6 4 -T h e p o s i t i o n of e l u t i o n of the g l y c i n e a c c e p t o r a c t i v i t y i n the p r e s e n c e a n d a b s e n c e of m a g n e s i u m i l l u s t r a t e s the p r o f o u n d i n f l u e n c e of m a g n e s i u m on the e l u t i o n c h a r a c t e r i s t i c s of s - R N A . T h u s w h e n m a g -n e s i u m i o n s w e r e i n c l u d e d i n the e l u t i n g s o l v e n t at c o n c e n t r a t i o n s b e t w e e n 0. 005 a n d 0. 02 M , t w o p e a k s of g l y c i n e a c c e p t o r a c t i v i t y w e r e o b s e r v e d ( F i g s 8 a n d 7 r e s p e c t i v e l y ) . T h e f i r s t p e a k ( g l y c i n e - I ) a p p e a r e d at the l e a d i n g edge of the e l u t i o n p r o f i l e i n b o t h c a s e s . The s e c o n d p e a k of g l y c i n e a c c e p t o r a c t i v i t y ( g l y c i n e - ! ! ) a p p e a r e d f u r t h e r a l o n g the e l u t i o n p r o f i l e a n d i n the p r e s e n c e of 0. 02 M m a g n e s i u m ( F i g . 7) w a s c l e a r l y s e p a r a t e d f r o m the f i r s t . W h e n , a m a g n e s i u m c h l o r i d e g r a d i e n t w a s e m p l o y e d f o r e l u t i o n ( F i g . 10), o r w h e n 0.10 M m a g n e s i u m c h l o r i d e w a s i n c l u d e d i n the e l u t i o n s o l v e n t ( F i g . 6), o n l y one b r o a d peak of g l y c i n e a c c e p t o r a c t i v i t y w a s o b s e r v e d , a n d t h i s a p p e a r e d b e h i n d the l e a d i n g edge of the e l u t i o n p r o f i l e . I n a d d i t i o n , the h i s t i d i n e a c c e p t o r a c t i v i t y e l u t e d c l o s e r to the g l y c i n e a c c e p t o r a c t i v i t y w h e n m a g n e s i u m c h l o r i d e w a s u s e d to d e v e l o p , the c o l u m n ( F i g . 10). It a p p e a r e d t h e n , that m a g n e s i u m i o n s w e r e i n f l u e n c i n g the i n t e r a c t i o n s b e t w e e n the e x c h a n g e r and the d i f f e r e n t s p e c i e s of s - R N A m o l e c u l e s a n d that t h i s i n f l u e n c e w a s g r e a t e r f o r s o m e s p e c i e s t h a n f o r o t h e r s . It f u r t h e r a p p e a r e d that t h o s e s - R N A m o l e c u l e s e l u t e d ' l a t e f r o m , the c o l u m n w e r e a f f e c t e d b y m a g n e s i u m to a g r e a t e r extent t h a n t h o s e - 1 65-e l u t e d e a r l y f r o m the c o l u m n . T h e ef fect of m a g n e s i u m i o n s on the s e c o n d a r y s t r u c t u r e of s - R N A i s , of c o u r s e , w e l l d o c u m e n t e d i n the l i t e r a t u r e ( for e x a m p l e , see P a r t I of t h i s t h e s i s ) . It w a s a n t i c i p a t e d that t h i s d i f f e r e n t i a l e f fect of m a g n e s i u m on the e l u t i o n p o s i t i o n of the a c c e p t o r a c t i v i t i e s w o u l d be a u s e f u l v a r i a b l e d u r i n g r e c h r o m a t o g r a p h y of f r a c t i o n s f r o m a p r e p a r a t i v e c o l u m n . T o f u r t h e r e m p h a s i z e the d i f f e r e n c e s i n the d e g r e e of i n t e r a c t i o n b e t w e e n the e x c h a n g e r a n d the v a r i o u s s - R N A s p e c i e s i t s h o u l d be n o t e d that the p h e n y l a l a n i n e a c c e p t o r a c t i v i t y w a s not e l u t e d f r o m B D E A E -c e l l u l o s e e v e n by c h l o r i d e i o n c o n c e n t r a t i o n s a b o v e 1. 0 M . T h e a c c e p t o r p r o f i l e f o r p h e n y l a l a n i n e , s h o w n i n F i g u r e 8, w a s o n l y a f r a c t i o n of the p h e n y l a l a n i n e a c c e p t o r a c t i v i t y a p p l i e d to the c o l u m n a n d e i t h e r r e p r e -s e n t e d m u l t i p l e a c c e p t o r a c t i v i t i e s f o r p h e n y l a l a n i n e o r d e g r a d e d p h e n y l a l a n i n e s - R N A ( m i s s i n g the t e r m i n a l a d e n o s i n e m o i e t y ) . T h e l a t t e r i n t e r p r e t a t i o n i s f a v o u r e d , f o r r e a s o n s w h i c h w i l l be d i s c u s s e d b e l o w . T o e l u t e the m a j o r p h e n y l a l a n i n e a c c e p t o r R N A i t w a s n e c e s s a r y to i n c l u d e i n the e l u t i n g s o l v e n t a l i p o p h i l i c b o n d b r e a k e r s u c h as u r e a , m e t h o x y e t h a n o l , o r e t h a n o l , (see F i g . 12). W h e n t h i s s t e p w a s i n c l u d e d at the end of a s o i U u m c h l o r i d e g r a d i e n t , 95-100% of the a p p l i e d m a t e r i a l w a s r e c o v e r e d . D i f f e r e n c e s i n the c h r o m a t o g r a p h i c b e h a v i o u r of s - R N A o n B D E A E - c e l l u l o s e , c o m p a r e d to D E A E - c e l l u l o s e have b e e n m e n t i o n e d . - 1 6 6 -The r e a s o n s f o r t h e s e d i f f e r e n c e s may b e c o m e c l e a r w h e n the n a t u r e of the i n t e r a c t i o n s i n v o l v e d b e t w e e n the n u c l e o t i d e s a n d D E A E - o r b e n z o y l a t e d - D E A E - c e l l u l o s e i s m o r e f u l l y u n d e r s t o o d . S e c o n d a r y b i n d i n g f o r c e s ( n o n - i o n i c ) b e t w e e n o l i g o n u c l e o t i d e s a n d D E A E - c e l l u l o s e have b e e n a t t r i b u t e d p r i m a r i l y to h y d r o g e n b o n d i n g b e t w e e n the i o n - e x c h a n g e r and the c o n s t i t u e n t n u c l e o t i d e s ( T o m l i n s o n and T e n e r , 1963a). S e p a r a t i o n of m i x e d o l i g o n u c l e o t i d e s o n D E A E -c e l l u l o s e has a l s o b e e n s h o w n to be a f u n c t i o n of the c o n s t i t u e n t p u r i n e -p y r i m i d i n e r a t i o ( B a r t o s ^ e t a l . , 1963). T h e s e f o r c e s c a n be d i m i n i s h e d by c h r o m a t o g r a p h y o n D E A E - c e l l u l o s e i n the p r e s e n c e of 7 M u r e a ( T o m l i n s o n and T e n e r , 1963a, 1963b) o r e l i m i n a t e d b y c h r o m a t o g r a p h y on D E A E - S e p h a d e x A - 2 5 i n the p r e s e n c e of 7 M u r e a ( R u s h i z k y et a l . , 1964). T h e s e forces" were p r o b a b l y r e s p o n s i b l e f o r the s l i g h t r e s o l u t i o n of r a t l i v e r l e u c y l - s - R N A on D E A E - c e l l u l o s e ( N i s h i y a m a et a l . , 1961). T h e i m p r e s s i v e f r a c t i o n a t i o n of s - R N A on D E A E - c e l l u l o s e a n d D E A E - S e p h a d e x r e p o r t e d b y C h e r a y i l a n d B o c k (1965) w a s not a c o n s e q u e n c e of s e c o n d a r y i n t e r a c t i o n s . T h e s e i n v e s t i g a t o r s u s e d h i g h c o n c e n t r a t i o n s of u r e a i n t h e i r e l u t i n g s y s t e m s a n d u r e a i s k n o w n to r r e d u c e the s e c o n d a r y f o r c e s b e t w e e n n u c l e o t i d e s a n d D E A E - c e l l u l o s e ( T o m l i n s o n a n d T e n e r , 1963a). It s e e m s l i k e l y that u r e a opens the s - R N A s t r u c t u r e a l l o w i n g i n c r e a s e d i n t e r a c t i o n b e t w e e n the c o n s t i t u e n t n u c l e o t i d e s a n d the i o n - e x c h a n g e g r o u p s of the c e l l u l o s e . - 1 6 7 -A s i m i l a r o p e n i n g of the s - R N A s t r u c t u r e o c c u r s at e l e v a t e d t e m p e r a t u r e s . T h i s has b e e n e x p l o i t e d b y B a g u l e y et a l . , (1965a, 1965b) a n d b y B e r g q u i s t (1966) f o r the f r a c t i o n a t i o n of y e a s t s - R N A . T h e s e w o r k e r s f r a c t i o n a t e d s - R N A on D E A E - c e l l u l o s e at 6 5 ° u t i l i z i n g a n e x p o n e n t i a l l y i n c r e a s i n g s a l t g r a d i e n t to e l u t e the s - R N A . T h e y a l s o s h o w e d that s - R N A c o u l d be f r a c t i o n a t e d on D E A E - S e p h a d e x by e m p l o y i n g a d e c r e a s i n g t e m p e r a t u r e g r a d i e n t at c o n s t a n t s a l t c o n c e n t r a t i o n . A s the t e m p e r a t u r e i s l o w e r e d , the s - R N A m o l e c u l e s p r e s u m a b l y r e n a t u r e , thus e x p o s i n g f e w e r of the c o n s t i t u e n t n u c l e o t i d e s to the i o n - e x c h a n g e r . H o w e v e r s e c o n d a r y b o n d i n g f o r c e s m i g h t w e l l p l a y a n i m p o r t a n t r o l e i n t h i s m e t h o d . s - R N A m o l e c u l e s a r e s i m i l a r i n s i z e a n d h a v e s i m i l a r n u m b e r s of p h o s p h a t e d i s s o c i a t i o n s . T h u s i t w o u l d be d i f f i c u l t to f r a c t i o n a t e t h e m by u t i l i z i n g o n l y t h e i r i o n - e x c h a n g e p r o p e r t i e s o t h e r t h a n at a c i d i c p H ' s w h e r e c y t o s i n e i s p r o t o n a t e d . T h e above d i s c u s s i o n h o w e v e r , s t r o n g l y s u g g e s t s that the c o n s t i t u e n t n u c l e o t i d e s of m i x e d s - R N A a r e a v a i l a b l e f o r i n t e r a c t i o n w i t h i o n - e x c h a n g e r s to v a r i a b l e e x t e n t s . In a d d i t i o n i t w a s s u g g e s t e d that the f o r c e s p r i m a r i l y r e s p o n s i b l e f o r f r a c t i o n a t i o n of s - R N A on M A K c o l u m n s w e r e a f u n c t i o n of the c o n s t i t u e n t b a s e s r a t h e r t h a n the p h o s p h a t e d i s s o c i a t i o n s . T h e f o r c e s i n v o l v e d i n the f r a c t i o n a t i o n of s - R N A on b e n z o y l a t e d D E A E - c e l l u l o s e a r e p r o b a b l y h y d r o p h o b i c i n n a t u r e , r e s u l t i n g f r o m the - 1 6 8 -i n t e r a c t i o n of the e x p o s e d n u c l e o t i d e s and the a r o m a t i c r i n g s of the B D E A E - c e l l u l o s e . T h e i o n - e x c h a n g e f o r c e s w o u l d be r e l e g a t e d to h o l d i n g the s - R N A m o l e c u l e s i n c l o s e c o n t a c t w i t h the c e l l u l o s e m a t r i x so that the r e l a t i v e l y s h o r t r a n g e h y d r o p h o b i c i n t e r a c t i o n s c a n be e f f e c t i v e . T h a t the s e c o n d a r y b o n d i n g f o r c e s i n D E A E - c e l l u l o s e a n d b e n z o y l a t e d D E A E - c e l l u l o s e a r e d i f f e r e n t , i s c l e a r f r o m the o r d e r of e l u t i o n of the m o n o r i b o n u c l e o t i d e s f r o m B D E A E - c e l l u l o s e ( F i g s 4 a n d 5). T h e o r d e r of e l u t i o n i n F i g u r e 4 i s p C , U p , G p a n d A p . S o m e s e p a r a t i o n of the 2' a n d 3' i s o m e r s i s e v i d e n t i n F i g u r e 4 , w i t h the 3' i s o m e r s e l u t i n g e a r l i e r t h a n the 2' i s o m e r s . T h e o r d e r of e l u t i o n of the m o n o r i b o n u c l e o t i d e s f r o m D E A E - c e l l u l o s e i s C p , U p , A p a n d G p ( S t a e h l i n , 1961). S e v e r a l c o m m e n t s c a n be m a d e about the e l u t i o n of the r i b o n u c l e o s i d e - 5 ' p h o s p h a t e s f r o m B D E A E - c e l l u l o s e ( F i g . 5). F i r s t l y , the p y r i m i d i n e s , as a c l a s s , a r e l e s s s t r o n g l y h e l d t h a n the p u r i n e s a l t h o u g h the o r d e r of e l u t i o n i s the s a m e as f o r the r i b o n u c l e o s i d e -2'(3') p h o s p h a t e s . S e c o n d l y , the p u r i n e - 5 ' p h o s p h a t e s ( F i g . 5) a r e not as c l e a r l y s e p a r a t e d as the p u r i n e - 2 ' ( 3 1 ) p h o s p h a t e s ( F i g . 4 ) . T h i r d l y , the c y t i d i n e n u c l e o t i d e e l u t e s as the s a m e s a l t c o n c e n t r a t i o n i n b o t h e x p e r i m e n t s (as i t s h o u l d s i n c e c y t o s i n e - 5 ' m o n o p h o s p h a t e w a s u s e d i n b o t h e x p e r i m e n t s ) , w h e r e a s the 5' p h o s p h a t e s of u r i d i n e , g u a n o s i n e a n d a d e n o s i n e a l l e l u t e d m u c h e a r l i e r i n the s a l t g r a d i e n t t h a n t h e i r 2 '(3 ') p h o s p h a t e c o u n t e r p a r t s . It i s d i f f i c u l t to see how the d i f f e r e n c e i n the - 1 6 9 -e l u t i o n p r o f i l e s c o u l d be e x p l a i n e d on s t e r e o c h e m i c a l g r o u n d s . It i s e q u a l l y d i f f i c u l t to e x p l a i n the d i f f e r e n c e s i n t e r m s of e l e c t r o n i c i n t e r a c t i o n s b e t w e e n the 5' p h o s p h a t e a n d the b a s e m o i e t y . If s u c h a n i n t e r a c t i o n e x i s t e d i n the r i b o n u c l e o s i d e - 5 ' p h o s p h a t e s to a n a p p r e c i a b l e extent , i t s h o u l d be r e f l e c t e d i n the u l t r a v i o l e t s p e c t r a of the i s o m e r s . N o c o r r e l a t i o n s c o u l d be d e t e c t e d . D u r i n g e a r l y s t u d i e s o n the c h r o m a t o g r a p h i c b e h a v i o u r of s - R N A on B D E A E - c e l l u l o s e i t w a s f o u n d by c o n d u c t i v i t y m e a s u r e m e n t s ( R a d i o -m e t e r , C o p e n h a g e n ) that the e a r l y p a r t of the s a l t g r a d i e n t w a s g o i n g t h r o u g h the c o l u m n i n a s t e p w i s e m a n n e r . T h i s p h e n o m e n o n o c c u r r e d only, w h e n t h e r e w a s a l a r g e d i f f e r e n c e i n d e n s i t i e s b e t w e e n the m i x e r and the r e s e r v o i r v e s s e l ( e . g . , 0.1 to 1. 0 M s o d i u m c h l o r i d e ) a n d c o u l d be p r e v e n t e d by u s i n g c a p i l l a r y t u b i n g f o r the U - t u b e , o r by d r a w i n g out one s i d e (the m i x e r s i d e ) of the U - t u b e to a 1 to 2 m m b o r e . It w a s s u s p e c t e d that t h i s p u l s i n g p h e n o m e n o n , o b s e r v e d w h e n the m i x e r a n d r e s e r v o i r w e r e c o n n e c t e d by 6 m m g l a s s tubing, w a s p a r t l y r e s p o n s i b l e f o r the i m p r e s s i v e f ine s t r u c t u r e of the e l u t i o n p r o f i l e s r e p r o d u c e d i n F i g u r e s 7 a n d 8. O n the s u s p i c i o n that p u l s e g r a d i e n t s m i g h t g i v e i m p r o v e d r e s o l u t i o n of a c c e p t o r a c t i v i t i e s , a n a p p r a t u s w a s d e v i s e d w h i c h w o u l d d e l i v e r 200 m l p u l s e s f r o m a l i n e a r g r a d i e n t of i n c r e a s i n g s o d i u m c h l o r i d e ( F i g . 11). T h e r e s o l u t i o n of g l y c i n e a c c e p t o r a c t i v i t y , w h e n . s - R N A w a s c h r o m a t o g r a p h e d on B D E A E - c e l l u l o s e u s i n g - 1 7 0 -the p u l s e g r a d i e n t ( F i g . 12) w a s no b e t t e r t h a n that o b t a i n e d u s i n g a s i m p l e l i n e a r g r a d i e n t . T h u s the f ine s t r u c t u r e i n the e l u t i o n p r o f i l e s of F i g u r e s 7 a n d 8 c a n be a t t r i b u t e d m a i n l y to the i n t r i n s i c s u p e r i o r i t y of B D E A E - c e l l u l o s e r a t h e r t h a n to the m e c h a n i c s of e l u t i o n . A s m e n t i o n e d p r e v i o u s l y , the p h e n y l a l a n i n e a c c e p t o r s - R N A w a s not e l u t e d f r o m B D E A E - c e l l u l o s e u n l e s s a l i p o p h i l i c b o n d b r e a k i n g s u b s t a n c e s u c h as e t h a n o l , m e t h o x y e t h a n o l , u r e a , e t c . , w a s i n c o r p o r a t e d i n the e l u t i n g s o l v e n t . W h e n the c o l u m n , d e s c r i b e d i n F i g u r e 12 w a s w a s h e d w i t h 1. 0 M s o d i u m c h l o r i d e , 15% m e t h o x y e t h a n o l ( v / v ) , v i a the p u l s e g r a d i e n t d e v i c e , t h r e e p e a k s of O. D . w e r e e l u t e d . W h e n the p e a k s w e r e a s s a y e d f o r a c c e p t o r a c t i v i t y t o w a r d p h e n y l a l a n i n e , m o s t of the a c t i v i t y w a s c o n t a i n e d i n the s e c o n d a n d t h i r d p e a k s ( F r a c t i o n s X V I , F i g . . 12). T h i s o b s e r v a t i o n s u g g e s t s that the s t r o n g l y h e l d p h e n y l a l a n i n e a c c e p t o r s - R N A m i g h t be e l u t e d i n high p u r i t y i f c h r o m a t o g r a p h e d by a n i n c r e a s i n g g r a d i e n t of e t h a n o l o r m e t h o x y e t h a n o l i n the p r e s e n c e of 1. 0 M s o d i u m c h l o r i d e . H o w e v e r , u n p u b l i s h e d e x p e r i m e n t s by D r . I . M a x w e l l i n d i c a t e d that the u s e of a n e t h a n o l g r a d i e n t gave no b e t t e r p u r i f i c a t i o n t h a n s t e p w i s e e l u t i o n w i t h e t h a n o l . W h e n F r a c t i o n s X V a n d X V I ( F i g . 12) w e r e e x a m i n e d by g e l f i l f r a t i on ( F i g s 1 3 - A a n d 1 3 - B , r e s p e c t i v e l y ) , 34% of the O . D . f r o m F r a c t i o n X V a n d 27% of the O. D . f r o m F r a c t i o n X V I d i d not show a c c e p t o r a c t i v i t y t o w a r d p h e n y l a l a n i n e . It i s i n t e r e s t i n g to note that - 1 7 1 -a l t h o u g h the m a j o r p e a k s f r o m the g e l f i l t r a t i o n s t u d i e s ( P e a k a i n F i g s 1 3 - A a n d B ) r e p r e s e n t 6 0 % and 7 0 % of the O . D . f r o m F r a c t i o n s X V and X V I r e s p e c t i v e l y , t h e y n o w a c c e p t t w i c e as m u c h p h e n y l a l a n i n e p e r O. D . u n i t as d i d the s a m e f r a c t i o n s b e f o r e g e l f i l t r a t i o n . T h e n a t u r e of the m a t e r i a l e x c l u d e d by the G-100 S e p h a d e x has not b e e n i n v e s t i g a t e d . T h e m e l t i n g p r o f i l e s of the s - R N A f r o m r e p r e s e n t a t i v e f r a c t i o n s ( F r a c t i o n s I , V , I X , X I I I , and X V I , F i g . 12) f r o m a B D E A E - c e l l u l o s e c o l u m n a n d a s a m p l e of m i x e d s - R N A a l l had the s a m e T j ^ ' s ( ' m e l t i n g ' t e m p e r a t u r e s ) . F r a c t i o n X V I , w h i c h w a s e l u t e d by m e t h o x y e t h a n o l , a n d the m i x e d s - R N A s a m p l e s h o w e d m o r e e a r l y m e l t i n g t h a n t h o s e e l u t e d by the s a l t g r a d i e n t . H o w e v e r , i t w a s not p o s s i b l e to d e t e c t s i g n i f i c a n t d i f f e r e n c e s i n the s e c o n d a r y s t r u c t u r e of the s - R N A ' s by t h i s m e t h o d . A s p o i n t e d out e a r l i e r i n t h i s d i s c u s s i o n , m a g n e s i u m i o n s s e e m e d to a f fect the s - R N A m o l e c u l e s e l u t e d l a t e f r o m the c o l u m n , to a g r e a t e r extent t h a n t h o s e w h i c h w e r e e l u t e d e a r l i e r . T h a t i s , the o r d e r of e l u t i o n of s - R N A f r o m B D E A E - c e l l u l o s e s e e m e d to be a f u n c t i o n of the e x p o s e d b a s e s c a p a b l e of i n t e r a c t i n g w i t h the e x c h a n g e r . It i s f u r t h e r a r g u e d , that the n u m b e r of i n t e r a c t i o n s p e r s - R N A m o l e c u l e i s p r o b a b l y s m a l l a n d that t h e y a r e h y d r o p h o b i c i n n a t u r e . It m i g h t be e x p e c t e d that the n u c l e o t i d e t r i p l e t r e p r e s e n t i n g the a n t i - c o d o n r e g i o n of s - R N A w o u l d be a m o n g t h o s e e x p o s e d . T h i s r e a s o n i n g l e d , i n the e a r l y s t u d i e s , t o a c o r r e l a t i o n b e t w e e n the o r d e r of e l u t i o n of the s - R N A ' s a n d t h e i r - 1 7 2 -p r o p o s e d a n t i - c o d o n c o m p o s i t i o n . R e s u l t s of the c h r o m a t o g r a p h y of the m o n o n u c l e o t i d e s a d d e d m o r e w e i g h t to the c o r r e l a t i o n . H o w e v e r , a t t e m p t s to p l a c e t h i s c o r r e l a t i o n on a q u a n t i t a t i v e b a s i s w e r e u n s u c c e s s -f u l . I n a d d i t i o n , a d e t a i l e d a n a l y s i s of the a c c e p t o r a c t i v i t i e s e l u t e d d u r i n g p r e p a r a t i v e c o l u m n c h r o m a t o g r a p h y of s - R N A (see G i l l a m et a l . , 1967) s o o n r e v e a l e d e x c e p t i o n s to t h i s h y p o t h e s i s . It i s w o r t h n o t i n g that the s - R N A u s e d i n the s t u d i e s to t h i s p o i n t w a s b a k e r s ' y e a s t s - R N A ( C a l b i o c h e m . ). B r e w e r ' s ' y e a s t s - R N A ( B o e h r i n g e r a n d Soehne) w a s u s e d f o r the r e m a i n d e r of the s t u d y . C h r o m a t o g r a p h y of a 5 g s a m p l e of b r e w e r s ' y e a s t s - R N A w a s c a r r i e d out by D r . I . G i l l a m i n t h i s l a b o r a t o r y . T h e d e t a i l e d r e s u l t s of t h i s a n d o t h e r s t u d i e s w i l l be p u b l i s h e d ( G i l l a m et_ a l . , 1967). T h e e l u t i o n p r o f i l e a n d the r e g i o n s of the g l y c i n e - I a n d g l y c i n e - I I a c c e p t o r a c t i v i t i e s a r e s h o w n i n F i g u r e 14. T h e g l y c i n e - I a n d the g l y c i n e - I I f r a c t i o n s f r o m t h i s c h r o m a t o g r a p h y w e r e u s e d as a s o u r c e of m a t e r i a l f o r f u r t h e r i n v e s t i g a t i o n s o n the c h r o m a t o g r a p h i c b e h a v i o u r of the g l y c i n e s - R N A ' s . T h e e x p e r i m e n t a l p r o c e d u r e s u s e d f o r the r e c h r o m a t o g r a p h y of the g l y c i n e - I a n d g l y c i n e - I I p e a k s a r e s u m m a r i z e d i n C h a r t s I a n d II, r e s p e c t i v e l y . T h e g l y c i n e - I p e a k (see F i g . 14, tube n u m b e r s 73 to 92) c o n t a i n e d , i n . a d d i t i o n to g l y c i n e - I a c c e p t o r a c t i v i t y , m a j o r p e a k s of m e t h i o n i n e a n d i s o l e u c i n e a c c e p t o r a c t i v i t i e s a n d m i n o r s h o u l d e r s of v a l i n e a n d p r o l i n e - 1 7 3 -C H A R T I F r a c t i o n a t i o n of G l y c i n e - I A c c e p t o r R N A on B D E A E - C e l l u l o s e 5 g m i x e d b r e w e r s ' y e a s t s - R N A C h r o m a t o g r a p h y 1 ( F i g u r e 14) 0. 01 M M g S 0 4 , N a C l g r a d i e n t , no b u f f e r ( P a r t I I - C , S e c t i o n 7 - G ) T u b e s 73 to 92 c o n t a i n g l y c i n e - I , m e t h i o n i n e , i s o l e u c i n e , v a l i n e a n d p r o l i n e a c c e p t o r R N A ' s C h r o m a t o g r a p h y 2 ( F i g u r e 15) 0. 05 M M g C l 2 , N a C l g r a d i e n t , p_H 3. 5 ( P a r t I I - C , S e c t i o n 7-H(i)) M a j o r P e a k M i n o r P e a k g l y c i n e - I , m e t h i o n i n e , i s o l e u c i n e a n d v a l i n e o r p r o l i n e a c c e p t o r R N A ' s ( F r a c t i o n I, F i g . 15) v a l i n e o r p r o l i n e a c c e p t o r R N A C h r o m a t o g r a p h y 3 ( F i g u r e 16) 1 m M E D T A , N a C l g r a d i e n t , p H 4. 0 ( P a r t I I - C , S e c t i o n 7 - H ( i i ) ) M a j o r P e a k s M i n o r P e a k m e t h i o n i n e a c c e p t o r R N A g l v c i n e - I , i s o l e u c i n e a c c e p t o r R N A v a l i n e o r p r o l i n e a c c e p t o r R N A - 1 7 4 -a c c e p t o r a c t i v i t i e s . W h e n the g l y c i n e - I p e a k w a s r e c h r o m a t o g r a p h e d o n B D E A E - c e l l u l o s e at p_H 3. 5 i n the p r e s e n c e of 0. 05 M m a g n e s i u m i o n s two p e a k s a p p e a r e d i n the e l u t i o n p r o f i l e ( F i g . 15). T h e m i n o r p e a k c o r r e s p o n d e d , p r e s u m a b l y , to one of the m i n o r , c o n t a m i n a t i n g a c c e p t o r a c t i v i t i e s ( v a l i n e o r p r o l i n e a c c e p t o r R N A ) . T h e m a j o r p e a k c o n t a i n e d two r e g i o n s of g l y c i n e a c c e p t o r a c t i v i t y . T h e s m a l l e r of the t w o , w h i c h a p p e a r e d at the l e a d i n g edge of the e l u t i o n p r o f i l e , i s p r o b a b l y g l y c i n e - I s - R N A m i s s i n g i t s t e r m i n a l a d e n o s i n e . T h e m a i n g l y c i n e a c c e p t o r r e g i o n w a s d i s p l a c e d f r o m the c e n t r e of the m a j o r p e a k i n the e l u t i o n p r o f i l e . T h i s o b s e r v a t i o n s u g g e s t e d t h a t we w e r e a p p r o a c h i n g the c o n d i t i o n s r e q u i r e d to r e m o v e the m a j o r c o n t a m i n a n t s f r o m the g l y c i n e -I a c c e p t o r R N A . It i s k n o w n that l o w e r i n g the jpH t e n d s to o p e n the s - R N A s t r u c t u r e thus e x p o s i n g m o r e n u c l e o t i d e s to the e x c h a n g e r . S m i t h (1966), has r e c e n t l y e x p l o i t e d t h i s f o r the f r a c t i o n a t i o n of s - R N A o n ' D E A E - S e p h a d e x c o l u m n s . It i s not u n r e a s o n a b l e to a s s u m e that m a g n e s i u m w o u l d a n t a g o n i z e t h i s a c t i o n of p H on the s - R N A m o l e c u l e s . T h u s w h e n the m a j o r p e a k of s - R N A ( F r a c t i o n I, F i g . 15) w a s r e c h r o m a t o g r a p h e d at p H 4 . 0 a n d i n the p r e s e n c e of E D T A , i t e l u t e d as t w o m a j o r a n d one m i n o r p e a k ( F i g . 16). T h e m i n o r p e a k p r e s u m a b l y r e p r e s e n t s the r e m o v a l of the o t h e r m i n o r c o n t a m i n a n t r e f e r r e d to e a r l i e r ( v a l i n e o r p r o l i n e a c c e p t o r R N A ) . M e t h i o n i n e a c c e p t o r a c t i v i t y c o i n c i d e d w i t h the l e a d i n g p e a k a n d the g l y c i n e a c c e p t o r a c t i v i t y w a s d i s p l a c e d to the -175-t a i l i n g edge of the s e c o n d p e a k of the e l u t i o n p r o f i l e . T h e i s o l e u c i n e a c c e p t o r a c t i v i t y (not a s s a y e d i n d e t a i l ) w a s c o n c e n t r a t e d b e t w e e n the m e t h i o n i n e a n d the g l y c i n e a c c e p t o r R N A ' s . T h u s , a l t h o u g h the l e a d i n g edge of the g l y c i n e - I a c c e p t o r p e a k i s s t i l l c o n t a m i n a t e d w i t h i s o l e u c i n e a c c e p t o r a c t i v i t y , f r a c t i o n s 86 to 98 ( F i g . 16) w e r e 95 to 100% g l y c i n e - I s - R N A as d e t e r m i n e d by the i n c o r p o r a t i o n of ^ 4 C - g l y c i n e i n t o T C A -i n s o l u b l e p r o d u c t . T h e g l y c i n e - I I p e a k (see F i g . 14, tube n u m b e r s 103 to 120) c o n t a i n e d , i n a d d i t i o n to g l y c i n e - I I s - R N A , m a j o r p e a k s of a l a n i n e a n d p r o l i n e a c c e p t o r a c t i v i t y a n d m i n o r a c t i v i t i e s t o w a r d v a l i n e , t h r e o n i n e , g l u t a m i c a c i d . a n d g l u t a m i n e . S i n c e the g l y c i n e - I I f r a c t i o n c o n t a i n e d so m a n y , o t h e r a c t i v i t i e s i t w a s c o n s i d e r e d a d v i s a b l e to a l t e r the c o n d i t i o n s o n l y s l i g h t l y d u r i n g e a c h r e c h r o m a t o g r a p h y . T h e e x p e r i m e n t a l p r o c e d u r e s a r e s u m m a r i z e d i n C h a r t I I . W h e n the g l y c i n e - I I f r a c t i o n , , f r o m the p r e p a r a t i v e c o l u m n , w a s r e c h r o m a t o g r a p h e d at p H 5 a n d i n the p r e s e n c e of 0. 01 M m a g n e s i u m i o n s , two p e a k s a p p e a r e d i n the e l u t i o n p r o f i l e ( F i g . 17). T h e g l y c i n e a c c e p t o r a c t i v i t y , w h i c h w a s a s s o c i a t e d w i t h the f i r s t p e a k ( F r a c t i o n I, F i g . 17), w a s u n c h a r a c t e r i s t i c a l l y b r o a d a n d , i n r e t r o s p e c t at l e a s t , w a s s u g g e s t i v e of m o r e t h a n one g l y c i n e a c c e p t o r a c t i v i t y . W h e n F r a c t i o n I ( F i g . 17) w a s r e c h r o m a t o g r a p h e d at p H 3. 5, i n the p r e s e n c e of E D T A , t w o p e a k s a p p e a r e d i n the e l u t i o n p r o f i l e ( F i g . 18). -176-C H A R T II F r a c t i o n a t i o n of G l y c i n e - I I A c c e p t o r R N A ' s on B D E A E - C e l l u l o s e 5 g b r e w e r s ' y e a s t s - R N A C h r o m a t o g r a p h y 1 ( F i g u r e 14) 0. 01 M M g S 0 4 , N a C l g r a d i e n t , no b u f f e r ( P a r t I I - C , S e c t i o n 7 - G ) T u b e s 103 to 120 c o n t a i n g l y c i n e - I I , a l a n i n e p r o l i n e , v a l i n e , t h r e o n i n e , g l u t a m i c a c i d a n d g l u t a m i n e a c c e p t o r R N A 1 s C h r o m a t o g r a p h y 2 ( F i g u r e 17) 0. 01 M M g C l 2 , N a C l g r a d i e n t , p H 5 . 0 M a j o r p e a k ( F r a c t i o n I) M i n o r p e a k c o n t a i n s . g l y c i n e - I I not a n a l y z e d a c c e p t o r a c t i v i t i e s C h r o m a t o g r aphy, 3 ( F i g u r e 18) 1 m M E D T A , N a C l g r a d i e n t , p H 3. 5 g l y c i n e - I P - l \ , g l y c i n e - I I - / 3 a n d " two m i n o r p e a k s of g l y c i n e a c c e p t o r a c t i v i t y -177-The g l y c i n e a c c e p t o r a c t i v i t y a p p e a r e d as t w o m a j o r a n d t w o m i n o r p e a k s . A l s o s h o w n i n F i g u r e 18 i s the a c c e p t o r a c t i v i t y f o r a l a n i n e . T h e p o s i t i o n of e l u t i o n of the o t h e r c o n t a m i n a t i n g s - R N A ' s w a s not d e t e r m i n e d . T h u s the g l y c i n e - I I f r a c t i o n f r o m the p r e p a r a t i v e c o l u m n c o n s i s t e d of at l e a s t t w o d i s t i n c t s - R N A ' s (g lycine-II-OC and g l y c i n e - I I - y3 » F i g . 18). T h e m i n o r p e a k of g l y c i n e a c c e p t o r a c t i v i t y on the l e a d i n g edge of the g l y c i n e - I I - ft p e a k m i g h t be glycine-11-^5 s - R N A m i s s i n g i t s t e r m i n a l a d e n o s i n e . T h e q u e s t i o n as to w h e t h e r o r not a n a c c e p t o r R N A h a s a n i n t a c t s e q u e n c e of n u c l e o t i d e s c a n be a n s w e r e d by a s s a y i n g the c o l u m n f r a c t i o n s w i t h the: p u r i f i e d a m i n o a c y l - s - R N A s y n t h e t a s e e n z y m e of i n t e r e s t . T h i s t e s t has not b e e n a p p l i e d . T h e s e s t u d i e s show that m i x e d s - R N A f r o m b r e w e r s ' y e a s t c o n t a i n s t h r e e m a j o r a n d f o u r o r f i v e m i n o r a c c e p t o r R N A ' s f o r g l y c i n e . T w o of the m i n o r a c c e p t o r R N A ' s f o r g l y c i n e a r e e v i d e n t f r o m F i g u r e 14. A n o t h e r one a p p e a r e d d u r i n g r e c h r o m a t o g r a p h y of the g l y c i n e - I p e a k (see F i g . 15). a n d i t w a s s u g g e s t e d that t h i s m i n o r p e a k w a s g l y c i n e -I s - R N A m i s s i n g i t s t e r m i n a l a d e n o s i n e . T w o m o r e m i n o r p e a k s of g l y c i n e a c c e p t o r a c t i v i t y a p p e a r e d d u r i n g r e c h r o m a t o g r a p h y of the g l y c i n e - I I p e a k (see F i g . 18). H o w e v e r , i t s h o u l d be p o i n t e d out that b o t h of t h e s e e x i s t by v i r t u e of s i n g l e e x p e r i m e n t a l p o i n t s . F o u r g l y c y l - s - R N A ' s h a v e b e e n i s o l a t e d r e c e n t l y f r o m b r e w e r s ' y e a s t by B e r g q u i s t (1966). S e q u e n c e s t u d i e s on t h e s e s - R N A ' s h a v e - 1 7 8 -s h o w n that o n l y s m a l l d i f f e r e n c e s o c c u r i n t h e i r b a s e c o m p o s i t i o n . S e v e r a l of t h e s e d i f f e r e n c e s i n v o l v e d the s u b s t i t u t i o n of u r i d y l i c , p s e u d o -u r i d y l i c o r r i b o s y l t h y m i d y l i c a c i d f o r e a c h o t h e r i n s m a l l g u a n y l i c a c i d -c o n t a i n i n g o l i g o n u c l e o t i d e s . A n o t h e r s i t e of h e t e r o g e n e i t y a p p e a r e d to be at the 5 ' - t e r m i n u s of the m o l e c u l e s . It i s r e m a r k a b l e that s u c h c l o s e l y r e l a t e d m o l e c u l e s c a n be r e s o l v e d i n the c h r o m a t o g r a p h i c s y s t e m s . T h e i r s e p a r a t i o n , p a r t i c u l a r l y w h e n the d i f f e r e n c e s i n v o l v e o n l y p y r i m i d i n e d e r i v a t i v e s ( r e c a l l the c h r o m a t o g r a p h y of the p y r i m i d i n e m o n o n u c l e o t i d e s ) , m a y be e x p l a i n e d by d i f f e r e n c e s i n t h e i r s e c o n d a r y s t r u c t u r e . T h e c o n d i t i o n s f o r f r a c t i o n a t i n g s - R N A o n B D E A E - c e l l u l o s e m u s t be d e t e r m i n e d e m p i r i c a l l y . H o w e v e r , the g e n e r a l s c h e m e d e s c r i b e d i n t h i s p a r t of the t h e s i s w h i c h e m p l o y s a n i n i t i a l c h r o m a t o g r a p h y at n e u t r a l p H i n the p r e s e n c e of 0. 01 M m a g n e s i u m i o n s f o l l o w e d by r e c h r o m a t o g r a p h y ' at a d i f f e r e n t p_H a n d i n the p r e s e n c e o r a b s e n c e of m a g n e s i u m i o n s ( E D T A ) s e e m s to be the b e s t a p p r o a c h . O n e p a r a m e t e r w h i c h has not b e e n i n v e s t i g a t e d i n d e t a i l i s the u s e of e l e v a t e d t e m p e r a t u r e o r t e m p e r a t u r e g r a d i e n t s . A p a r t i c u l a r l y u s e f u l f e a t u r e of B D E A E - c e l l u l o s e i s that the c h r o m a t o g r a p h y c a n be c a r r i e d out at l o w p H (down to p H 3. 5). T h u s , f o r a n a l y t i c a l p u r p o s e s , s - R N A c a n be c h a r g e d w i t h a p a r t i c u l a r l a b e l l e d a m i n o a c i d , t h e n c h r o m a t o g r a p h e d at p H 4 to 5, on B D E A E -- 1 7 9 -c e l l u l o s e . D e t e r m i n a t i o n of the r a d i o a c t i v i t y i n the c o l u m n e l u a t e w o u l d t h e n r e v e a l the p o s i t i o n of e l u t i o n of the a m i n o a c y l - s - R N A ( G i l l a m et a l . , 1967). I n the p a s t , t h i s t y p e of a n a l y s i s h a s b e e n l i m i t e d p r i n c i p a l l y to M A K c o l u m n c h r o m a t o g r a p h y ( S u e o k a a n d Y a m a n e , 1962). U n s u i t e d to t h i s type of a n a l y s i s o n B D E A E - c e l l u l o s e a r e the a r o m a t i c a m i n o a c y l -s - R N A ' s ( t r y p t o p h a n e , t y r o s i n e , a n d p h e n y l a l a n i n e ) . F o r e x a m p l e , t y r o s i n e a c c e p t o r R N A i s n o r m a l l y e l u t e d t o w a r d the end of the s a l t g r a d i e n t . H o w e v e r , D r . E . W i m m e r , w h i l e w o r k i n g i n the l a b o r a t o r y of D r . G . M . T e n e r , o b s e r v e d that w h e n t y r o s i n e w a s e s t e r i f i e d to i t s a c c e p t o r R N A a n d s u b s e q u e n t l y c h r o m a t o g r a p h e d o n B D E A E - c e l l u l o s e , s o m e a l c o h o l h a d to be i n c l u d e d i n the e l u t i n g s o l v e n t i n o r d e r to e l u t e the t y r o s y l - s - R N A . A f t e r h y d r o l y s i n g the e s t e r a n d r e c h r o m a t o g r a p h y , the t y r o s i n e a c c e p t o r R N A a g a i n e l u t e d f r o m the c o l u m n at the e n d of the s a l t g r a d i e n t . F r o m t h e s e o b s e r v a t i o n s a g e n e r a l m e t h o d f o r the i s o l a t i o n of p o p u l a t i o n s of s - R N A ' s s p e c i f i c f o r a g i v e n a m i n o a c i d h a s b e e n d e v e l o p e d . T h e p r i n c i p l e of the m e t h o d i n v o l v e s the a t t a c h m e n t of a ' l i p o p h i l i c h a n d l e ' to the d e s i r e d a m i n o a c y l - s - R N A . T h u s , m i x e d s - R N A i s c h a r g e d w i t h a p a r t i c u l a r a m i n o a c i d , t h e n r e a c t e d w i t h the p h e n o x y -a c e t y l e s t e r of N - h y d r o x y s u c c i n i m i d e . T h e r e s u l t i n g m i x t u r e of s - R N A ' s and N ( p h e n o x y a c e t y l ) - a m i n o a c y l - s - R N A i s a p p l i e d to a c o l u m n of B D E A E - c e l l u l o s e . T h e n o n p h e n o x y a c e t y l a t e d m a t e r i a l i s e l u t e d b y w a s h i n g the c o l u m n w i t h 0. 8 M s o d i u m c h l o r i d e . T h e p h e n o x y a c e t y l a t e d - 1 8 0 -m a t e r i a l i s t h e n e l u t e d by i n c l u d i n g s o m e a l c o h o l i n the e l u t i n g s o l v e n t . The c h r o m a t o g r a p h y i s c a r r i e d out at p H 4. 5 to s t a b i l i z e the e s t e r l i n k a g e b e t w e e n the a m i n o a c i d a n d i t s c o r r e s p o n d i n g s - R N A . T h e d e r i v a t i z e d a m i n o a c y l - s - R N A i s h y d r o l y z e d by i n c u b a t i n g the s o l u t i o n at p H 9. 0 f o r 2 h r at r o o m t e m p e r a t u r e . T h e r e g e n e r a t e d s - R N A i s t h e n r e c o v e r e d a n d c h r o m a t o g r a p h e d o n B D E A E - c e l l u l o s e u s i n g a l i n e a r g r a d i e n t of s o d i u m c h l o r i d e . A n o u t l i n e of the r e a c t i o n s i s s h o w n i n C h a r t III. P a r t I I - D of t h i s t h e s i s d e m o n s t r a t e s the f e a s i b i l i t y of the m e t h o d u s i n g g l y c i n e a c c e p t o r R N A as an e x a m p l e . T h e m a j o r d i s a d v a n t a g e to any c h e m i c a l p r o c e d u r e i n v o l v i n g a m i n o a c y l - s - R N A ' s l i e s i n the r e q u i r e m e n t f o r the a m i n o a c y l - s - R N A s y n t h e t a s e e n z y m e s w h i c h a r e r e q u i r e d i n q u a n t i t y . In a d d i t i o n , the s y n t h e t a s e s s h o u l d be f r e e f r o m n u c l e a s e s a n d p e p t i d a s e s . P a r t III of t h i s t h e s i s d e s c r i b e s the s o l u t i o n to t h i s p r o b l e m f o r m o s t of the a m i n o a c y l - s - R N A s y n t h e t a s e s . T h e u s e of the p h e n o x y a c e t y l e s t e r of N - h y d r o x y s u c c i n i m i d e f o r a c y l a t i n g the fif - a m i n o g r o u p of a m i n o a c y l - s - R N A ' s w a s p r e f e r r e d b e c a u s e of i t s g r e a t e r s e l e c t i v i t y c o m p a r e d to the a c y l c h l o r i d e s . G l y c i n e - I s - R N A , p r e v i o u s l y e n r i c h e d 6 f o l d by c h r o m a t o g r a p h y on 14 B D E A E - c e l l u l o s e ( P a r t I I - C , S e c t i o n 7 - G ) , w a s c h a r g e d w i t h C - g l y c i n e , t h e n p h e n o x y a c e t y l a t e d as s h o w n i n C h a r t III . W h e n the N - ( p h e n o x y a c e t y l ) -^ C - g l y c y l - s - R N A w a s c h r o m a t o g r a p h e d on B D E A E - c e l l u l o s e b y s t e p -181-C H A R T III S y n t h e s i s of N - ( P h e n o x y a c e t y l ) - a m i n o a c y l - s - R N A m i x e d s - R N A a m i n o a c y l a t i o n R - C H - C O O " ( e n z y m a t i c N H + s y n t h e s i s ) O R - C H - C - O - s - R N A + s - R N A N H ^ phe n o x y a c e t y l a t i on £ ^ C H 2 < ? - 0 - N > t o R - C H - C - O - s - R N A N H i + s - R N A o=c N C H 2 0 - 0 c h r o m a t o g r a p h y B D E A E - c e l l u l o s e 0. 8 M N a C l 1. 0 M N a C l + a l c o h o l s - R N A N - ( p h e n o x y a c e t y l ) - a m i n o a c y l - s- R N A - 1 8 2 -w i s e e l u t i o n , about 66% of the a p p l i e d m a t e r i a l w a s e l u t e d by 0. 8 M s o d i u m c h l o r i d e ( F i g . 19). T h e e t h a n o l - s a l t w a s h , w h i c h r e m o v e d the r e m a i n i n g 34% of the a p p l i e d m a t e r i a l c o n t a i n e d e s s e n t i a l l y a l l of the a p p l i e d r a d i o a c t i v i t y ( F r a c t i o n II , F i g . 19). T h u s i t w o u l d a p p e a r that the r e a c t i o n c o n d i t i o n s u s e d (10 m i n , p H 8. 0, 0 ° ; S e c t i o n 2 - C ) w e r e s u f f i c i e n t f o r c o m p l e t e a c y l a t i o n of the * 4 C - g l y c y l - s - R N A . T h e c o n d i t i o n s u s e d by de G r o o t et a l . , (1966), f o r the a c e t y l a t i o n of g l y c y l -and p h e n y l a l a n y l - s - R N A w i t h the a c e t y l e s t e r of N - h y d r o x y sue c i n i m i d e , i n v o l v e d a r e a c t i o n t i m e of 15 h r at pjH 5. 0 a n d at r o o m t e m p e r a t u r e . T h e d e r i v a t i z e d a m i n o a c y l - s - R N A , e l u t e d b y the e t h a n o l - s a l t w a s h ( P e a k II, F i g . 19), w a s c o m p l e t e l y s t r i p p e d of N - ( p h e n o x y a c e t y l ) -l 4 C - g l y c i n e i n t w o h r at p H 9 a n d at r o o m t e m p e r a t u r e . W h e n the s - R N A f r o m t h i s p e a k w a s r e c o v e r e d a n d a s s a y e d f o r g l y c i n e a c c e p t o r a c t i v i t y i t s h o w e d a 9 . 4 f o l d e n r i c h m e n t i n g l y c i n e a c c e p t o r a c t i v i t y c o m p a r e d to c r u d e s - R N A . T h i s r e p r e s e n t s o n l y a 3 . 4 f o l d e n r i c h m e n t o v e r the s t a r t i n g m a t e r i a l . The m a j o r r e a s o n f o r t h i s s m a l l i n c r e a s e i n p u r i t y w a s the f a c t that F r a c t i o n I ( e l u t e d by 0. 8 M s o d i u m c h l o r i d e ) w a s s t i l l 2. 6 f o l d e n r i c h e d i n g l y c i n e a c c e p t o r a c t i v i t y c o m p a r e d to c r u d e s - R N A . T h i s m e a n s e i t h e r that the s - R N A w a s i n c o m p l e t e l y c h a r g e d w i t h g l y c i n e , o r that c o n s i d e r a b l e h y d r o l y s i s h a d o c c u r r e d b e t w e e n the t i m e of p r e p a r a t i o n of the ^ 4 C - g l y c y l - s - R N A a n d the p h e n o x y a c e t y l a t i o n r e a c t i o n . T h e r e c o v e r y of r a d i o a c t i v i t y f o l l o w i n g p h e n o x y a c e t y l a t i o n w a s 68% ( S e c t i o n 2 - C ) . H o w e v e r , t h i s does not m e a n that the -183-r e m a i n d e r w a s l o s t d u r i n g the p h e n o x y a c e t y l a t i o n r e a c t i o n b e c a u s e the * 4 C - g l y c y l - s - R N A h a d b e e n s t o r e d as a pe l le t f o r about t w o w e e k s at -18° p r i o r to the d e r i v a t i z a t i o n . In f a c t , the r e c o v e r y of r a d i o a c t i v i t y f r o m the p h e n o x y a c e t y l a t i o n r e a c t i o n w h e n the g l y c y l - s ^ R N A w a s d e r i v a t i z e d i m m e d i a t e l y w a s 84% ( S e c t i o n 3). T h e f o l d e n r i c h m e n t s q u o t e d a b o v e w e r e c a l c u l a t e d i n c o m p a r i s o n to the g l y c i n e a c c e p t o r a c t i v i t y of c r u d e ( o r w h o l e ) s - R N A . It m u s t be e m p h a s i z e d t h e r e f o r e , that i t w a s the g l y c i n e - I s - R N A f r a c t i o n that w a s u s e d i n the e x p e r i m e n t s d i s c u s s e d a b o v e a n d that g l y c i n e - I r e p r e s e n t s r o u g h l y 6 0 % of the t o t a l g l y c i n e a c c e p t o r a c t i v i t y i n w h o l e s - R N A . W h e n p r e p a r i n g a m i n o a c y l - s - R N A ' s i t i s i m p o r t a n t to d e t e r m i n e b e f o r e h a n d the c o n d i t i o n s f o r m a x i m u m c h a r g i n g of the s - R N A w i t h any g i v e n a m i n o a c i d . A n u m b e r of the c o - f a c t o r s s u c h as p o t a s s i u m c h l o r i d e , b u f f e r , m e r c a p t o e t h a n o l ( i f r e q u i r e d b y the e n z y m e ) , a n d a m i n o a c i d , c a n be p r e s e n t i n a r e a s o n a b l e e x c e s s w i t h o u t a f f e c t i n g t o t a l i n c o r p o r a t i o n . T h e a d d i t i o n of A T P , i n e x c e s s of that r e q u i r e d f o r a c t i v a t i o n of the a m i n o a c i d a n d any r e p a i r p r o c e s s e s , i s i m p o r t a n t by v i r t u e of i t s a b i l i t y t o c h e l a t e m a g n e s i u m . E D T A w a s r o u t i n e l y i n c l u d e d i n the i n c u b a t i o n m i x t u r e s at v e r y l o w c o n c e n t r a t i o n t o r e m o v e h e a v y m e t a l s . A g a i n , E D T A c a n n o t be a d d e d i n d i s c r i m i n a t e l y b e c a u s e of i t s a b i l i t y to c h e l a t e m a g n e s i u m . W h e n e x p e r i m e n t s w e r e c a r r i e d out to d e t e m i n e the a m o u n t of m a g n e s i u m r e q u i r e d to a c h i e v e m a x i m u m - 1 8 4 -s y n t h e s i s of g l y c y l - s - R N A , a n u n e x p e c t e d r e s u l t w a s o b t a i n e d . It i s c l e a r f r o m the r e s u l t s s h o w n i n F i g u r e s 20 a n d 21 that t h e r e i s a n o p t i m u m m a g n e s i u m r e q u i r e m e n t f o r the f o r m a t i o n of g l y c y l - s - R N A . C o n c e n t r a t i o n s above o r b e l o w the o p t i m u m r e s u l t i n a r a p i d l y d e c r e a s i n g r e a c t i o n v e l o c i t y . H e l e (1964) has d e s c r i b e d s i m i l a r r e s u l t s f o r the a f fect of m a g n e s i u m i o n s o n the a m i n o a c i d - d e p e n d e n t p y r o p h o s p h a t e e x c h a n g e r e a c t i o n u s i n g r a t l i v e r as a s o u r c e of e n z y m e s a n d r a t l i v e r o r y e a s t as a s o u r c e of s - R N A . T h i s i n v e s t i g a t o r e x p l a i n e d h e r r e s u l t s i n t e r m s of a n " a l l o s t e r i c c o n t r o l m e c h a n i s m " i n w h i c h the e n z y m e s c a t a l y z i n g the A T P - p y r o p h o s p h a t e e x c h a n g e r e a c t i o n a g g r e g a t e i n the p r e s e n c e of m a g n e s i u m a n d R N A . F r a s e r (1963) has a l s o r e p o r t e d s i m i l a r o b s e r v a t i o n s o n the e f fect of m a g n e s i u m i o n s on b o t h the 14 A T P - p y r o p h o s p h a t e e x c h a n g e r e a c t i o n a n d the i n c o r p o r a t i o n of C -g l y c i n e i n t o r a t l i v e r s - R N A . I n a d d i t i o n , o b s e r v a t i o n s s i m i l a r t o t h o s e r e p o r t e d h e r e h a v e b e e n m a d e f o r y e a s t p h e n y l a l a n y l - s - R N A ( R o y a n d T e n e r , 1967). T h u s i t i s p a r t i c u l a r l y i m p o r t a n t to o b s e r v e the r e s t r i c t i o n s i m p o s e d o n t h i s r e a c t i o n by m a g n e s i u m s i n c e i t i s d e s i r a b l e to a c h i e v e m a x i m u m c h a r g i n g of s - R N A i n the s h o r t e s t p e r i o d of t i m e to a v o i d the a c t i o n of i n t e r f e r i n g e n z y m e s . S t a r t i n g f r o m m i x e d (or w h o l e ) s - R N A i t w a s p o s s i b l e t o o b t a i n g l y c i n e - I s - R N A i n h i g h p u r i t y . T h e e x p e r i m e n t a l p r o c e d u r e s e m p l o y e d a r e s u m m a r i z e d i n C h a r t I V . 185-C H A R T I V I s o l a t i o n of G l y c i n e - I s - R N A f r o m W h o l e s - R N A ( P a r t I I - D , S e c t i o n 3 - B ) w h o l e s - R N A S t e p 1 S t e p w i s e e l u t i o n f r o m B D E A E - c e l l u l o s e 0. 9 M N a C l e l u a t e 1. 0 M N a C l , a l c o h o l e l u a t e s - R N A p h e n y l a l a n i n e s - R N A + o t h e r ' R N A ' S t e p 2 1 1. P h e n o x y a c e t y l a t i o n 2. S t e p w i s e e l u t i o n f r o m B D E A E - c e l l u l o s e 0. 9 M N a C l e l u a t e s - R N A S t e p 3 S t e p 4 1. 0 M N a C l , a l c o h o l e l u a t e ' p h e n o x y a c e t y l a t e d m a t e r i a l ' A m i n o a c y l a t i o n ( e n z y m a t i c ) a m i n o a c y l - s - R N A + s - R N A P h e n o x y a c e t y l a t i o n N - ( p h e n o x y a c e t y l ) - a m i n o a c y l - s - R N A + s - R N A S t e p 5 ( F i g . 22) •< S t e p w i s e e l u t i o n f r o m B D E A E - c e l l u l o s e 0. 8 M N a C l e l u a t e 1. 0 M N a C l , a l c o h o l e l u a t e s - R N A p h e n o x y a c e t y l a t e d a m i n o a c y l - s - R N A p H 9. 0, 2 h r , r o o m t e m p e r a t u r e R e g e n e r a t e d s - R N A S t e p 6 ( F i g . 23) G r a d i e n t e l u t i o n f r o m B D E A E - c e l l u l o s e P e a k I P e a k II P e a k III ( g l y c i n e I) ( g l y c i n e - I I ? ) ? - 1 8 6 -T h e f i r s t s t e p r e m o v e d the p h e n y l a l a n i n e a c c e p t o r R N A a n d o t h e r R N A ' s (non s - R N A ) w h i c h w e r e o n l y e l u t e d w h e n e t h a n o l w a s i n c l u d e d i i i the e l u t i n g s o l v e n t . T h i s w a s e a s i l y a n d r a p i d l y a c c o m p l i s h e d b y s t e p w i s e e l u t i o n of w h o l e s - R N A f r o m B D E A E - c e l l u l o s e (see S e c t i o n 3 - B , S t e p 1). S t e p 2 r e m o v e d a n y m a t e r i a l w h i c h w a s r e a d i l y p h e n o x y a c e t y l a t e d by the r e a g e n t p r i o r to c h a r g i n g the s - R N A w i t h the d e s i r e d a m i n o a c i d . F o r e x a m p l e , the a m i n o a c y l a d e n y l a t e n u c l e o t i d e s d e s c r i b e d b y H a l l (1964) m i g h t be r e m o v e d i n t h i s s t e p . W h e n the p h e n y l a l a n i n e a c c e p t o r R N A i s not d e s i r e d , Steps 1 a n d 2 c a n be c o m b i n e d . T h e s - R N A e l u t e d f r o m B D E A E - c e l l u l o s e b y the 0. 9 M s o d i u m c h l o r i d e w a s h (Step 2, S e c t i o n 3 - B ) i s now f r e e of p h e n y l a l a n i n e a c c e p t o r a c t i v i t y , o t h e r s t r o n g l y h e l d n o n - a c c e p t o r R N A a n d any n u c l e o t i d e m a t e r i a l w h i c h i s r e a d i l y p h e n o x y a c e t y l a t e d u n d e r the c o n d i t i o n s d e s c r i b e d i n the e x p e r i m e n t a l . T h e m a t e r i a l o b t a i n e d f r o m S t e p 2 w a s c h a r g e d w i t h g l y c i n e (Step 3) u s i n g a p u r i f i e d e n z y m e ( S e c t i o n 3 - A ) , t h e n p h e n o x y a c e t y l a t e d (Step 4 ) . T h e r e c o v e r y of r a d i o a c t i v i t y f o l l o w i n g a c y l a t i o n w a s 8 4 % . It i s r e c o m m e n d e d that the a m o u n t of p h e n o x y a c e t y l a t i n g a g e n t u s e d i n S t e p 4 ( S e c t i o n 3 - B ) be r e d u c e d f r o m 1 g to 100 m g , s i n c e the a c y l a t i n g agent i s o n l y s p a r i n g l y s o l u b l e i n an a q u e o u s m e d i u m a n d v a s t e x c e s s e s t e n d to o c c l u d e the s - R N A . The N - ( p h e n y o x y a c e t y l ) ^ C - g l y c y l - s - R N A w a s i s o l a t e d by c h r o m a t o g r a p h y - 1 8 7 -on B D E A E - c e l l u l o s e (Step 5, F i g . 22). A l l of the r a d i o a c t i v i t y w a s r e c o v e r e d i n the e l u a t e of the e t h a n o l - s a l t w a s h . T h e s - R N A i n p e a k I ( F i g . 22) w a s c o m p l e t e l y s t r i p p e d of p r e c i p i t a b l e r a d i o a c t i v i t y i n t w o h o u r s at jaH 9 a n d at r o o m t e m p e r a t u r e . C h r o m a t o g r a p h y of the r e g e n e r a t e d s - R N A on B D E A E - c e l l u l o s e u s i n g a n i n c r e a s i n g l i n e a r g r a d i e n t of s o d i u m c h l o r i d e a n d 0 . 0 1 M m a g n e s i u m c h l o r i d e r e s u l t e d i n the e l u t i o n of a m a j o r ( P e a k I) a n d a m i n o r p e a k ( P e a k II) of s - R N A , ( F i g . 23, S t e p 6). A b o u t 4 6 % of the r e g e n e r a t e d s - R N A w a s e l u t e d b y the e t h a n o l - s a l t w a s h ( P e a k III) . T h i s r e l a t i v e l y h i g h p e r c e n t a g e of O . D . u n i t s e l u t e d by the e t h a n o l - s a l t w a s h c a n p r o b a b l y be r e d u c e d by u s i n g l e s s p h e n o x y a c e t y l a t i n g agent t h a n w a s u s e d i n t h i s e x p e r i m e n t (as, r e c o m m e n d e d a b o v e ) a n d b y c a r r y i n g out the p h e n o x y a c e t y l a t i o n r e a c t i o n i n t r i e t h a n o l a m i n e b u f f e r . P e a k I, i n F i g u r e 23 r e p r e s e n t s 2. 8 6 % of the O . D . u n i t s o b t a i n e d f r o m S t e p 2 ( p r i o r to a m i n o a c y l a t i o n ) , w h i l e P e a k II ( f r a c t i o n s 78 to 90) r e p r e s e n t s 0 . 7 6 % t o g i v e a c o m b i n e d t o t a l of 3. 6 2 % . T h e t h e o r e t i c a l r e c o v e r y b a s e d on the a m o u n t of g l y c i n e - I p l u s g l y c i n e - I I s - R N A i n w h o l e s - R N A w a s 4 . 7 5 % ( w i t h g l y c i n e - I s - R N A c o n t r i b u t i n g 2. 8 5 % , a n d g l y c i n e -II c o n t r i b u t i n g 1. 9 0 % of the O . D . u n i t s ) . T h u s the r e c o v e r y of g l y c i n e a c c e p t o r R N A f r o m S t e p 6 w a s 7 6% of the g l y c i n e a c c e p t o r R N A p r e s e n t i n w h o l e s - R N A . - 1 8 8 -H o w e v e r i t m u s t be e m p h a s i z e d that the ^ C - g l y c y l - s - R N A w a s p r e p a r e d u s i n g a p u r i f i e d e n z y m e . T h e p u r i f i c a t i o n of the g l y c y l - s - R N A s y n t h e t a s e on h y d r o x y a p a t i t e ( d e s c r i b e d i n P a r t 111, S e c t i o n 6 - C ) c l e a r l y shows the p r e s e n c e of t w o g l y c y l - s - R N A s y n t h e t a s e a c t i v i t i e s . It i s p o s s i b l e t h a t the s y n t h e t a s e u s e d i n t h i s e x p e r i m e n t w i l l o n l y c h a r g e the g l y c i n e - I a c c e p t o r R N A a n d e i t h e r the g l y c i n e - I I - 0 6 o r g l y c i n e - I I - f$ a c c e p t o r R N A ' s (see P a r t I I - C , S e c t i o n 7 - G a n d 7 J ) . T h e v a l i d i t y of P e a k I i n F i g u r e 23, b e i n g the g l y c i n e - I f r a c t i o n has b e e n e s t a b l i s h e d by r e c h r o m a t o g r a p h i n g the s - R N A f r o m P e a k I u n d e r c o n d i t i o n s w h i c h a r e k n o w n to s p l i t the g l y c i n e - I I a c c e p t o r a c t i v i t y i n t o g l y c i n e - I I - O d a n d g l y c i n e - I I - s - R N A p e a k s (see P a r t I I -C , S e c t i o n 7 - J ) . O n l y one p e a k of O . D . w a s e l u t e d a n d t h i s c o n t a i n e d a l l the g l y c i n e a c c e p t o r a c t i v i t y . T h e O . D . u n i t s i n P e a k s I a n d II ( F i g u r e 23) a r e i n the c o r r e c t r a t i o i f P e a k I i s g l y c i n e - I a c c e p t o r R N A and P e a k II i s e i t h e r g l y c i n e -II-OC o r g l y c i n e - I I - ^ $ a c c e p t o r R N A ' s . T h i s p o s s i b i l i t y i s b e i n g e x p l o r e d by c h a r g i n g w h o l e s - R N A w i t h ^ C - g l y c i n e u s i n g one o r the o t h e r g l y c y l - s - R N A s y n t h e t a s e s , t h e n a n a l y z i n g the p r o d u c t s o n B D E A E - c e l l u l o s e u s i n g a l i n e a r g r a d i e n t of i n c r e a s i n g s o d i u m c h l o r i d e to d e v e l o p the c o l u m n ( R . W a r r i n g t o n , e x p e r i m e n t s i n p r o g r e s s ) . T h e p r e s e n c e of m o r e t h a n one a m i n o a c y l - s - R N A s y n t h e t a s e e n z y m e f o r a g i v e n a m i n o a c i d i s not n e w . B a r n e t t a n d B r o w n (1967) -189-have shown that mitochondrial preparations f r o m N. c r a s s a have their own seemingly unique set of s-RNA's and also that N. c r a s s a contains two synthetase enzymes for both phenylalanine and aspartic acid. Alternative explanations for the results obtained i n Figure 23 have not been excluded. It i s possible, for example, that either the glycine-II- OL or the glycine-II-16 acceptor RNA's contains a group which i s p a r t i c u l a r l y reactive with the acylating agent. In this case that p a r t i c u l a r acceptor would be removed by the 'sham' phenoxy-acetylation step (Step 2). This p o s s i b i l i t y could be tested by f i r s t c a r r y i n g out the 'sham' phenoxyacetylation on mixed s-RNA followed by chromatography on B D E A E - c e l l u l o s e by the stepwise procedure. The s-RNA eluted i n the 0. 9 M sodium chloride eluate could then be 14 charged with C-glycine using a crude enzyme preparation and the product subsequently chromatographed on B D E A E - c e l l u l o s e by gradient elution. Determination of the radioactivity i n the column eluate would reveal a change i n the r a t i o of the two peaks corresponding to glycine acceptor RNA. -190-B I B L I O G R A P H Y Albertsson, P. A. 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PART III PREPARATION AND PARTIAL PURIFICATION OF AMINOACYL-s-RNA SYNTHETASES FROM BAKERS' YEAST - 1 9 6 -T A B L E O F C O N T E N T S PAGE NUMBER LIST OF FIGURES 19 9 LIST OF TABLES AND CHARTS 200 INTRODUCTION 201 EXPERIMENTAL 205 Abbreviations Used 205 Materials 205 Methods 206 1. Preparation of Aminoacyl-s-RNA Synthetases from Yeast: Method 1 207 2. Preparation of Aminoacyl-s-RNA Synthetases from Yeast: Method 2 208 A. Assay Procedure 208 B. Lysis of Yeast Cells with Toluene at 37° 209 C. Ammonium Sulfate Fractionation of the Cel l -Free Extract 210 D. Preparation of Aminoacyl-s-RNA Synthetase Enzymes Used in Part II-D, Section 2-B 212 E. Chromatography of Fraction B on Hydroxy-apatite 213 F. Radiochromatographic Analysis of the Ammonium Sulfate Eluate 215 3. Chromatography of Yeast Cel l-Free Extract on Hydroxy apatite by Stepwise Elution 216 A. Preparation of the Yeast Cell-Free Extract 216 - 1 9 7 -P A G E N U M B E R B . C h r o m a t o g r a p h y 1 218 C . C h r o m a t o g r a p h y 2 219 D . C h r o m a t o g r a p h y 3 219 E f f e c t of A m m o n i u m S u l f a t e a n d D i - i s opr o p y l -f l u o r o p h o s p h a t e on the A m i n o a c y l - s - R N A S y n t h e t a s e A c t i v i t y 220 A . E f f e c t of A m m o n i u m S u l f a t e 220 B . E f f e c t of D i - i s o p r o p y l f l u o r o p h o s p h a t e 221 P r e p a r a t i o n a n d P a r t i a l P u r i f i c a t i o n of Y e a s t A m i n o a c y l - s - R N A S y n t h e t a s e E n z y m e s : M e t h o d 3 221 A . P r e p a r a t i o n of the C e l l - F r e e E x t r a c t 221 B . A m m o n i u m S u l f a t e F r a c t i o n a t i o n of the C e l l - F r e e E x t r a c t 221 C . D e t e c t i o n of A m i n o a c y l - s - R N A S y n t h e t a s e s i n the A m m o n i u m S u l f a t e F r a c t i o n s 223 D . C h r o m a t o g r a p h y of A m m o n i u m S u l f a t e F r a c t i o n III on H y d r o x y a p a t i t e 224 E . D e t e c t i o n of A m i n o a c y l - s - R N A S y n t h e t a s e s i n the H y d r o x y a p a t i t e C o l u m n E l u a t e 226 F . D e t e r m i n a t i o n of A m i n o a c y l - s - R N A S y n t h e t a s e A c t i v i t i e s 230 P r e p a r a t i o n a n d P a r t i a l P u r i f i c a t i o n of A m i n o a c y l -s - R N A S y n t h e t a s e s f r o m Y e a s t : M e t h o d 3 ( M o d i f i e d ) 230 A . P r e p a r a t i o n of the C e l l - F r e e P r o t e i n E x t r a c t 230 - 1 9 8 -P A G E N U M B E R B . P a r t i a l P u r i f i c a t i o n of the A m i n o a c y l -s - R N A S y n t h e t a s e s 232 C . D e t e c t i o n of A m i n o a c y l - s - R N A S y n t h e t a s e i n the H y d r o x y a p a t i t e C o l u m n E l u a t e 233 D I S C U S S I O N 238 B I B L I O G R A P H Y 254 - 1 9 9 -F I G U R E 1 2 3 4 5 L I S T O F F I G U R E S P A G E N U M B E R E l u t i o n P r o f i l e f r o m the C h r o m a t o g r a p h y of F r a c t i o n B o n H y d r o x y a p a t i t e 214 R e s u l t s of the R a d i o c h r o m a t o g r a p h y A n a l y s i s of the A m m o n i u m S u l f a t e E l u a t e 217 E l u t i o n P r o f i l e f r o m the C h r o m a t o g r a p h y of F r a c t i o n III o n H y d r o x y a p a t i t e 227 C h r o m a t o g r a p h y of the 0 - 8 0 % P r o t e i n F r a c t i o n o n H y d r o x y a p a t i t e 234 E l u t i o n P r o f i l e s of A m i n o a c y l - s - R N A S y n t h e t a s e s f r o m H y d r o x y a p a t i t e 236 6 E l u t i o n P r of i l e s of A m i n o a c y l - s - R N A S y n t h e t a s e s f r o m H y d r o x y a p a t i t e 237 - 2 0 0 -L I S T O F T A B L E S & C H A R T S T A B L E P A G E N U M B E R I P r o p e r t i e s of A m m o n i u m S u l f a t e F r a c t i o n s A - D 211 II G l y c y l - s - R N A S y n t h e t a s e A c t i v i t y i n F r a c t i o n s A a n d B 212 III P r o t o c o l f o r A m m o n i u m S u l f a t e F r a c t i o n a t i o n 222 I V P r o p e r t i e s of A m m o n i u m S u l f a t e F r a c t i o n s II -I V 224 V A m i n o a c y l - s - R N A S y n t h e t a s e A c t i v i t i e s i n the A m m o n i u m S u l f a t e F r a c t i o n s 225 V I A m i n o a c y l - s - R N A S y n t h e t a s e s P r e s e n t i n the A m m o n i u m S u l f a t e E l u a t e s 229 V I I S p e c i f i c A c t i v i t i e s of S e v e r a l A m i n o a c y l - s - R N A S y n t h e t a s e s 231 V I I I A m i n o a c y l - s - R N A S y n t h e t a s e s P r e s e n t i n the W h o l e - p r o t e i n E x t r a c t 235 C H A R T I S u m m a r y of E x p e r i m e n t a l ( M e t h o d 3) 245 II S u m m a r y of E x p e r i m e n t a l ( M e t h o d 3, m o d i f i e d ) 250 - 2 0 1 -I N T R O D U C T I O N T h e a m i n o a c y l - s - R N A s y n t h e t a s e s o c c u p y a s i g n i f i c a n t p o s i t i o n i n the p a t h w a y l e a d i n g f r o m f r e e a m i n o a c i d s to- p r o t e i n s by v i r t u e of t h e i r a b i l i t y to r e c o g n i z e b o t h a p a r t i c u l a r a m i n o a c i d a n d the c o r r e s p o n -d i n g s - R N A a c c e p t o r . T h u s the a m i n o a c y l - s - R N A s y n t h e t a s e s a r e p r o t e i n s w h i c h c a n d i s c r i m i n a t e b e t w e e n s t r u c t u r a l l y s i m i l a r n u c l e i c a c i d m o l e c u l e s . T h e m o l e c u l a r b a s i s f o r t h i s s t r i c t s p e c i f i c i t y r e m a i n s one of the c e n t r a l p r o b l e m s i n u n d e r s t a n d i n g the t r a n s l a t i o n of the g e n e t i c m e s s a g e . It i s o b v i o u s that a m i s t a k e at the a m i n o a c y l - s - R N A s y n t h e t a s e l e v e l w o u l d l e a d to a n e r r o r i n the t r a n s l a t i o n of the g e n e t i c m e s s a g e d u r i n g r i b o s o m a l p r o t e i n s y n t h e s i s . T h u s the s t u d y of the r e l a t i o n s h i p b e t w e e n s - R N A a n d the a m i n o a c y l - s - R N A s y n t h e t a s e s has b e c o m e the s u b j e c t of c o n s i d e r a b l e i n t e r e s t . B e s i d e s t h e i r i n t r i n s i c p r o p e r t i e s a n d the e l u c i d a t i o n of the m o l e c u l a r b a s i s of t h e i r u n i q u e i n t e r a c t i o n s w i t h n u c l e i c a c i d s , i n t e r e s t i n the a m i n o a c y l - s - R N A s y n t h e t a s e s d e r i v e s f r o m t h e i r i n d i s p e n s a b l e r o l e i n the d e v e l o p m e n t of c h e m i c a l m e t h o d s f o r p u r i f y i n g a m i n o a c i d -s p e c i f i c s - R N A ' s ( d i s c u s s e d i n P a r t I I - D ) . M e t h o d s f o r p r e p a r i n g s p e c i f i c s - R N A ' s w h i c h i n v o l v e the p r e f o r m a t i o n of the a m i n o a c y l - s - R N A w i l l o b v i o u s l y r e q u i r e r e l a t i v e l y l a r g e a m o u n t s of the s y n t h e t a s e e n z y m e s . It i s a l s o i m p o r t a n t to h a v e s y n t h e t a s e e n z y m e s f r e e f r o m c o n t a m i n a t i n g e n z y m e s s u c h a s , r i b o n u c l e a s e s , d i e s t e r a s e s a n d p e p t i d a s e s . - 2 0 2 -M c C o r q u o d a l e (1964), d e s c r i b e d a s i m p l e p r o c e d u r e f o r the p r e p a r a t i o n a n d p a r t i a l p u r i f i c a t i o n of a m i n o a c y l - s - R N A s y n t h e t a s e s f r o m E . c o l i . H i s p r o c e d u r e i n v o l v e d g r i n d i n g , the c e l l s w i t h a l u m i n a f o l l o w e d by u l t r a c e n t r i f u g a t i o n a n d c h r o m a t o g r a p h y on t w o c o l u m n s . A n o t h e r m e t h o d f o r the p r e p a r a t i o n . a n d p a r t i a l p u r i f i c a t i o n of the a m i n o -a c y l - s - R N A s y n t h e t a s e s f r o m ; E . c o l i i s d e s c r i b e d . i n . t h e t r e a t i s e of C a n t o n i a n d D a v i e s . (1966). T h i s t r e a t i s e a l s o d e s c r i b e s p r o c e d u r e s f o r t h e - p u r i f i c a t i o n of t y r o s y l - , i s o l e u c y l - , g l u t a m y l - , g l u t a m i n y l - , l y s y l -and' p r o l y l - s - R N A s y n t h e t a s e s f r o m E . c o l i . A l s o c o n t a i n e d i n . t h e t e x t a r e - p r o c e d u r e s f o r the p u r i f i c a t i o n of s e r y l - , p h e n y l a l a n y l - , a r g i n y l -a n d . l e u c y l - s - R N A s y n t h e t a s e s f r o m y e a s t . , In a d d i t i o n to E . c o l i , a n d y e a s t , s o m e of the a m i n o a c y l - s - R N A s y n t h e t a s e s f r o m a v a r i e t y of o t h e r o r g a n i s m s h a v e a l s o b e e n s t u d i e d , e . g . r a t l i v e r ( B u b l i t z , 1966a), c h i c k e m b r y o ( B u b l i t z , . 1966b), c a l f l i v e r a n d s p l e e n ( W e v e r s , ' et a l . , 1966), bee f p a n c r e a s ( L e m a i r e et a l . , 1967), N . c r a s s a ( B a r n e t t a n d E p l e r , 1966), S a l m o n e l l a , t y p h i m u r i u m ( R o t h and A m e s , 1966), w h e a t g e r m a n d p e a s e e d ( M o u s t a f a , . 1966), and t o b a c c o l e a v e s ( A n d e r s o n a n d R o w a n , 1966). The r e a d y a v a i l a b i l i t y of y e a s t i s a d e s i r a b l e f e a t u r e of t h i s o r g a n i s m but the d i f f i c u l t y e n c o u n t e r e d i n d i s r u p t i n g y e a s t c e l l s , c o m p a r e d w i t h t h o s e of b a c t e r i a l o r a n i m a l o r i g i n , i s a m a j o r d i s -advantage f o r the p r e p a r a t i o n of l a r g e q u a n t i t i e s of y e a s t e n z y m e s . T h e - 2 0 3 -u s u a l m e t h o d e m p l o y e d f o r - d i s r u p t i o n of y e a s t c e l l s c o n s i s t s of g r i n d i n g the c e l l s w i t h g l a s s b e a d s at h i g h s p e e d i n a; h o m o g e n i z e r . M a k m a n a n d C a n t o n i (1965) h a v e u s e d t h i s m e t h o d - f o r d i s r u p t i n g 100 g b a t c h e s of y e a s t c e l l s . I n t h i s l a b o r a t o r y , g r i n d i n g : y e a s t c e l l s w i t h g l a s s b e a d s p r o v i d e d s u f f i c i e n t q u a n t i t i e s of y e a s t c e l l - f r e e e x t r a c t f o r a s s a y i n g s - R N A . How-e v e r the p r e p a r a t i o n of a m i n o a c y l - s - R N A s y n t h e t a s e s by t h i s m e t h o d , i n a m o u n t s s u f f i c i e n t to' c h a r g e g r a m q u a n t i t i e s of s - R N A , w a s t e d i o u s . In a s e a r c h . f o r a l t e r n a t i v e p r o c e d u r e s , the u s e of t o l u e n e to l y s e y e a s t c e l l s w a s i n v e s t i g a t e d . K u n i t z (1952),- (see a l s o ' H e p p e l , 1955) h a d r e p o r t e d that t o l u e n e e f f e c t i v e l y l y s e d y e a s t c e l l s by w a r m i n g to 38 to 4 0 ° t h e n l e a v i n g the m i x t u r e at 20 to 2 5 ° f o r 3 h r . A m o d i f i c a t i o n of t h i s m e t h o d w a s e m p l o y e d s u c c e s s f u l l y f o r the p u r i f i c a t i o n of y e a s t h i s t i d y l - s - R N A s y n t h e t a s e ( v o n T i g e r s t r o m a n d T e n e r , 1967). H o w e v e r the a m i n o a c y l - s - R N A s y n t h e t a s e a c t i v i t i e s f o r a m i n o a c i d s o t h e r t h a n h i s t i d i n e w e r e e i t h e r absent , , o r p r e s e n t i n v e r y , l o w a m o u n t s i n the c e l l -f r e e e x t r a c t . P r o c e d u r e s d e s c r i b e d : i n t h i s t h e s i s h a v e s i g n i f i c a n t l y o v e r c o m e t h i s l o s s of a c t i v i t y by. c o n t r o l l i n g ; the p H of the y e a s t s u s p e n s i o n d u r i n g l y s i s w i t h t o l u e n e i n the p r e s e n c e of b u f f e r . L a z a r u s et al.- (1966) i n a. r e p o r t o n the p u r i f i c a t i o n of y e a s t h e x o k i n a s e d e s c r i b e d the l y s i s of y e a s t c e l l s i n a t o l u e n e - d r y i c e m i x t u r e . T h i s p r o c e d u r e h a s b e e n s u c c e s s f u l l y e m p l o y e d i n the p r e s e n t - 2 0 4 -s t u d i e s , f o r the p r e p a r a t i o n of l a r g e q u a n t i t i e s of y e a s t c e l l - f r e e e x t r a c t s . A p a r t i a l p u r i f i c a t i o n of the a m i n o a c y l - s - R N A s y n t h e t a s e s by c h r o m a t o -g r a p h y on h y d r o x y a p a t i t e i s a l s o r e p o r t e d . - 2 0 5 -E X P E R I M E N T A L I. I. .1. M IM ... •• • M I II II - • •" ' — ^ *•'•*" A B B R E V I A T I O N S U S E D A T P : a d e n o s i n e - 5 ' - t r i p h o s p h a t e . C T P : c y t i d i n e - 5 ' - t r i p h o s p h a t e . E D T A : e t h y l e n e d i a m i n e t e t r a a c e t i c a c i d . P P O : 2, 5 - d i p h e n y l o x a z o l e . D i m e t h y l P O P Q P : 1, 4 - b i s - 2 - ( 4 - m e t h y l - 5 - p h e n y l o x a z o l y l ) -b e n z e n e . D F P : d i - i s o p r o p y l f l u o r o p h o s p h a t e . T h e s t a n d a r d a b b r e v i a t i o n s w e r e u s e d f o r the a m i n o a c i d s . M A T E R I A L S B a k e r s ' y e a s t w a s o b t a i n e d l o c a l l y f r o m S t a n d a r d B r a n d s L t d . Y e a s t e x t r a c t w a s " B a c t o - Y e a s t E x t r a c t " o b t a i n e d . f r o m D i f c o L a b o r a t o r i e s , D e t r o i t , M i c h i g a n . A T P a n d C T P w e r e o b t a i n e d as the d i s o d i u m s a l t s f r o m P - L B i o c h e m i c a l s I n c . , M i l w a u k e e , . W i s c o n s i n . H y d r o x y a p a t i t e ( B i o - G e l H T P ) w a s p u r c h a s e d as the d r y p o w d e r f r o m B i o - R a d L a b o r a t o r i e s , R i c h m o n d , C a l i f o r n i a . T h e D i a f l o a p p a r a t u s w a s p u r c h a s e d f r o m A m i c o n C o r p o r a t i o n , C a m b r i d g e , ! M a s s . - 2 0 6 -S u p e r b r i t e g l a s s b e a d s , T y p e 110-5005, w e r e p u r c h a s e d f r o m M i n n e s o t a M i n i n g a n d M a n u f a c t u r i n g C o . 14 T h e - C - a m i n o a c i d s w e r e p u r c h a s e d f r o m N e w E n g l a n d N u c l e a r C o r p . , B o s t o n , M a s s a c h u s e t t s . T h e ^ C - a m i n o a c i d m i x t u r e w a s a u n i f o r m l y l a b e l l e d A l g a l p r o t e i n h y d r o l y s a t e p u r c h a s e d f r o m N e w E n g l a n d N u c l e a r C o r p o r a t i o n , B o s t o n , M a s s a c h u s e t t s . P P O ( S c i n t i l l a t i o n G r a d e ) w a s o b t a i n e d f r o m K e n t C h e m i c a l s L t d . , V a n c o u v e r , C a n a d a . D i m e t h y l P O P O P ( S c i n t i l l a t i o n G r a d e ) w a s o b t a i n e d f r o m the P a c k a r d I n s t r u m e n t C o m p a n y , . I n c . , L a G r a n g e , . I l l i n o i s . O p t i c a l d e n s i t y m e a s u r e m e n t s w e r e m a d e u s i n g a C a r y M o d e l 11 s p e c t r o p h o t o m e t e r . One o p t i c a l d e n s i t y (O. D . ) u n i t i s d e f i n e d . a s that a m o u n t of m a t e r i a l p e r m l w h i c h , i n a 1 - c m l i g h t p a t h at the s p e c i f i e d w a v e l e n g t h , g i v e s a s p e c t r o p h o t o m e t r i c r e a d i n g of one. M E T H O D S A l l b u f f e r s u s e d i n t h i s s tudy w e r e p a s s e d t h r o u g h t a T y p e H A -0. 45>J- M i l l i p o r e f i l t e r p r i o r to u s e . T h i s w a s p a r t i c u l a r l y i m p o r t a n t w h e n the b u f f e r s c o n t a i n e d g l y c e r o l . F l o w r a t e s d u r i n g c h r o m a t o g r a p h y on h y d r o x y a p a t i t e w e r e m a i n t a i n e d w i t h the a i d of a n A c c u - F l o P u m p ( B e c k m a n I n s t r u m e n t s ) . - 2 0 7 -1. P r e p a r a t i o n of A m i n o a c y l - s - R N A S y n t h e t a s e s f r o m Y e a s t :  M e t h o d 1 C r u m b l e d y e a s t c a k e (9 g) w a s m i x e d i n a 500 m l E r l e n m e y e r c o n t a i n i n g 200 m l of d i s t i l l e d w a t e r , 16 g of s u c r o s e , 4 g of y e a s t e x t r a c t , 3 g of ( N H ^ H P O , , 100 m g of M g ( O A e ) 2 ' H 2 ° » and.1. 5 m l of l a c t i c . a c i d . T h e p H of the m e d i u m w a s a d j u s t e d to 3 . 5 w i t h l a c t i c a c i d a n d i n c u b a t e d at 3 3 ° f o r 17 h r w i t h a c o n t i n u o u s s t r e a m of a i r b u b b l i n g t h r o u g h the m e d i u m . T h e c e l l s w e r e h a r v e s t e d b y c e n t r i f u g a t i o n in* an I n t e r n a t i o n a l r e f r i g e r a t e d . p r e p a r a t i v e c e n t r i f u g e and w a s h e d t w i c e w i t h 0. 05 M T r i s -(acetate) , . p H 7 . 4 , 0. 005 M M g j O A c j ^ . T h e c e l l s w e r e s u s p e n d e d i n a s m a l l a m o u n t of the s a m e b u f f e r t h e n t r a n s f e r r e d t o the p r e - c o o l e d j a r of a V i r t i s " 4 5 " h o m o g e n i z e r c o n t a i n i n g about 40 g of g l a s s b e a d s , a d r o p of o c t y l a l c o h o l a n d 0. 5 m l of 5% m e r c a p t o e t h a n o l . T h e j a r w a s p a c k e d i n i c e a n d the h o m o g e n i z e r t u r n e d to f u l l s p e e d f o r 5 m i n , s t o p p e d a n d c o o l e d f o r 3 m i n a n d the p r o c e s s w a s r e p e a t e d t w i c e . T h e h o m o g e n a t e w a s d e c a n t e d f r o m the g l a s s b e a d s a n d c e n t r i f u g e d at 11, 000 r p m i n the S o r v a l l , T y p e SS-1 c e n t r i f u g e at 3 - 5 ° . T h e s u p e r n a t e w a s d e c a n t e d and t h e n c e n t r i f u g e d i n the " 4 0 " r o t o r of a B e c k m a n M o d e l L p r e p a r a t i v e u l t r a c e n t r i f u g e at 100, 000 x g f o r 2 h r . T h e m i d d l e , c l e a r p o r t i o n of the s u p e r n a t e w a s d i a l y z e d f o r 12 h r a g a i n s t 3 l i t r e s of 0 . 0 5 M T r i s ( a c e t a t e . ) , p H 7. 4 , 0. Q 0 5 ' M M g ( O A c ) 2 , c o n t a i n i n g ; 0. 5 m l of - 2 0 8 -m e r c a p t o e t h a n o l . T h e d i a l y s a t e w a s t h e n t r a n s f e r r e d to a m p o u l e s , w h i c h w e r e t h e n s e a l e d and s t o r e d i n . l i q u i d n i t r o g e n u n t i l ready, f o r u s e . 2. P r e p a r a t i o n of A m i n o a c y l - s - R N A S y n t h e t a s e s f r o m . Y e a s t : M e t h o d 2 A . A s s a y / P r o c e d u r e T h e ; p r e s e n c e of a m i n o a c y l - s - R N A s y n t h e t a s e e n z y m e s can•be 14 d e t e c t e d by i n c u b a t i n g the t e s t s o l u t i o n w i t h the a p p r o p r i a t e C - l a b e l l e d a m i n o a c i d , s - R N A , . a n d c o - f a c t o r s . T h e a m i n o a c y l - s - R N A s y n t h e t a s e e n z y m e s c o n v e r t the T C A - s o l u b l e ^ C - a m i n o a c i d to T C A - i n s o l u b l e 14 C - a m i n o a c y l - s - R N A . T h e r e a c t i o n r n i x t u r e ( t o t a l v o l u m e , 0.15 m l ) c o n t a i n e d 27 ^ o r i o l e s s o d i u m c a c o d y l a t e , p H 7. 3, 6. 7 / j - m o l e s K C l , 3 . 7^_i__moles M g C l 2 , 0 . 5 j j . m o l e s o d i u m A T P , , O. lOyumole s o d i u m C T P , 0.12yumole m e r c a p t o e t h a n o l , 0. lO^onole E D T A , 75 r m x m o l e s g l y c i n e , 0. 2 Ju-Ci ^ C - g l y c i n e , 1. 4 m g s - R N A a n d 50 JJ! of e n z y m e s o l u t i o n o r p h o s p h a t e b u f f e r i n . t h e c a s e of the b l a n k . T h e r e a c t i o n m i x t u r e w a s i n c u b a t e d at 25°-and 20y-U a l i q u o t s . w e r e r e m o v e d at i n t e r v a l s a n d a p p l i e d to "Whatman 3 M M p a p e r s (2 cm- , ) . T h e f i l t e r p a p e r s w e r e a l l o w e d t o d r y f o r 30 s e c o n d s b e f o r e p l u n g i n g t h e m i n t o c o l d . 1 0 % T C A . T h e f i l t e r p a p e r s r e m a i n e d at l e a s t 15 m i n , w i t h o c c a s i o n a l s t i r r i n g , i n e a c h of t h r e e 10% T C A b a t h s . E a c h . b a t h c o n t a i n e d 10 m l of 10% T C A p e r f i l t e r p a p e r . A f t e r the T C A w a s h e s , the f i l t e r p a p e r s w e r e w a s h e d i n c o l d e t h a n o l t h e n i n e t h y l e t h e r , d r i e d a n d p l a c e d - 2 0 9 -i n s t a n d a r d c o u n t i n g v i a l s . T o e a c h v i a l w a s a d d e d 5 m l of s c i n t i l l a t i o n f l u i d ( c o n t a i n i n g 3 g P P O a n d 0. 3 g d i m e t h y l P O P O P p e r l i t r e of t o l u e n e ) and the r a d i o a c t i v i t y d e t e r m i n e d i n a s c i n t i l l a t i o n s p e c t r o m e t e r . A u n i t 14 of e n z y m e c a t a l y z e s the f o r m a t i o n of 1 r r y o m o l e of C - a m i n o a c y l - s - R N A p e r m i n . o B . L y s i s of Y e a s t C e l l s w i t h T o l u e n e at 37 T o a 1. l i t r e b e a k e r , , c o n t a i n i n g 200 m l of t o l u e n e i n a 4 5 ° w a t e r b a t h , w a s a d d e d 4 0 0 g of c r u m b l e d b a k e r s ' y e a s t f o l l o w e d by 120 m l of 0. 6 5 ' M T r i s ( a c e t a t e ) , " p H 8 . 0 . T h e m i x t u r e w a s w a r m e d to 3 7 0 ' w i t h s l o w s t i r r i n g then, the b e a k e r w a s t r a n s f e r r e d . t c a 3 8 0 , w a t e r b a t h a n d the s t i r r i n g c o n t i n u e d . T h e v i s c o s i t y of the m i x t u r e d e c r e a s e d a n d the e v o l u t i o n of gas w a s a p p a r e n t as the t e m p e r a t u r e of the m i x t u r e a p p -r o a c h e d 3 7 ° . A d r o p . i n : t h e p_H of the s o l u t i o n ; w a s o b s e r v e d c o n c u r r e n t w i t h the e v o l u t i o n of g a s . The-p_H w a s m o n i t o r e d w i t h a L e e d s a n d N o r t h r o p g l a s s e l e c t r o d e a n d w a s m a i n t a i n e d at about 7 b y the a d d i t i o n of I N N a O H . . The l y s i s w a s e s s e n t i a l l y c o m p l e t e 40 m i n . a f t e r the m i x t u r e h a d r e a c h e d 3 7 ° , as i n d i c a t e d by the c o n s u m p t i o n of a l k a l i (50 m l ) . The t e m p e r a t u r e . of the m i x t u r e w a s m a i n t a i n e d at 3 7 ° f o r a. t o t a l t i m e of 60 m i n d u r i n g w h i c h 52 m l of I N N a O H w a s c o n s u m e d . T h e m i x t u r e w a s t h e n c o o l e d to about 5 ° i n . a n . i c e b a t h and 100 m l of 0. 65 M T r i s ( a c e t a t e ) , p H 8 . 0 , , w a s a d d e d . A l l s u b s e q u e n t o p e r a t i o n s w e r e c a r r i e d out b e t w e e n 0 a n d 5 ° . T h e m i x t u r e w a s c e n t r i f u g e d at -2000 r p m - 2 1 0 -f o r 15 m i n i n . a r e f r i g e r a t e d I n t e r n a t i o n a l C e n t r i f u g e . T h e a q u e o u s l a y e r w a s r e m o v e d f r o m b e n e a t h t h e t o l u e n e l a y e r and c e n t r i f u g e d at 11, 000 r p m f o r 20 : m i n i n a S o r v a l l , T y p e SS-1 c e n t r i f u g e . T h e m i d d l e , aqueous l a y e r ( t o t a l v o l u m e , . 340 m l ) ; w a s r e m o v e d w i t h the a i d of a l a r g e s y r i n g e a n d c e n t r i f u g e d at 100, 0 0 0 : x g i n the T y p e - 3 0 r o t o r of the B e c k m a n , M o d e l L u l t r a c e n t r i f u g e f o r 3. 5 h r . A b o u t 230 m l of s u p e r n a t a n t f l u i d , c o n t a i n i n g 0. 27 m g of p r o t e i n p e r m l w a s r e c o v e r e d . T h e 2 8 0 : 2 6 0 my, r a t i o of the c e l l - f r e e e x t r a c t w a s 0 . 6 6 . C . A m m o n i u m S u l f a t e F r a c t i o n a t i o n of the C e l l - F r e e E x t r a c t To,180 m l of the c o l d , c e l l - f r e e e x t r a c t o b t a i n e d a b o v e , w a s a d d e d 68. 9 g of a m m o n i u m s u l f a t e . T h e m i x t u r e w a s s t i r r e d u n t i l a l l the a m m o n i u m s u l f a t e h a d d i s s o l v e d t h e n l e f t f o r an. a d d i t i o n a l 20 m i n at 0 ° . T h e p r e c i p i t a t e w a s c o l l e c t e d by c e n t r i f u g a t i o n ( a m m o n i u m s u l f a t e f r a c t i o n A ) . T o the s u p e r n a t e (192 m l ) w a s a d d e d 12. 1 g of a m m o n i u m sul fa te a n d the m i x t u r e t r e a t e d as d e s c r i b e d above ( a m m o n i u m s u l f a t e f r a c t i o n B ) . A m m o n i u m s u l f a t e f r a c t i o n s C and D w e r e o b t a i n e d i n a n a n a l o g o u s m a n n e r by a d d i n g 13.1 g a n d 14. 0 g of a m m o n i u m s u l f a t e to the s u p e r n a t e s f r o m f r a c t i o n s B (190 m l ) , a n d C ( 1 9 5 m l ) , r e s p e c t i v e l y . The s u p e r n a t e f r o m a m m o n i u m s u l f a t e f r a c t i o n D w a s d i s c a r d e d . P e l l e t s A t o ' D w e r e d i s s o l v e d . i n 0. 10 M s o d i u m p h o s p h a t e , p H 7. 4 t h e n g l y c e r o l w a s a d d e d to 35% (v/v) a n d the s o l u t i o n s s t o r e d at - 1 8 ° u n t i l r e a d y f o r u s e . T a b l e I l i s t s the d a t a r e l a t i n g to the a m m o n i u m s u l f a t e f r a c t i o n s A t o D . - 2 1 1 -T A B L E I P r o p e r t i e s of A m m o n i u m S u l f a t e F r a c t i o n s A - D F r a c t i o n V o l u m e a ( m l ) T o t a l O. U n i t s (280 D . rryj_) T o t a l , p r o t e i n (g) 280:260 b • r a t i o C r u d e 1 4 , 7 0 0 . 9. 685 0. 85 A 111 4, 470 3. 881 1. 10 B 30 : 1, 920 1. 097 0 . 7 7 C 15. 5 550 . 0. 289 - 0 . 74 D 9. 2 277 0. 146 0. 74 S u p e r n a t e of D 195 7, 520 a V o l u m e of 0. 10 M s o d i u m p h o s p h a t e p l u s g l y c e r o l a d d e d to p e l l e t s 2 8 0 : 2 6 0 r a t i o s w e r e d e t e r m i n e d a f t e r p a s s a g e t h r o u g h G - 2 5 S e p h a d e x as d e s c r i b e d i n S e c t i o n 2 - C . P o r t i o n s of the c r u d e c e l l - f r e e e x t r a c t a n d f r a c t i o n s A t o ' D w e r e d e s a l t e d on G - 2 5 S e p h a d e x e q u i l i b r a t e d w i t h 0. 10 M s o d i u m p h o s p h a t e , p H 7 . 4 , p r i o r to 1 a s s a y i n g f o r g l y c y l - s - R N A s y n t h e t a s e a c t i v i t y . T h e r e a c t i o n m i x t u r e ( S e c t i o n 2 - A ) f o r a s s a y i n g the c r u d e e n z y m e c o n t a i n e d 0. 018 O. D . u n i t s (280 rmj.) (12yu_g of p r o t e i n ) of the S e p h a d e x t r e a t e d c r u d e e x t r a c t . The ' s y n t h e s i s of ^ C - g l y c y l - s - R N A w a s l i n e a r o v e r 20 m i n . A m m o n i u m s u l f a t e f r a c t i o n s A a n d B w e r e a s s a y e d f o r - 2 1 2 -g l y c y l - s - R N A s y n t h e t a s e a c t i v i t y u s i n g 0. 020 ; a n d 0. 010 O . D . u n i t s (at 280 m y u ) o f e n z y m e r e s p e c t i v e l y . F r a c t i o n s C a n d D c o n t a i n e d n e g l i g i b l e g l y c y l - s - R N A s y n t h e t a s e a c t i v i t y . The d a t a a r e s u m m a r i z e d i n T a b l e II . T A B L E II G l y c y l - s - R N A S y n t h e t a s e A c t i v i t y i n F r a c t i o n s A a n d B r F r a c t i o n S p e c i f i c A c t i v i t y , T o t a l b  1 U n i t s R e c o v e r y . % C r u d e 0 . 7 6 7 . 3 5 100 A 0. 18 . 0 . 70 9. 5 B 5. 04 5. 53 7 5 . 2 • S p e c i f i c A c t i v i t y i s u n i t s of e n z y m e a c t i v i t y p e r m g of p r o t e i n b T o t a l u n i t s a r e o b t a i n e d by m u l t i p l y i n g s p e c i f i c a c t i v i t y / b y the t o t a l m g of p r o t e i n D . P r e p a r a t i o n . o f A m i n o a c y l - s - R N A S y n t h e t a s e E n z y m e s U s e d i n  P a r t I I - D , S e c t i o n 2 - B A 2. 0 m l p o r t i o n of a m m o n i u m s u l f a t e f r a c t i o n B ( S e c t i o n C ) w a s d e s a l t e d on G - 2 5 S e p h a d e x e q u i l i b r a t e d w i t h 0. 01 M p o t a s s i u m p h o s p h a t e , p H 7 . 5 , , 0 . 0 0 1 M m e r c a p t o e t h a n o l , 35% g l y c e r o l ( v / v ) . T h e c o l u m n f r a c t i o n s w e r e a s s a y e d f o r g l y c y l - s - R N A s y n t h e t a s e a c t i v i t y - a s d e s c r i b e d i n S e c t i o n 3 - E . . T h e f r a c t i o n s c o n t a i n i n g the d e s i r e d e n z y m a t i c a c t i v i t y w e r e p o o l e d a n d s t o r e d at - 1 8 ° u n t i l r e a d y f o r u s e . - 2 1 3 -E . C h r o m a t o g r a p h y of F r a c t i o n B o n H y d r o x y a p a t i t e T h e h y d r o x y a p a t i t e p o w d e r ( B i o - G e l H T P ) iWas s u s p e n d e d o v e r -- 2 2 n i g h t i n 10" M p o t a s s i u m p h o s p h a t e , 10" M m e r c a p t o e t h a n o l 35% g l y c e r o l ( v / v ) , p_H 6. 8 ( s t a r t i n g ; b u f f e r ) t h e n . p a c k e d u n d e r p r e s s u r e i n a c h r o m a t o g r a p h i c c o l u m n m a i n t a i n e d : a t 3 - 5 ° . T h e c o l u m n (14 x 3 . 0 c m d i a m . ) w a s e q u i l i b r a t e d w i t h 500 m l of s t a r t i n g : b u f f e r . T h e f l o w r a t e w a s c o n t r o l l e d by m e a n s of a n A c c u - F l o p u m p ( B e c k m a n I n s t r u m e n t s I n c . ) . A 10 m l p o r t i o n of f r a c t i o n B (3 66 m g p r o t e i n ) w a s d i l u t e d to 100 m l w i t h 35% a q u e o u s g l y c e r o l t h e n a p p l i e d t o ' t h e h y d r o x y a p a t i t e c o l u m n . A f t e r w a s h i n g the c o l u m n w i t h : a few m i s of s t a r t i n g ; b u f f e r , a 2 l i t r e l i n e a r g r a d i e n t of p o t a s s i u m p h o s p h a t e , f r o m 10 M to 0. 20 M w a s a p p l i e d , w i t h b o t h - v e s s e l s c o n t a i n i n g 10" M m e r c a p t o e t h a n o l a n d 35% g l y c e r o l ( v / v ) . T h e f l o w r a t e w a s about 1 m l p e r m i n a n d 15 m l f r a c t i o n s w e r e c o l l e c t e d . W h e n the 2 l i t r e l i n e a r g r a d i e n t f i n i s h e d , a 4 0 0 m l l i n e a r g r a d i e n t of a m m o n i u m s u l f a t e f r o m 0-to 10% (w/v) w a s a p p l i e d - 2 w i t h b o t h v e s s e l s c o n t a i n i n g 0. 20 M p o t a s s i u m p h o s p h a t e 10 M m e r c a p t o e t h a n o l , . p H 6. 8 a n d 35% g l y c e r o l ( v / v ) . T h e O . D . at 280 myu. w a s r e c o r d e d f o r e a c h . f r a c t i o n ( F i g . 1). T h e f r a c t i o n s w e r e a s s a y e d f o r g l y c y l - s - R N A s y n t h e t a s e a c t i v i t y a c c o r d i n g to the p r o c e d u r e d e s c r i b e d i n S e c t i o n A e x c e p t the r e a c t i o n m i x t u r e ( t o t a l v o l u m e , . 0. 20 m l ) c o n t a i n e d 2. 8 m g of s - R N A , a n d a 0. 10 m l a l i q u o t of the c o l u m n f r a c t i o n s . T h e i n c u b a t i o n p e r i o d w a s 60< m i n . I I 1 o P P . Phosphate Buffer(M) and cpm(per0.1 ml eluate)incorporated in 2,8mg s-RNA(x10~3) - 2 1 5 -F . R a d i o c h r o m a t o g r a p h i c A n a l y s i s of the A m m o n i u m Sulfate: E l u a t e The r e a c t i o n m i x t u r e ( f i n a l v o l u m e 0. 30 m l ) c o n t a i n e d 7. 3yuimoles M g C ^ , 13. 3 ^umoles K C l , 40^juxmoles s o d i u m c a c o d y l a t e , p H 7. 3, 1 j U m o l e s o d i u m A T P , 0. 2 / u m o l e s o d i u m C T P , 0. 24 yurnole m e r c a p t o e t h a n o l , 0. 2 yUmole E D T A , 173 m / x m o l e s m i x e d a m i n o a c i d s a n d 0. 67^^uCi of m i x e d 14 C - a m i n o a c i d s , 5. 6 . m g of s - R N A and.lOOyial of c o l u m n e l u a t e . T h e r e a c t i o n . m i x t u r e w a s i n c u b a t e d f o r 60 m i n . a t r o o m t e m p e r a t u r e . A 20 Jul a l i q u o t w a s r e m o v e d a n d a p p l i e d to a W h a t m a n 3 M M f i l t e r p a p e r (2 2 ' c m ). The f i l t e r p a p e r was. w a s h e d a n d the r a d i o a c t i v i t y d e t e r m i n e d as i d e s c r i b e d i n S e c t i o n A . T h e r e m a i n d e r of the r e a c t i o n m i x t u r e w a s c o o l e d i n i c e a n d 0.10 m l 0. 5 M s o d i u m f o r m a t e , p H 3. 0, 0.10 m l 5 M N a C l a n d 1. 50 m l e t h a n o l w e r e a d d e d . A f t e r 10 m i n i n i c e , the m i x t u r e w a s c e n t r i f u g e d a n d the s u p e r n a t e d e c a n t e d . T h e p e l l e t w a s w a s h e d w i t h 0. 5 m l e t h a n o l - s a l t s o l u t i o n (2:1, e t h a n o l : 1. 5>M N a C l ) , t h e n c e n t r i f u g e d a n d d r a i n e d . f r e e of e x c e s s w a s h s o l v e n t . T h e n the p e l l e t w a s d i s s o l v e d . i n . 0 . 5 m l 0.10 M M g C l 2 , 0. 005 M N a O A c , - p H 5. O a n d p r e c i p i t a t e d a g a i n w i t h 1. 5 m l of c o l d e t h a n o l . The p e l l e t , o b t a i n e d by c e n t r i f u g a t i o n , w a s w a s h e d as d e s c r i b e d a b o v e , d r a i n e d f r e e of e x c e s s w a s h s o l v e n t a n d d i s s o l v e d i n . 0. 15 m l of 1. 5 N N H ^ O H . A f t e r i n c u b a t i n g the m i x t u r e at 3 7 ° f o r 30 m i n , 0. 60 m l of c o l d e t h a n o l w a s a d d e d a n d the m i x t u r e c e n t r i f u g e d . M o s t of the s u p e r n a t e w a s a p p l i e d to W h a t m a n 1 p a p e r a n d c h r o m a t o g r a p h e d 26 h r i n n - b u t a n o l : a c e t i c a c i d : H ^ O (60: 15:25). T h e c h r o m a t o g r a m w a s cut i n t o s t r i p s a n d s c a n n e d i n the - 2 1 6 -A c t i g r a p h ( N u c l e a r C h i c a g o , M o d e l 1036). T h e r e s u l t s a r e s h o w n i n ' F i g u r e 2. 3. C h r o m a t o g r a p h y of Y e a s t C e l l - F r e e E x t r a c t on H y d r o x y a p a t i t e b y  S t e p w i s e E l u t i o n A l l o p e r a t i o n s w e r e c a r r i e d out b e t w e e n 0 and. 5 ° . A . P r e p a r a t i o n of the Y e a s t C e l l - F r e e E x t r a c t T h e c e l l - f r e e e x t r a c t w a s p r e p a r e d . f r o m 400 g of b a k e r s ' y e a s t a c c o r d i n g to M e t h o d 2 , ( S e c t i o n 2 - B ) e x c e p t that 150 m l of T r i s ( a c e t a t e ) , p H 8. 0, w a s a d d e d to the c r u m b l e d y e a s t - t o l u e n e m i x t u r e p r i o r to l y s i s . A l s o , the a d d i t i o n of 100 m l of T r i s ( a c e t a t e ) , p H 8. 0, f o l l o w i n g l y s i s w a s o m i t t e d . T h e ; u l t r a c e n t r i f u g a t i o n s t e p w a s a l s o o m i t t e d . T o the s u p e r n a t e (300 m l ) o b t a i n e d f r o m the S o r v a l l , T y p e SS-1 c e n t r i f u g a t i o n w a s a d d e d 115 g of a m m o n i u m s u l f a t e (about 2. 9 M ) , s l o w l y w i t h s t i r r i n g and the m i x t u r e l e f t i n i c e f o r 20 m i n a f t e r a l l the a m m o n i u m s u l f a t e h a d d i s s o l v e d . O n e - h a l f of the p e l l e t s o b t a i n e d by c e n t r i f u g a t i o n w a s d i s s o l v e d i n 40 m l of 0. 01 M p o t a s s i u m p h o s p h a t e , p H 6. 8, 10" 3 M m e r c a p t o e t h a n o l , 35% g l y c e r o l ( v / v ) . T h e f i n a l v o l u m e w a s 5 0 . 0 m l and. c o n t a i n e d 2400 O . D . u n i t s (280 m ^ ) : (1. 26 g of p r o t e i n ) . T h e 280: 260 rryj. r a t i o of the s o l u t i o n w a s 0 . 7 4 . F I G U R E 2. R e s u l t s of the R a d e l u a t e . i o c h r o m a t o g r a p h i c a n a l y s i s of the a m m o n i u m s u l f a t e - 2 1 8 -B . C h r o m a t o g r a p h y 1 T h e 50 m l of c r u d e e x t r a c t w e r e a p p l i e d to' a h y d r o x y a p a t i t e c o l u m n (13. 2 x 3 . 0 c m d i a m . ) p r e v i o u s l y e q u i l i b r a t e d w i t h 0. 20 M p o t a s s i u m - 3 p h o s p h a t e , p_H 6. 8, 10 M m e r c a p t o e t h a n o l , 35% g l y c e r o l ( v / v ) • ( s t a r t i n g ; b u f f e r ) . A f t e r w a s h i n g the c o l u m n w i t h 250 m l of the s a m e buf fer a 4 0 0 m l l i n e a r g r a d i e n t of a m m o n i u m s u l f a t e , f r o m 0 to 10% ( w / v ) , w a s a p p l i e d . B o t h v e s s e l s c o n t a i n e d 0. 2 M p o t a s s i u m p h o s p h a t e , 1 0 " 3 M m e r c a p t o e t h a n o l a n d 35% g l y c e r o l ( v / v ) . A f l o w r a t e of about 1 m l p e r m i n w a s m a i n t a i n e d w i t h the a i d of a n A c c u - F l o p u m p . T h e c o l u m n f r a c t i o n s w e r e a s s a y e d f o r a m i n o a c y l - s - R N A s y n t h e t a s e a c t i v i t y u s i n g m i x e d ^ C - a m i n o a c i d s . To>2.4 c m W h a t m a n 3 M M f i l t e r d i s k s , s u s p e n d e d on. s t a i n l e s s s t e e l p i n s , w a s a d d e d 40 JJLI of a r e a c t i o n m i x t u r e c o n t a i n i n g 8 yoxnoles s o d i u m c a c o d y l a t e , p H 7. 3, 1.47 yu.rn.oles M g C L , 2. 66 y u m o l e s K C l , 0. 2 ^ uunole s o d i u m A T P , 0. 04 ylxmole s o d i u m C T P , 0. 05 y U m o l e m e r c a p t o e t h a n o l , 0. 04 / u.mole E D T A , 14 34. 5 m y u m o l e s a n d 133 m y u C i of C - a m i n o a c i d s , a n d 1.12 m g of s - R N A . A 2 0 ^ a l i q u o t of the c o l u m n f r a c t i o n s w a s a d d e d a n d the d i s k s t h e n i n c u b a t e d f o r 30 m i n at r o o m t e m p e r a t u r e . T h e b l a n k c o n t a i n e d a 20 pi a l i q u o t of the s t a r t i n g ; b u f f e r i n s t e a d of c o l u m n e l u a t e . T h e s t y r o f o a m b l o c k s u p p o r t i n g ; the s t a i n l e s s s t e e l p i n s w a s c o n t a i n e d i n a g l a s s d i s h c o n t a i n i n g w a t e r a n d c o v e r e d w i t h a t i g h t f i t t i n g c o v e r to m a i n t a i n , a m o i s t a t m o s p h e r e . A f t e r the 30 m i n i n c u b a t i o n p e r i o d the f i l t e r d i s k s w e r e w a s h e d a n d the r a d i o a c t i v i t y d e t e r m i n e d as d e s c r i b e d i n S e c t i o n 2 - A . A l l the s y n t h e t a s e a c t i v i t y a n d a l l the a p p l i e d O . D . u n i t s w e r e e l u t e d by the s t a r t i n g b u f f e r . T h e a p p l i e d O . D . u n i t s w e r e r e c o v e r e d . i n 84 m l of s t a r t i n g , b u f f e r . C . C h r o m a t o g r a p h y 2 A 20 m l p o r t i o n (570 O . D . u n i t s ) of the 84 m l r e c o v e r e d f r o m c h r o m a t o g r a p h y 1 w a s r e c h r o m a t o g r a p h e d a n d the c o l u m n f r a c t i o n a s s a y e d u n d e r the c o n d i t i o n s d e s c r i b e d above . (Sect ion 3 - B ) . A l l the a p p l i e d O . D . u n i t s a n d a l l the s y n t h e t a s e a c t i v i t y w e r e a g a i n e l u t e d b y the s t a r t i n g b u f f e r . D . • C h r o m a t o g r a p h y 3 A 20 m l p o r t i o n (570 O . D . u n i t s ) of the 84 m l r e c o v e r e d f r o m c h r o m a t o g r a p h y 1 w a s d i a l y z e d f o r 5 h r a g a i n s 1 l i t r e of 10 M p o t a s s i u m p h o s p h a t e , . p H 7 . 0 , 1 0 " 3 M m e r c a p t o e t h a n o l , 35% g l y c e r o l ( v / v ) . T h e d i a l y s a t e ( 3 6 . 5 m l , 510 O . D . units)* w a s a p p l i e d to a h y d r o x y a p a t i t e c o l u m n (13. 2 x 3. 0 c m d i a m . ), p r e v i o u s l y e q u i l i b r a t e d w i t h 10" M p o t a s s i u m p h o s p h a t e , , p H 6. 8, 10" M m e r c a p t o e t h a n o l , 35% - 2 g l y c e r o l ( v / v ) . T h e c o l u m n w a s w a s h e d f i r s t w i t h 180 m l 10 M p o t a s s i u m - 3 phosphate , . p H 6 . 8 , 10 M m e r c a p t o e t h a n o l , 35% g l y c e r o l ( v / v ) , t h e n 3 w i t h 340 m l of 0. 2 M p o t a s s i u m p h o s p h a t e , p H 6. 8, 10" M m e r c a p t o -e t h a n o l , 35% g l y c e r o l ( v / v ) g l y c e r o l a n d f i n a l l y w i t h 0. 2 M p o t a s s i u m - 2 2 0 -p h o s p h a t e , p H 6. 8, 10 M m e r c a p t o e t h a n o l , 35% g l y c e r o l ( v / v ) , 10% a m m o n i u m s u l f a t e ( w / v ) . T h e c o l u m n f r a c t i o n s w e r e a s s a y e d as d e s c r i b e d i n S e c t i o n 3 - B . T h e 10" 2 M p h o s p h a t e w a s h e l u t e d 4 . 6% of the a p p l i e d O . D . u n i t s but none of the s y n t h e t a s e a c t i v i t y . T h e 0. 2 M p h o s p h a t e s t e p e l u t e d 91 .4% of the a p p l i e d O . D . u n i t s a n d m o s t of the s y n t h e t a s e a c t i v i t y . T h e 10% a m m o n i u m s u l f a t e s t e p e l u t e d 3. 9% of the a p p l i e d O . D . u n i t s a n d a s m a l l a m o u n t of the s y n t h e t a s e a c t i v i t y . 4 . E f f e c t of A m m o n i u m S u l f a t e a n d D i - i s o p r , o p y l f l u o r o p h o s p h a t e on the  A m i n o a c y l - s - R N A S y n t h e t a s e A c t i v i t y A . E f f e c t of A m m o n i u m S u l f a t e T h e a s s a y s w e r e c a r r i e d out as d e s c r i b e d i n S e c t i o n 2 - A . T h e r e a c t i o n m i x t u r e (0.15 m l ) c o n t a i n e d 5OJJI of the a m m o n i u m s u l f a t e f r a c t i o n B ( b e f o r e S e p h a d e x t r e a t m e n t ) . A s s a y s w e r e c a r r i e d out at e n z y m e d i l u t i o n s of 1, 2, 4 , a n d 8 f o l d . T h e e n z y m e w a s d i l u t e d w i t h 0. 05 M p o t a s s i u m p h o s p h a t e , 10" m e r c a p t o e t h a n o l , 35% g l y c e r o l ( v / v ) . T h e f o u r a s s a y s e a c h s h o w e d m a x i m u m c h a r g i n g i n l e s s t h a n 20 14 m i n w h i l e the i n c o r p o r a t i o n of C - g l y c i n e i n t o a T C A i n s o l u b l e f o r m i n c r e a s e d w i t h i n c r e a s i n g d i l u t i o n of the e n z y m e . T h e r e a c t i o n m i x t u r e s c o n t a i n i n g the 2, 4 a n d 8 f o l d d i l u t e d e n z y m e s h o w e d 25%, 31% a n d 33% 14 m o r e C - g l y c i n e i n c o r p o r a t i o n t h a n the u n d i l u t e d e n z y m e . - 2 2 1 -B . E f f e c t of D i - i s o p r o p y l f l u o r o p h o s p h a t e T w o a s s a y s w e r e c a r r i e d out u n d e r the c o n d i t i o n s d e s c r i b e d i n S e c t i o n 2 - A e x c e p t that one of t h e m c o n t a i n e d 0.15 j u m o l e of d i - i s o p r o p y l -f l u o r o p h o s p h a t e ( D F P ) . T h e p r e s e n c e of D F P d i d not e f fect the s y n t h e s i s of 1 4 C - g l y c y l - s - R N A . 5. P r e p a r a t i o n a n d P a r t i a l P u r i f i c a t i o n of Y e a s t A m i n o a c y l - s - R N A  S y n t h e t a s e E n z y m e s : M e t h o d 3 A . P r e p a r a t i o n of the C e l l - F r e e E x t r a c t T w o p o u n d s of f r e s h , p r e s s e d b a k e r s ' y e a s t w e r e t h o r o u g h l y c r u m b l e d , t h e n a d d e d to 2 l i t r e s of t o l u e n e c o n t a i n i n g e x c e s s d r y i c e . T h e m i x t u r e w a s l e f t i n the c o l d r o o m (-18°) f o r 6 h r by. w h i c h t i m e the m i x t u r e h a d w a r m e d to about - 2 0 ° . T h e t o l u e n e w a s t h e n d e c a n t e d a n d the f r o z e n y e a s t l e f t at 3 - 5 ° f o r 44 h r . A 0. 10 m l a l i q u o t of 1 M D F P ( i n i s o p r o p a n o l ) w a s a d d e d to the y e a s t p a s t e . T h e p a s t e w a s t h e n c e n t r i f u g e d f o r 30 m i n at 14, 600 x g i n the G S A h e a d of a n R C - 2 B o r e f r i g e r a t e d (0 ) c e n t r i f u g e . T h e s u p e r n a t e s w e r e c o m b i n e d to y i e l d 210 m l of c e l l - f r e e e x t r a c t . B . A m m o n i u m S u l f a t e F r a c t i o n a t i o n of the C e l l - F r e e E x t r a c t T o a p o r t i o n , of the c e l l - f r e e e x t r a c t (180 m l , . p H 5. 6) w a s a d d e d 50 m l of 0. 65 M T r i s ( a c e t a t e ) , p H 8. 0 a n d the r e s u l t i n g s o l u t i o n w a s - 2 2 2 -p l a c e d i n , a n i c e b a t h . T h e f i n a l v o l u m e of the s o l u t i o n w a s 230 m l a n d the p H w a s 7 . 3 . T h e p r o t o c o l f o r the a m m o n i u m s u l f a t e f r a c t i o n a t i o n i s s h o w n i n T a b l e III a n d the p r o c e d u r e w a s as f o l l o w s : the r e q u i r e d a m o u n t of s o l i d a m m o n i u m s u l f a t e w a s a d d e d s l o w l y , w i t h s t i r r i n g , to the c o l d s o l u t i o n . A f t e r a l l the a m m o n i u m s u l f a t e h a d d i s s o l v e d , the T A B L E III P r o t o c o l f o r A m m o n i u m S u l f a t e F r a c t i o n a t i o n F r a c t i o n V o l u m e a ( m l ) A m m o n i u m S u l f a t e A d d e d (g) B u f f e r A d d e d ( m l ) O . D . u n i t s (280 myx) b I C 230 32. 2 -II i 232 35. 2 24 3600 III 210 d 1 6. 0 21 1980 • I V 210 ; 17. 8 24 2400 a V o l u m e b e f o r e a d d i t i o n of a m m o n i u m s u l f a t e O. D . . u n i t s w e r e d e t e r m i n e d a f t e r p a s s a g e t h r o u g h G - 2 5 S e p h a d e x as d e s c r i b e d i n S e c t i o n 5 - B c F r a c t i o n I w a s d i s c a r d e d A few m i s w e r e l o s t due to d i s t u r b a n c e of the p e l l e t s o l u t i o n w a s l e f t f o r a f u r t h e r 20 m i n i n the i c e b a t h . T h e s o l u t i o n w a s t h e n c e n t r i f u g e d f o r 30 m i n at 11, 000 r p m , i n a S o r v a l l , T y p e SS-1 c e n t r i f u g e at 3 - 5 ° . T h e p e l l e t s w e r e - d i s s o l v e d i n a m i n i m u m of 10" 2 M p o t a s s i u m p h o s p h a t e , 1 0 " 3 M m e r c a p t o e t h a n o l , 35% g l y c e r o l ( v / v ) , p H - 2 2 3 -7. 5 a n d e x c e s s a m m o n i u m s u l f a t e r e m o v e d by g e l f i l t r a t i o n on G - 2 5 S e p h a d e x e q u i l i b r a t e d w i t h the s a m e b u f f e r . F r a c t i o n s f r o m the S e p h a d e x c o l u m n c o n t a i n i n g u l t r a v i o l e t a b s o r b i n g (at 280 rryj.) m a t e r i a l e x c l u d e d by the S e p h a d e x b e a d s w e r e c o m b i n e d a n d s t o r e d at - 1 8 ° u n t i l r e a d y f o r u s e . C . D e t e c t i o n of A m i n o a c y l - s - R N A S y n t h e t a s e s i n the A m m o n i u m  S u l f a t e F r a c t i o n s T h e a s s a y s w e r e c a r r i e d out i n 1 m l g l a s s t u b e s c o n t a i n i n g ( i n a f i n a l v o l u m e of 0. 10 m l ) ; 5' to 10 u m o l e s s o d i u m c a c o d y l a t e , p H 7. 5, 1 yjumole M g C l ,• 0 to 1 /pumole K C l , 0. 25 to 1 .0 y u m o l e s o d i u m A T P , 0 to 0. 04 ^ u n o l e E D T A , 1. 25 m g s - R N A , 5 m/jmaoles of a g i v e n a m i n o a c i d c o n t a i n i n g 20 yu_Ci p e r y U m o l e e x c e p t m e t h i o n i n e (8 m / u m o l e s c o n t a i n i n g 12. 2 y x C i p e r ^ o r i o l e ) a n d p h e n y l a l a n i n e (4. 9 m y u m o l e s c o n t a i n i n g 37. 5 ^LtCi p e r yumole) , a n d 50 jud of e n z y m e (or p h o s p h a t e b u f f e r , i n the c a s e of the b l a n k ) s o l u t i o n . T h e a s s a y m i x t u r e f o r cy s t e i n y l - s - R N A s y n t h e t a s e , c o n t a i n e d , i n a d d i t i o n , 1 yumole of m e r c a p t o e t h a n o l . D a t a r e l a t i n g t o t r e a t m e n t of the a m m o n i u m s u l f a t e f r a c t i o n s on G - 2 5 S e p h a d e x a n d s u b s e q u e n t d i l u t i o n s m a d e p r i o r to a s s a y i n g a r e s u m m a r i z e d i n T a b l e I V . A f t e r 30 m i n at r o o m t e m p e r a t u r e 25 yul a l i q u o t s w e r e r e m o v e d a n d a p p l i e d to W h a t m a n 3 M M , 2 . 4 c m d i a m . f i l t e r d i s k s . E a c h d i s k w a s w a s h e d once f o r a m i n i m u m of 10 m i n i n 10% T C A , t w i c e f o r 10 m i n i n 5% T C A , 10 m i n i n E t O H t h e n i n e t h e r . T h e d i s k s w e r e t h e n d r i e d a n d the r a d i o a c t i v i t y d e t e r m i n e d as d e s c r i b e d i n S e c t i o n 2 - A . - 2 2 4 -T A B L E I V P r o p e r t i e s of A m m o n i u m S u l f a t e F r a c t i o n s II - I V A m m o n i u m S u l f a t e F r a c t i o n N o . 2 8 0 : 2 6 0 a r a t i o F i n a l m g of p r o t e i n p e r m l A s s a y b d i l u t i o n m g p r o t e i n s p e r 50Jul II 0. 65 10. 6 1:10 0. 053 III 0. 94 19. 8 1:20 0. 050 I V 0. 99 2 4 . 4 1:20 0. 061 S u p e r n a t e ° 0. 88 1. 20 1 0. 060 a T h e 2 8 0 : 2 6 0 r a t i o w a s d e t e r m i n e d a f t e r S e p h a d e x t r e a t m e n t . D i l u t i o n s m a d e w i t h 10 M p o t a s s i u m p h o s p h a t e , 10" ^ M m e r c a p t o e t h a n o l , 35% g l y c e r o l ( v / v ) , p H 7 . 5 . c S u p e r n a t e of a m m o n i u m s u l f a t e F r a c t i o n I V . T a b l e V l i s t s the myLtmoles of the v a r i o u s a m i n o a c i d s i n c o r p o r -a t e d i n t o s - R N A u n d e r the c o n d i t i o n s d e s c r i b e d a b o v e . D . C h r o m a t o g r a p h y of A m m o n i u m S u l f a t e F r a c t i o n III on  H y d r oxy a p a t i t e O n e - t h i r d of the a m m o n i u m s u l f a t e F r a c t i o n III w a s d i s s o l v e d i n a - 2 - 3 m i n i m u m of 10 M p o t a s s i u m p h o s p h a t e , 10 M m e r c a p t o e t h a n o l , p H 7. 5 a n d c h r o m a t o g r a p h e d o n G - 2 5 S e p h a d e x e q u i l i b r a t e d w i t h the s a m e b u f f e r . T h e a b s o r b a n c e (at 280 rryj_) of the f r a c t i o n s f r o m the G - 2 5 S e p h a d e x c o l u m n w a s r e c o r d e d a n d the f r a c t i o n s c o m p r i s i n g the l e a d i n g - 2 2 5 -T A B L E V A m i n o a c y l - s - R N A S y n t h e t a s e A c t i v i t i e s i n the A m m o n i u m S u l f a t e F r a c t i o n s A m i n o m ^ x m o l e s of a m i n o acid, i n c o r p o r a t e d a A c i d F r a c t i o n II F r a c t i o n III F r a c t i o n I V S u p e r n a t e a l a 0. 05 0. 25 0. 22 0 a r g 0. 15 0. 31 1. 10 0. 22 a s p 0. 23 0. 96 0. 31 0 a s n 0 . 0 2 0. 07 0. 002 0 c y s 0. 27 0 . 2 0 0. 01 0. 01 g l u 0. 01 0. 01 0. 002 0 g i n 0. 02 0. 08 0. 04 0 g l y 0. 15 0. 57 0 . 4 9 0 h i s 0 . 1 0 0. 23 0. 39 0. 04 i l e 0 . 3 9 1. 23 0. 37 0 ' l e u 0. 08 0. 34 0. 18 0 l y s 1. 15 1. 04 0. 01 0 m e t 0. 11 0. 25 0. 15 0. 01 phe 0. 10 0 . 5 6 0. 09 0 p r o 0. 02 0. 05 0. 01 0. 005 s e r 0. 17 0. 71 0. 74 0. 01 t h r 0. 07 0. 36 0 . 0 3 0 t r y 0. 08 0. 23 0. 16 0 . 0 1 t y r 0. 50 0. 81 0. 18 0 v a l 0. 08 0. 27 0. 01 0 a m ^ i m o l e s of a m i n o a c i d i n c o r p o r a t e d i n 1. 25 m g s - R N A b y 0. 05 to 0. 06 m g p r o t e i n u n d e r the c o n d i t i o n s d e s c r i b e d i n S e c t i o n 5 - C . s u p e r n a t e of F r a c t i o n I V . p e a k , w h i c h w e r e e l u t e d a f t e r one c o l u m n v o l u m e of b u f f e r h a d p a s s e d t h r o u g h the c o l u m n , w e r e c o m b i n e d to y i e l d 27 m l of s o l u t i o n a n d 817 O . D . u n i t s (280 m u ) . A 3. 0 m l p o r t i o n of the s o l u t i o n w a s put a s i d e - 2 2 6 -( d e s a l t e d a m m o n i u m s u l f a t e F r a c t i o n III) a n d the r e m a i n i n g 24 m l w e r e a p p l i e d to a h y d r o x y a p a t i t e c o l u m n (17 x 3 c m d i a m . ) w h i c h h a d b e e n - 2 - 3 p r e v i o u s l y e q u i l i b r a t e d w i t h 10 M p o t a s s i u m p h o s p h a t e , 10" M m e r c a p t o e t h a n o l , p H 7. 5. T h e c o l u m n w a s w a s h e d w i t h a few m i s of the e q u i l i b r a t i o n b u f f e r t h e n a 2 l i t r e l i n e a r g r a d i e n t of p o t a s s i u m p h o s p h a t e , - 2 3 f r o m 10 M to 0. 2 M w a s a p p l i e d . B o t h v e s s e l s c o n t a i n e d 10 M m e r c a p t o e t h a n o l but no g l y c e r o l . W h e n the l i n e a r g r a d i e n t of p o t a s s i u m p h o s p h a t e f i n i s h e d , the c o l u m n w a s w a s h e d w i t h 90 m l of 0. 20 M _ 3 p o t a s s i u m p h o s p h a t e , p H 7. 5, 10 M m e r c a p t o e t h a n o l . T h e n the h e a d of l i q u i d above the c o l u m n p a c k i n g w a s r e m o v e d a n d the c o l u m n w a s _ 3 w a s h e d w i t h 0. 20 M p o t a s s i u m p h o s p h a t e , 10" M m e r c a p t o e t h a n o l , 5% a m m o n i u m s u l f a t e . The c o l u m n e l u a t e w a s c o l l e c t e d as 11 m l f r a c t i o n s a n d the a b s o r b a n c e at 280 mpu w a s r e c o r d e d . T h e e l u t i o n p r o f i l e i s s h o w n i n F i g u r e 3. E . D e t e c t i o n of A m i n o a c y l - s - R N A S y n t h e t a s e s i n the H y d r o x y a p a t i t e  C o l u m n E l u a t e T h e a s s a y s w e r e c a r r i e d out as d e s c r i b e d i n S e c t i o n C . A l i q u o t ' s of the f r a c t i o n s f r o m the p h o s p h a t e g r a d i e n t w e r e . a s s a y e d d i r e c t l y . T h e e l u a t e f r o m the 5% a m m o n i u m s u l f a t e w a s h ( test t u b e s 200 to 215 i n c l u s i v e , F i g . 3) w a s c o m b i n e d (174 m l , 23. 5 O . D . u n i t s ) a n d c o n c e n -t r a t e d by u l t r a f i l t r a t i o n ( B l a t t est a l . , 1965) to 5. 5 m l i n a D i a f l o a p p a r a t u s ( A m i c o n C o r p ; ), u s i n g a U M - 1 m e m b r a n e , at 0 ° . T h e c o n c e n t r a t e w a s A m m o n i u m S u l f a t e ( 5 ° / o ) E o oo 30.6 > i -00 g 0 . 4 O < Q 2 CL O 0 S O 8 0 100 120 140 160 180 2 0 0 F r a c t i o n No. 2 0 4 0 F I G U R E 3. E l u t i o n p r o f i l e f r o m the C h r o m a t o g r a p h y of F r a c t i o n III on h y d r o x y a p a t i t e JO.1 - 2 2 8 -f r e e d f r o m e x c e s s a m m o n i u m s u l f a t e b y p a s s a g e t h r o u g h a G - 2 5 S e p h a d e x 2 - 3 c o l u m n , p r e v i o u s l y e q u i l i b r a t e d w i t h 10" M p o t a s s i u m p h o s p h a t e , 10 M m e r c a p t o e t h a n o l , 4 0 % g l y c e r o l ( v / v ) , p H 7. 5. F r a c t i o n s f r o m the S e p h a d e x c o l u m n c o m p r i s i n g the l e a d i n g p e a k of a b s o r b a n c e (at 280 mp_) w e r e c o m b i n e d a n d s t o r e d at - 1 8 ° u n t i l r e a d y f o r u s e ( F r a c t i o n A ) . - 2 A l i q u o t s of F r a c t i o n A w e r e d i l u t e d 1:10 w i t h 10 M p o t a s s i u m _ 3 p h o s p h a t e , 10 M m e r c a p t o e t h a n o l , 4 0 % g l y c e r o l ( v / v ) , p H 7. 5 t h e n a s s a y e d f o r the v a r i o u s a m i n o a c y l - s - R N A s y n t h e t a s e a c t i v i t i e s . T h e r e s u l t s a r e s h o w n i n T a b l e V I a l o n g w i t h the c o r r e s p o n d i n g s y n t h e t a s e a c t i v i t i e s c o n t a i n e d i n the d e s a l t e d a m m o n i u m s u l f a t e . F r a c t i o n III (see S e c t i o n D) a f t e r i t h a d b e e n l e f t at 3 - 5 ° d u r i n g the p e r i o d of c h r o m a t o g r a p h y . T h e a s s a y s w e r e c a r r i e d out at a 1:20 d i l u t i o n e x c e p t w h e r e n o t e d . T h e c o l u m n f r a c t i o n s c o n t a i n i n g the a r g i n y l - s - R N A s y n t h e t a s e a c t i v i t y ( tes t tubes 170 to 199 i n c l u s i v e , F i g . 3) w e r e c o m b i n e d a n d c o n c e n t r a t e d f r o m 312 m l (about 12 O . D . u n i t s at 280 m^i) to about 2 m l on a D i a f l o U M - 1 m e m b r a n e ' a t 0 ° . T h e c o n c e n t r a t e w a s p a s s e d t h r o u g h a G - 2 5 S e p h a d e x c o l u m n as d e s c r i b e d a b o v e . T h e S e p h a d e x e l u a t e c o n t a i n i n g a r g i n y l - s - R N A s y n t h e t a s e a c t i v i t y w a s s t o r e d at - 1 8 ° ( F r a c t i o n B ) p r i o r to d e t e r m i n a t i o n of a r g i n y l - s - R N A s y n t h e t a s e a c t i v i t y ( S e c t i o n F ) . - 2 2 9 -T A B L E V I a A m i n o a c y l - s - R N A S y n t h e t a s e s P r e s e n t i n the A m m o n i u m S u l f a t e E l u a t e s A m i n o m ^ i m o l e s a m i n o a c i d i n c o r p o r a t e d ^ A c i d D e s a l t e d F r a c t i o n III F r a c t i o n A a l a 0. 18 0. 98 a r g 0. 28 2. 3 a s p 0. 68 1.7 a s n c 0. 03 0. 007 g i n c 0. 01 0. 05 g i y 0. 58 3. 5 i l e 0. 08 0. 24 l e u 0. 23 0 l y s 0. 67 5. 9 m e t 0. 23 0 . 4 2 phe 0. 43 0 . 0 4 s e r 0 . 4 7 0. 03 t h r 0. 24 1. 3 t r y 0. 09 0 . 0 3 t y r 0. 66 9. 2 v a l 0. 03 0 a V a l u e s u n d e r l i n e d i n d i c a t e l o s s of a c t i v i t y . r r y x m o l e s of a m i n o a c i d i n c o r p o r a t e d i n 1. 25 m g s - R N A b y 0. 05 m g p r o t e i n u n d e r the c o n d i t i o n s d e s c r i b e d i n S e c t i o n 5 - C . a s n a n d g i n a s s a y e d u n d i l u t e d f o r F r a c t i o n A - 2 3 0 -F . D e t e r m i n a t i o n of A m i n o a c y l - s - R N A S y n t h e t a s e A c t i v i t i e s o T h e a s s a y s w e r e c a r r i e d out at 25 i n 1 m l g l a s s t u b e s c o n t a i n i n g ( i n a f i n a l v o l u m e of 0 . 20 m l ) 20yujmoles s o d i u m c a c o d y l a t e , p_H 7. 5, 4 ^ftmoles K C l , 2yi_moles M g C l 2 , 1. 5^jumoles A T P , 0 . 0 8 yuumole E D T A , about 50 m y u m o l e s of a m i n o a c i d w i t h s p e c i f i c a c t i v i t y 10 ^LlCi p e r ^jcrnole, 2. 5 m g s - R N A , a n d 50 pi of e n z y m e s o l u t i o n . T h e e n z y m e , a p p r o p r i a t e l y d i l u t e d , w a s a d d e d at z e r o t i m e . A l i q u o t s (25 ^ i l ) w e r e r e m o v e d at one m i n i n t e r v a l s , a p p l i e d to W h a t m a n 3 M M f i l t e r d i s k s a n d w a s h e d as d e s c r i b e d i n S e c t i o n C . R a d i o a c t i v i t y w a s a l s o d e t e r m i n e d as d e s c r i b e d i n S e c t i o n C . E n z y m e a c t i v i t i e s w e r e d e t e r m i n e d u n d e r c o n d i t i o n s w h i c h gave l i n e a r i n c o r p o r a t i o n of a g i v e n a m i n o a c i d d u r i n g 6 m i n . A u n i t of e n z y m e c a t a l y z e s the f o r m a t i o n of 1 r r y x m o l e of ^ C - a m i n o a c y l -s - R N A p e r m i n . T h e r e s u l t s f o r s e v e r a l a m i n o a c i d s a r e s u m m a r i z e d i n T a b l e V I I . 6. P r e p a r a t i o n a n d P a r t i a l P u r i f i c a t i o n of A m i n o a c y l - s - R N A  S y n t h e t a s e s f r o m Y e a s t : M e t h o d 3 ( M o d i f i e d ) A . P r e p a r a t i o n of the C e l l - F r e e P r o t e i n E x t r a c t T w o p o u n d s of f r e s h , p r e s s e d b a k e r s ' y e a s t w e r e t h o r o u g h l y c r u m b l e d t h e n a d d e d to 2 l i t r e s of t o l u e n e c o n t a i n i n g e x c e s s d r y i c e a n d 0. 20 m l of 1 M D F P . T h e m i x t u r e w a s l e f t at - 1 8 ° f o r one h o u r t h e n . t h e . t o l u e n e w a s d e c a n t e d a n d the f r o z e n y e a s t c e l l s l e f t at 3 - 5 ° f o r 72 h r . T A B L E V I I S p e c i f i c A c t i v i t i e s of S e v e r a l A m i n o a c y l - s - R N A S y n t h e t a s e s A m i n o A c i d T y r A s p G l y L y s A r g D e s a l t e d F r a c t i o n III m g p r o t e i n / a s s a y a S p e c i f i c a c t i v i t y b T o t a l u n i t s c 0. 005, 0. 01 6 . 4 3. 4 0. 055 , 0. 11 2. 0 1. 1 0. 014, 0. 028 1. 8 0. 94 0. 0 2 2 , 0. 044 2. 7 1. 4 0. 0 2 8 , 0 . 056 3 . 4 1. 8 F r a c t i o n A m g p r o t e i n / a s s a y a S p e c i f i c . a c t i v i t y b T o t a l u n i t s c P u r i f i c a t i o n 0 . 0 0 1 5 , 0 . 0 0 3 3 0 . 0 0. 67 4 . 7 0. 0225, 0. 045 3. 2 0. 069 1. 6 0. 0 2 2 5 , 0. 045 1. 0 0. 023 0. 56 0. 0 0 9 , 0. 018 6. 63 0. 15 2. 5 0. 011 , 0. 022 9 . 1 0 . 2 0 5 2. 7 F r a c t i o n B m g p r o t e i n / a s s a y a S p e c i f i c a c t i v i t y b T o t a l U n i t s c P u r i f i c a t i o n 0. 0 0 5 , 0 . 0024 5 0 . 0 0 . 012 15. % R e c o v e r y 20 6. 6 2. 5 11 12 a S p e c i f i c a c t i v i t y c a l c u l a t e d at two e n z y m e c o n c e n t r a t i o n s S p e c i f i c a c t i v i t y e x p r e s s e d as u n i t s of e n z y m e a c t i v i t y p e r m g of p r o t e i n T o t a l u n i t s o b t a i n e d by m u l t i p l y i n g s p e c i f i c a c t i v i t y by t o t a l m g of p r o t e i n - 2 3 2 -The f r o z e n y e a s t l i q u e f i e d d u r i n g the 72 h r at 3 - 5 ° a n d the p H of the p a s t e m e a s u r e d about 5. 8. T h e j>H w a s a d j u s t e d to 7, 2 by the a d d i t i o n of 200 m l of 1 M T r i s ( c h l o r i d e ) , p_H 8. 0. C e l l d e b r i s w a s r e m o v e d b y c e n t r i f u g a t i o n at 14, 600 x g f o r 30 m i n i n the G S A h e a d of a n R C - 2 B r e f r i g e r a t e d (0°) c e n t r i f u g e . T o the s u p e r n a t e (500 m l ) w e r e a d d e d 0.10 m l of 1 M D F P a n d 10 g s t r e p t o m y c i n s u l f a t e a n d the m i x t u r e l e f t f o r 20 m i n i n i c e . T h e s o l u t i o n w a s c e n t r i f u g e d as d e s c r i b e d a b o v e , a n d the s u p e r n a t e d e c a n t e d . T h e n a m m o n i u m s u l f a t e (56.1 g p e r 100 m l c e l l - f r e e e x t r a c t ) w a s a d d e d t o the s u p e r n a t e a n d , a f t e r 20 m i n i n i c e , the p r e c i p i t a t e d m a t e r i a l w a s c o l l e c t e d by c e n t r i f u g a t i o n . B . P a r t i a l P u r i f i c a t i o n of the A m i n o a c y l - s - R N A S y n t h e t a s e s O n e of the s i x p e l l e t s o b t a i n e d a b o v e ( S e c t i o n 6 - A ) w a s d i s s o l v e d 2 3 4 i n a m i n i m u m of 10 M s o d i u m p h o s p h a t e , 10 M m e r c a p t o e t h a n o l , 10" M E D T A a n d 4 0 % g l y c e r o l ( v / v ) , p H 7. 5. E x c e s s a m m o n i u m s u l f a t e w a s r e m o v e d b y p a s s i n g the s o l u t i o n t h r o u g h a G - 2 5 S e p h a d e x c o l u m n e q u i l i b r a t e d w i t h the s a m e b u f f e r . The e l u a t e f r o m the S e p h a d e x c o l u m n c o n t a i n i n g u l t r a v i o l e t a b s o r b i n g (at 280 mjm) m a t e r i a l e x c l u d e d by the S e p h a d e x b e a d s w a s c o m b i n e d (129 m l ) a n d s t o r e d at - 1 8 ° u n t i l r e a d y f o r the c h r o m a t o g r a p h y . A p o r t i o n of the c o m b i n e d e l u a t e (5 m l ) w a s set a s i d e a n d the r e m a i n i n g 124 m l w a s a p p l i e d to a h y d r o x y a p a t i t e c o l u m n (26 x 5 c m d i a m . ) _ 2 w h i c h h a d b e e n p r e v i o u s l y e q u i l i b r a t e d w i t h 10 M s o d i u m p h o s p h a t e , - 2 3 3 -10'° M m e r c a p t o e t h a n o l , 10 M E D T A , 4 0 % g l y c e r o l ( v / v ) , p _ H 7 . 5 . T h e c o l u m n w a s d e v e l o p e d w i t h a 3 l i t r e l i n e a r p h o s p h a t e g r a d i e n t f r o m 0. 01 to - 3 0. 20 M s o d i u m p h o s p h a t e , p H 7. 5, b o t h v e s s e l s c o n t a i n i n g 10 M m e r c -a p t o e t h a n o l , 1 0 " 4 M E D T A a n d 4 0 % g l y c e r o l ( v / v ) . A f l o w r a t e of about 1 m l p e r m i n w a s m a i n t a i n e d w i t h the a i d of a n A c c u - F l o p u m p a n d 18 m l f r a c t i o n s w e r e c o l l e c t e d . W h e n the p h o s p h a t e g r a d i e n t e x p i r e d , a 4 0 0 m l l i n e a r g r a d i e n t of a m m o n i u m s u l f a t e f r o m 0 to 10% (w/v) w a s a p p l i e d . B o t h v e s s e l s c o n t a i n e d 0. 2 M s o d i u m p h o s p h a t e , 10 M m e r c a p t o e t h a n o l , 1 0 ~ 4 M E D T A , 4 0 % g l y c e r o l ( v / v ) , p H 7. 5. The e l u t i o n p r o f i l e i s s h o w n i n F i g u r e 4 . C . D e t e c t i o n of A m i n o a c y l - s - R N A S y n t h e t a s e i n the H y d r o x y a p a t i t e  C o l u m n E l u a t e T h e a s s a y s w e r e c a r r i e d out as d e s c r i b e d i n S e c t i o n 5 - C e x c e p t that the s - R N A (1. 25 m g ) w a s a d d e d l a s t to the c o l d i n c u b a t i o n m i x t u r e and the t o t a l i n c u b a t i o n t i m e w a s 45 m i n . In a d d i t i o n , the f r a c t i o n f r o m the a m m o n i u m s u l f a t e g r a d i e n t ( f r a c t i o n s 187 to 210, F i g . 4) w e r e a s s a y e d i n d i v i d u a l l y . T h e b l a n k s c o n t a i n e d s t a r t i n g b u f f e r i n p l a c e of e n z y m e s o l u t i o n w h i l e the c o n t r o l s c o n t a i n e d the c r u d e a m m o n i u m s u l f a t e p r e c i p i t a t e d a n d d e s a l t e d c e l l - f r e e e x t r a c t d i l u t e d 20 f o l d w i t h s t a r t i n g b u f f e r . T a b l e V I I I l i s t s the m p m o l e s of 20 a m i n o a c i d s i n c o r p o r a t e d i n t o 1. 25 m g of s - R N A i n 45 m i n by 0. 05 m g of c r u d e p r o t e i n p r i o r to c h r o m a t o g r a p h y . OPTICAL DENSITY(280mu) . _ _ j 9 Ammonium o p pSulfate(%) r o Phosphate BuffeKM) - 2 3 5 -T A B L E V I I I A m i n o a c y l - s - R N A S y n t h e t a s e s P r e s e n t  i n the W h o l e - p r o t e i n : E x t r a c t A m i n o m y u m o l e s of a m i n o A c i d a c i d i n c o r p o r a t e d a a l a 0. 27 a r g 0. 80 a s p 0 . 5 2 a s n 0. 09 c y s 0. 22 g l u 0 . 0 3 g i n 0. 13 g i y 1.13 h i s 0. 72 i l e 0 . 2 8 l e u 1 .10 l y s 1. 65 m e t 0. 52 phe 0. 52 p r o 0. 003 s e r 0. 52 t h r 0. 21 t r y 0. 26 t y r 0. 20 v a l 0. 21 myjonoles of a m i n o a c i d i n c o r -p o r a t e d i n 1. 25 m g of s - R N A b y 0. 05 m g of w h o l e - p r o t e i n u n d e r the c o n d i t i o n s d e s c r i b e d i n S e c t i o n 6 - C . F i g u r e s 5 a n d 6 s h o w the p o s i t i o n of e l u t i o n of the v a r i o u s a m i n o a c y l - s - R N A s y n t h e t a s e s f r o m the h y d r o x y a p a t i t e c o l u m n . - 2 3 6-Ammon ium Sul fate 100 120 140 160 180 200 F rac t ion No. E l u t i o n p r o f i l e s of a m i n o a c y l - s - R N A s y n t h e t a s e s f r o m h y d r o x y a p a t i t e . -237-2.0 < z cr to cn E LO CO "O (D D i _ O Q. i _ O u c 1.0 O L \ \ g l u 190 200 210 190 200 210 190 F r a c t i o n N u m b e r 200 210 E eor o O C E • a • ±6 1 0 o jD O E a . 0 190 200 210 190 200 F r a c t i o n N u m b e r 210 F I G U R E 6. E l u t i o n p r o f i l e s of a m i n o a c y l - s - R N A s y n t h e t a s e s f r o m h y d r oxy a p a t i t e . - 2 3 8 -D I S C U S S I O N T h e n e e d to p r e p a r e a m i n o a c y l - s - R N A s y n t h e t a s e s i n r e l a t i v e l y l a r g e q u a n t i t i e s a r o s e as a r e s u l t of a t t e m p t s to d e v e l o p a c h e m i c a l p r o c e d u r e f o r the i s o l a t i o n of a m i n o a c i d - s p e c i f i c s - R N A ' s . T h u s the p r o c e d u r e d e s c r i b e d i n P a r t I I - D d e p e n d s o n the a v a i l a b i l i t y of the a m i n o -a c y l - s - R N A s y n t h e t a s e e n z y m e s i n q u a n t i t i e s s u f f i c i e n t f o r c h a r g i n g g r a m q u a n t i t i e s of s - R N A w i t h a s p e c i f i c a m i n o a c i d . In t h i s r e g a r d , y e a s t p r e s e n t s a p a r t i c u l a r p r o b l e m b e c a u s e of the d i f f i c u l t e n c o u n t e r e d i n d i s r u p t i n g the c e l l u n d e r c o n d i t i o n s w h i c h a r e s u f f i c i e n t l y m i l d to p r e s e r v e the d e s i r e d e n z y m e a c t i v i t i e s . T h e u s u a l m e t h o d e m p l o y e d to d i s r u p t y e a s t c e l l s i s by g r i n d i n g t h e m w i t h g l a s s b e a d s i n a h o m o g e n i z e r . T h i s p r o c e d u r e ( S e c t i o n 1, M e t h o d 1) w a s a d e q u a t e f o r p r e p a r i n g the s y n t h e t a s e e n z y m e s i n q u a n t i t i e s s u f f i c i e n t f o r the p u r p o s e of a s s a y i n g f o r a m i n o a c i d a c c e p t o r a c t i v i t y . A t t e m p t s to s c a l e u p the p r o c e d u r e such that 50 a n d 100 g b a t c h e s of y e a s t c e l l s c o u l d be d i s r u p t e d w e r e u n s u c c e s s f u l . A l t h o u g h the e n z y m e a c t i v i t i e s w e r e p r e s e n t i n the s u p e r n a t e a f t e r the u l t r a c e n t r i f u g a t i o n s t e p t h e y w e r e p r e s e n t i n r e l a t i v e l y l o w a m o u n t s . T h e l o w l e v e l s of s y n t h e t a s e a c t i v i t y p r o b a b l y r e f l e c t e d the i n e f f i c i e n c y of the g l a s s b e a d m e t h o d f o r d i s r u p t i n g the y e a s t c e l l s . It s h o u l d be p o i n t e d out h e r e , h o w e v e r , that M a k m a n (1966) has s u c c e s s f u l l y e m p l o y e d t h i s m e t h o d f o r the d i s r u p t i o n of 100 g b a t c h e s of y e a s t c e l l s f o r the p u r i f i c a t i o n of s e r y l - , p h e n y l a l a n y l - , - 2 3 9 -a r g i n y l - a n d l e u c y l - s - R N A s y n t h e t a s e s . U s e of c e r t a i n o t h e r p r o c e d u r e s f o r d i s r u p t i o n of y e a s t c e l l s , s u c h as t r e a t m e n t w i t h t o l u e n e , i n c u b a t i o n w i t h b i c a r b o n a t e a n d f r e e z i n g i n a d r y i c e - e t h a n o l b a t h have b e e n t r i e d but w e r e f o u n d to be u n s a t i s f a c t o r y f o r p r e p a r a t i o n of the s e r i n e e n z y m e ( M a k m a n a n d C a n t o n i , 1965). S o n i c d i s i n t e g r a t i o n , o r f r e e z i n g i n l i q u i d n i t r o g e n f o l l o w e d by h o m o g e n i z a t i o n w i t h g l a s s b e a d s ( B e n n e t t et a l . , 1963) r e s u l t e d i n c r u d e e x t r a c t s w i t h s e r y l - s - R N A s y n t h e t a s e a c t i v i t y c o m p a r a b l e to that o b t a i n e d b y the g l a s s b e a d m e t h o d . A n a l t e r n a t i v e p r o c e d u r e i n v e s t i g a t e d f o r d i s r u p t i n g l a r g e q u a n t i t i e s of y e a s t c e l l s u t i l i z e d the a b i l i t y of t o l u e n e to l y s e y e a s t c e l l s w h e n w a r m e d to 3 8 t o 4 0 ° ( K u n i t z , 1952; H e p p e l , 1955). T h i s p r o c e d u r e has b e e n e m p l o y e d by v o n T i g e r s t r o m and T e n e r (1967) f o r the p u r i f i c a t i o n of h i s t i d y l - s - R N A s y n t h e t a s e f r o m y e a s t . A l t h o u g h the y e a s t c e l l s w e r e e f f e c t i v e l y l y s e d w i t h t o l u e n e at 3 7 ° m o s t of the s y n t h e t a s e e n z y m e s w e r e d e s t r o y e d b y t h i s t r e a t m e n t . S t u d i e s d e s c r i b e d i n t h i s t h e s i s rshow that the i n a c t i v a t i o n of the s y n t h e t a s e e n z y m e s d u r i n g t o l u e n e l y s i s at 3 7 ° w a s due to a d r o p i n the p H of the m e d i u m . A s d e s c r i b e d i n S e c t i o n 2 - B , w h e n y e a s t c e l l s w e r e l y s e d w i t h t o l u e n e at 3 7 ° a d r o p i n the p H of the m e d i u m c o u l d be o b s e r v e d by m o n i t o r i n g the s o l u t i o n w i t h a p H m e t e r , u s i n g a g l a s s e l e c t r o d e . T h e d r o p i n p H of the m e d i u m w a s o b s e r v e d c o n c u r r e n t l y , w i t h the e v o l u t i o n of gas a n d - 2 4 0 -the l i q u e f a c t i o n of the y e a s t p a s t e . T h e p_H of the m i x t u r e w a s m a i n t a i n e d at about 7 by the a d d i t i o n of 1 N s o d i u m h y d r o x i d e a n d the c o n s u m p t i o n of a l k a l i w a s l i n e a r u p t o about 35 m i n a f t e r the t e m p e r a t u r e of the m i x t u r e h a d r e a c h e d 3 7 ° . U s i n g t h i s as a n i n d i c a t i o n of the extent of l y s i s , the y e a s t c e l l s w e r e e s s e n t i a l l y a l l d i s r u p t e d a f t e r 40 m i n at 3 7 ° . C e n t r i -f u g a t i o n of the m i x t u r e y i e l d e d a c e l l - f r e e e x t r a c t c o n t a i n i n g m o s t of the a m i n o a c y l - s - R N A s y n t h e t a s e e n z y m e s . P r o l y l - , g l u t a m i n y l - a n d g l u t a m y l - s - R N A s y n t h e t a s e s w e r e n o t a b l e e x c e p t i o n s . T h e c e l l - f r e e e x t r a c t w a s t h e n f r a c t i o n a t e d u s i n g a m m o n i u m s u l f a t e as d e s c r i b e d i n S e c t i o n 2 - C . M o s t of the g l y c y l - s - R N A s y n t h e t a s e a c t i v i t y w a s p r e c i p i t a t e d b e t w e e n 2, 4 a n d 2. 9 M a m m o n i u m s u l f a t e ( T a b l e I, A m m o n i u m S u l f a t e F r a c t i o n B ) . In a d d i t i o n to b e i n g a b l e to p r e p a r e l a r g e q u a n t i t i e s of y e a s t c e l l - f r e e e x t r a c t , i t w a s a l s o d e s i r a b l e to have a s i m p l e p r o c e d u r e f o r the p a r t i a l p u r i f i c a t i o n of the s y n t h e t a s e e n z y m e s . It w a s c o n s i d e r e d i m p o r t a n t to have p r e p a r a t i o n s of the s y n t h e t a s e e n z y m e s w h i c h w e r e f r e e f r o m n u c l e a s e s a n d p e p t i d a s e s f o r u s e i n the c o m b i n e d c h e m i c a l and c h r o m a t o g r a p h i c p r o c e d u r e d e s c r i b e d i n P a r t I I - D . T h e n u c l e a s e s w o u l d of c o u r s e d e g r a d e the s - R N A w h i l e the p e p t i d a s e s c o u l d s u p p l y a m i n o a c i d s o t h e r t h a n the one of i n t e r e s t w h i c h w o u l d t e n d to c o m p l i c a t e the p r o c e d u r e . - 2 4 1 -M u e n c h a n d B e r g (1966) have d e s c r i b e d the f r a c t i o n a t i o n of the a m i n o a c y l - s - R N A s y n t h e t a s e e n z y m e s f r o m E . c o l i o n h y d r o x y a p a t i t e . A l i n e a r g r a d i e n t of p h o s p h a t e b u f f e r f r o m 0. 01 M to 0. 20 M w a s u s e d to e l u t e a l l the a m i n o a c y l - s - R N A s y n t h e t a s e e n z y m e s . A f t e r d e s a l t i n g on S e p h a d e x , a p o r t i o n of the a m m o n i u m s u l f a t e f r a c t i o n B w a s a p p l i e d t o a h y d r o x y a p a t i t e c o l u m n and the l a t t e r d e v e l o p e d w i t h a l i n e a r p h o s p h a t e g r a d i e n t u p to 0. 20 M . T h e i n c o r p o r a t i o n of g l y c e r o l (35% v / v ) i n the d e v e l o p i n g s o l v e n t s p e r m i t t e d s t o r a g e of the c o l u m n e l u a t e at - 1 8 ° w i t h o u t f r e e z i n g . T h i s c o n d i t i o n g r e a t l y e n h a n c e d the s t a b i l i t y of the s y n t h e t a s e e n z y m e s . G l y c e r o l has b e e n u s e d to s t a b i l i z e o t h e r e n z y m e s d u r i n g s t o r a g e a n d p u r i f i c a t i o n ( J a r a b a k et a l . 1962). S i n c e the c h r o m a t o g r a p h y w a s c a r r i e d out i n the c o l d r o o m at 3 - 5 ° i n the p r e s e n c e of 35% g l y c e r o l , i t w a s e s s e n t i a l to use a p r e s s u r e p u m p i n o r d e r to m a i n t a i n a r e a s o n a b l e f l o w r a t e of the o r d e r of 1 m l p e r m i n . A l t h o u g h m o s t of the p r o t e i n w a s e l u t e d by the 0. 01 to 0. 20 M p h o s p h a t e g r a d i e n t (see F i g . 1), the e l u a t e s h o w e d no a c t i v i t y w h e n a s s a y e d f o r g l y c y l - s - R N A s y n t h e t a s e a c t i v i t y . T h e l a t t e r w a s e l u t e d w h e n the c o l u m n w a s w a s h e d w i t h a n i n c r e a s i n g c o n c e n t r a t i o n of a m m -o n i u m s u l f a t e f r o m 0 to 10% ( w / v ) . R a d i o c h r o m a t o g r a p h i c a n a l y s i s w a s c a r r i e d out on a l t e r n a t e f r a c t i o n s of the a m m o n i u m s u l f a t e e l u a t e to d e t e r m i n e i f a m i n o a c y l - s -- 2 4 2 -R N A s y n t h e t a s e s o t h e r t h a n the g l y c i n e e n z y m e w e r e p r e s e n t ( S e c t i o n 2 - F ) . F i g u r e 2 c l e a r l y s h o w s the p r e s e n c e of at l e a s t n i n e s y n t h e t a s e a c t i v i t i e s . T h u s m a n y of the a m i n o a c y l - s - R N A s y n t h e t a s e s a r e not e l u t e d f r o m h y d r o x y a p a t i t e by u p to 0. 2 M p h o s p h a t e b u f f e r . A l s o e v i d e n t f r o m F i g u r e 2 i s the f a c t that the a c t i v i t i e s a r e e l u t e d as r e l a t i v e l y s h a r p p e a k s as j u d g e d by the a p p e a r a n c e a n d d i s a p p e a r a n c e of the d i f f e r e n t p e a k s of r a d i o a c t i v i t y on the c h r o m a t o g r a m . T h e r e a s o n so m a n y of the s y n t h e t a s e e n z y m e s show s u c h a s t r o n g a f f i n i t y f o r h y d r o x y a p a t i t e i s f a r f r o m c l e a r . T h e r e a r e a few i n d i c a t i o n s that the a f f i n i t y m a y be a r e s u l t of the f o r m a t i o n of s o m e t y p e of n u c l e o t i d e - o r p o l y n u c l e o t i d e - e n z y m e c o m p l e x . F o r e x a m p l e , M a k m a n a n d C a n t o n i (1966) have d e m o n s t r a t e d a n i n c r e a s e d t h e r m a l s t a b i l i t y of c e r t a i n s y n t h e t a s e e n z y m e s i n the p r e s e n c e of A T P a n d m a g n e s i u m , w h i c h s u g g e s t s the f o r m a t i o n of a d e f i n i t e A T P - m a g n e s i u m - e n z y m e c o m p l e x . I n . a d d i t i o n t h e s e w o r k e r s ( M a k m a n a n d C a n t o n i , 1965) have s u g g e s t e d that the p u r i f i e d s e r y l - s - R N A s y n t h e t a s e f r o m y e a s t c o n t a i n s a n u c l e o t i d e , as y e t u n i d e n t i f i e d , a t t a c h e d to the e n z y m e m o l e c u l e . It i s a l s c i n t e r e s t i n g to note that n u c l e o t i d e p o l y p h o s p h a t e s a r e s t r o n g l y a d s o r b e d to the h y d r o x y a p a t i t e a n d r e q u i r e h i g h m o l a r i t i e s of p h o s p h a t e buf fer to be e l u t e d ( B e r n a r d i , 1964). S i n c e m a n y of the a m i n o a c y l - s - R N A s y n t h e t a s e a c t i v i t i e s w e r e not e l u t e d by the 0. 01 to 0. 2 0 M p h o s p h a t e g r a d i e n t a n a t t e m p t w a s m a d e - 2 4 3 -to r e m o v e the b u l k of the p r o t e i n by w a s h i n g the h y d r o x y a p a t i t e c o l u m n i n a s i n g l e s t e p w i t h 0. 20 M p h o s p h a t e b u f f e r ( S e c t i o n 3 B , C h r o m a t o g r a p h y 1). A l l of the a p p l i e d m a t e r i a l a n d a l l of the s y n t h e t a s e a c t i v i t y w e r e e l u t e d i n the 0. 2 M p h o s p h a t e b u f f e r . T o be s u r e the c o l u m n h a d not b e e n o v e r l o a d e d the c h r o m a t o g r a p h y w a s r e p e a t e d ( C h r o m a t o g r a p h y 2) by a p p l y i n g a n a m o u n t of c r u d e e x t r a c t e q u i v a l e n t to that u s e d i n the g r a d i e n t c h r o m a t o g r a p h y . A g a i n a l l the a p p l i e d m a t e r i a l a n d a l l of the s y n t h e t a s e a c t i v i t y w a s e l u t e d i n the 0. 2 M p h o s p h a t e b u f f e r . T h e c h r o m a t o g r a p h y w a s r e p e a t e d a g a i n e x c e p t the c r u d e e x t r a c t w a s . a p p l i e d i n , 0. 01 M p h o s p h a t e b u f f e r a n d the c o l u m n s u b s e q u e n t l y w a s h e d w i t h the s a m e b u f f e r . N o s y n t h e t a s e a c t i v i t y a n d o n l y a s m a l l a m o u n t o f the a p p l i e d m a t e r i a l w a s e l u t e d . W h e n the c o l u m n w a s w a s h e d w i t h 0. 20 M p h o s p h a t e b u f f e r , m o r e t h a n 9 0 % of the a p p l i e d m a t e r i a l w a s e l u t e d a n d the e l u a t e c o n t a i n e d m o s t of the o r i g i n a l s y n t h e t a s e a c t i v i t y . O n l y a s m a l l a m o u n t of the s y n t h e t a s e a c t i v i t y w a s d e t e c t e d w h e n the c o l u m n w a s s u b s e q u e n t l y w a s h e d w i t h 10% a m m o n i u m s u l f a t e ( w / v ) . It b e c a m e c l e a r that the c r u d e e x t r a c t h a d to be a p p l i e d to the c o l u m n i n l o w c o n c e n t r a t i o n s of p h o s p h a t e b u f f e r . In a d d i t i o n , the c o l u m n m u s t be d e v e l o p e d e i t h e r w i t h s m a l l i n c r e m e n t s ( i n c r e a s i n g i n 0 . 0 5 M s t e p s ) o r p r e f e r a b l y , w i t h a n i n c r e a s i n g l i n e a r g r a d i e n t of p h o s p h a t e b u f f e r , f o l l o w e d by an a m m o n i u m s u l f a t e (10% w / v ) w a s h . - 2 4 4 -L y s i s of y e a s t c e l l s w i t h t o l u e n e at 3 7 ° u n d e r c o n d i t i o n s of c o n t r o l l e d p H f o l l o w e d by c h r o m a t o g r a p h y on h y d r o x y a p a t i t e o f f e r e d a s i m p l e a n d u s e f u l p r o c e d u r e f o r o b t a i n i n g m o s t of the s y n t h e t a s e e n z y m e s . H o w e v e r t h e r e w e r e s t i l l a few s y n t h e t a s e s w h i c h w e r e a b s e n t . T h u s , m e t h o d s w e r e i n v e s t i g a t e d f o r l y s i n g the y e a s t c e l l s u n d e r m i l d e r c o n -d i t i o n s of t e m p e r a t u r e . A r e c e n t r e p o r t on the p r e p a r a t i o n a n d . p u r i f i c a t i o n of y e a s t h e x o k i n a s e ( L a z a r u s ^ t a l . , 1966) d e s c r i b e d the l y s i s of y e a s t c e l l s u s i n g t o l u e n e i n e x c e s s d r y i c e . S e c t i o n 5 - A d e s c r i b e s the p r e p a r a t i o n of a y e a s t c e l l - f r e e e x t r a c t by e s s e n t i a l l y the s a m e p r o c e d u r e . A s u m m a r y of the e x p e r i m e n t a l p r o c e d u r e s e m p l o y e d i s s h o w n i n C h a r t I. A l t h o u g h t w o p o u n d l o t s of c r u m b l e d y e a s t c a k e w e r e l y s e d by t o l u e n e c o n t a i n i n g e x c e s s d r y i c e , the m e t h o d i s e a s i l y - a p p l i e d to m u c h l a r g e r q u a n t i t i e s . In S e c t i o n 5 - A the y e a s t c e l l s w e r e l e f t i n the t o l u e n e - d r y i c e m i x t u r e f o r s i x h o u r s p r i o r to i n c u b a t i o n at 3 - 5 ° . H o w -e v e r , , as d e s c r i b e d i n S e c t i o n 6 - A , one h o u r i n the t o l u e n e - d r y i c e m i x t u r e i s s u f f i c i e n t . It s h o u l d a l s o be p o i n t e d out that the o p t i m u m t i m e of i n c u b a t i o n of the c e l l p a s t e at 3 - 5 ° a f t e r d e c a n t i n g the t o l u e n e has not b e e n r i g o r o u s l y e s t a b l i s h e d . A n a l i q u o t of the y e a s t p a s t e w h i c h w a s r e m o v e d a f t e r 12 h r i n c u b a t i o n at 3 - 5 ° w a s s h o w n to c o n t a i n a m i n o a c y l - s - R N A s y n t h e t a s e a c t i v i t y i n the s t a n d a r d a s s a y . In a d d i t i o n the 44 h r i n c u b a t i o n p e r i o d u s e d i n S e c t i o n 5 - A w a s b e t t e r t h a n the 72 h r 2 4 5 -C H A R T I S u m m a r y of E x p e r i m e n t a l ( M e t h o d 3) 2 l b b a k e r s ' y e a s t - t o l u e n e , d r y i c e (6 h r ) - i n c u b a t i o n at 3 - 5 ° , (44 h r ) J/ - c e n t r i f u g a t i o n C e l l - f r e e e x t r a c t - a m m o n i u m s u l f a t e f r a c t i o n a t i o n F r a c t i o n s I, II , III , I V , s u p e r n a t e of I V . ( T a b l e III) - G - 25 S e p h a d e x D e s a l t e d F r a c t i o n s II, III, I V , s u p e r n a t e of I V . ( T a b l e I V ) a s s a y e d w i t h 20 a m i n o a c i d s ( T a b l e V ) D e s a l t e d F r a c t i o n III H y d r o x y a p a t i t e C h r o m a t o g r a p h y (No g l y c e r o l ) ( F i g . 3) I-A p o r t i o n l e f t 3 d a y s at 3 - 5 ° t h e n a s s a y e d w i t h 16 a m i n o a c i d s ( T a b l e VI) Ir A m m o n i u m s u l f a t e e l u a t e - c o m b i n e d & c o n c e n t r a t e d •+• t h e n G - 2 5 S e p h a d e x (40% g l y c e r o l ) p h o s p h a t e g r a d i e n t e l u a t e - tubes 170 to 199 c o m b i n e d , c o n c e n t r a t e d t h e n G - 2 5 S e p h a d e x (40% g l y c e r o l ) ^ F r a c t i o n . A A s s a y e d w i t h 16 a m i n o a c i d s ( T a b l e VI) F r a c t i o n B C o n t a i n s a r g i n y l - s- R N A s y n t h e t a s e -246-i n c u b a t i o n p e r i o d used, i n S e c t i o n 6-A. T h u s i t s e e m s l i k e l y that i n c u b a t i o n p e r i o d s of l e s s than.44 h r at 3 - 5 ° w o u l d p r o v i d e j u s t as e f f i c i e n t l y s i s of the y e a s t c e l l s s i n c e the b r e a k d o w n of the c e l l w a l l p r o b a b l y o c c u r s a s a r e s u l t of f r e e z i n g at - 7 0 ° i n the p r e s e n c e of t o l u e n e . D i - i s o p r o p y l f l u o r o p h o s p h a t e ( D F P ) w a s a d d e d r o u t i n e l y d u r i n g p r e p a r a t i o n of the c e l l - f r e e e x t r a c t s to d e c r e a s e d e g r a d a t i o n of the s y n t h e t a s e e n z y m e s b y p r o t e o l y t i c e n z y m e s d u r i n g the e x t e n d e d i n c u b a t i o n s at 3 - 5 ° . T h i s p r e c a u t i o n w o u l d a l s o r e s u l t i n the f o r m a t i o n of f e w e r end g r o u p s f o r the p e p t i d a s e s , so that w h e n the e n z y m e p r e p a r a t i o n s a r e u s e d f o r c h a r g i n g s - R N A w i t h a p a r t i c u l a r a m i n o a c i d , e x t r a n e o u s a m i n o a c i d s w o u l d be p r e s e n t i n m i n i m a l a m o u n t s . T h e 14 p r e s e n c e of 0.15yu_mole of D F P d i d not af fect the s y n t h e s i s of C - g l y c y l -s - R N A u n d e r the c o n d i t i o n s d e s c r i b e d i n S e c t i o n 4-B. T h e c e l l - f r e e e x t r a c t , o b t a i n e d a f t e r r e m o v a l of the c e l l d e b r i s by c e n t r i f u g a t i o n , w a s f r a c t i o n a t e d by the a d d i t i o n of a m m o n i u m s u l f a t e (see C h a r t I ) . T h e r e s u l t i n g t h r e e f r a c t i o n s ( F r a c t i o n s II to I V ) a n d the s u p e r n a t e f r o m F r a c t i o n I V w e r e a s s a y e d f o r 20 a m i n o a c y l - s - R N A s y n t h e t a s e a c t i v i t i e s as d e s c r i b e d i n S e c t i o n 5-C. It w a s n e c e s s a r y to r e m o v e the e x c e s s a m m o n i u m s u l f a t e p r i o r t o a s s a y i n g s i n c e a m m o n i u m s u l f a t e d e c r e a s e s the l e v e l of m a x i m u m i n c o r p o r a t i o n of g l y c i n e i n t o s-R N A ( S e c t i o n 4-B). R e m o v a l of a m m o n i u m s u l f a t e w a s r e a d i l y a c h i e v e d by g e l f i l t r a t i o n on G-25 S e p h a d e x . A n a l i q u o t of the c o m b i n e d S e p h a d e x - 2 4 7 -e l u a t e w a s d i l u t e d p r i o r to a s s a y i n g , to about 0. 05 to 0. 06 m g of p r o t e i n p e r 50 y x l . T h i s a m o u n t of p r o t e i n w o u l d not f u l l y c h a r g e the s - R N A u n d e r the c o n d i t i o n s d e s c r i b e d i n S e c t i o n 5 - C . T h u s a r o u g h i n d i c a t i o n of the a m o u n t of e a c h a m i n o a c y l - s - R N A s y n t h e t a s e a c t i v i t y w a s o b t a i n e d f o r e a c h . f r a c t i o n . T h e r e s u l t s a r e t a b u l a t e d i n T a b l e V . E s s e n t i a l l y a l l the s y n t h e t a s e a c t i v i t i e s w e r e p r e c i p i t a t e d b e t w e e n 1. 0 a n d 3. 0 M a m m o n i u m s u l f a t e at 0 ° . O n l y a r g i n y l - s - R N A s y n t h e t a s e w a s p r e s e n t to an a p p r e c i a b l e extent i n the s u p e r n a t e of F r a c t i o n I V . W h i l e a l l the s y n t h e t a s e a c t i v i t i e s w e r e p r e s e n t i n F r a c t i o n III (2. 0 to 2. 5 M a m m o n i u m s u l f a t e ) o n l y c y s t e i n y l - and l y s y l - s - R N A s y n t h e t a s e s w e r e p r e s e n t to a g r e a t e r ex tent i n F r a c t i o n II (1. 0 to 2. 0 M a m m o n i u m s u l f a t e ) w h i l e a r g i n y l - , h i s t i d y l - a n d s e r y l - s - R N A s y n t h e t a s e s w e r e p r e s e n t to a g r e a t e r ex tent i n F r a c t i o n I V (2. 5 to 3. 0 M a m m o n i u m s u l f a t e ) . A p o r t i o n of F r a c t i o n III w a s c h r o m a t o g r a p h e d on h y d r o x y a p a t i t e i n the a b s e n c e of g l y c e r o l (see F i g u r e III). T h e e l u a t e f r o m the 5% a m m o n i u m s u l f a t e w a s h w a s c o m b i n e d , c o n c e n t r a t e d , a n d f i n a l l y d e s a l t e d on S e p h a d e x ( F r a c t i o n A ) i n the p r e s e n c e of 4 0 % g l y c e r o l ( v / v ) . The d e s a l t e d a m m o n i u m s u l f a t e F r a c t i o n III w h i c h h a d b e e n set a s i d e p r i o r t o c o l u m n . c h r o m a t o g r a p h y ( S e c t i o n 5 - D ) a n d w h i c h c o n t a i n e d no g l y c e r o l , w a s a s s a y e d , a l o n g - w i t h F r a c t i o n A . T a b l e V I l i s t s the a m o u n t of the v a r i o u s a m i n o a c i d s i n c o r p o r a t e d i n t o s - R N A b y the d e s a l t e d a m m o n i u m s u l f a t e F r a c t i o n III a n d b y F r a c t i o n A . It m u s t be e m p h a s i z e d - 2 4 8 -that the d e s a l t e d a m m o n i u m s u l f a t e F r a c t i o n III w a s s t o r e d at the s a m e t e m p e r a t u r e at w h i c h the c h r o m a t o g r a p h y w a s c a r r i e d out ( 3 - 5 ° ) a n d a l s o that g l y c e r o l w a s not u s e d i n e i t h e r c a s e . C o m p a r i s o n of the v a l u e s l i s t e d i n T a b l e V I f o r F r a c t i o n III w i t h t h o s e l i s t e d i n T a b l e V f o r F r a c t i o n III ( w h e n s t o r e d at - 1 8 ° i n 4 0 % g l y c e r o l ) c l e a r l y s h o w a l a r g e l o s s of e n z y m e a c t i v i t y w h e n the e n z y m e s a r e not s t o r e d at - 1 8 ° i n 4 0 % g l y c e r o l . T h e o n l y a c t i v i t i e s w h i c h d c not a p p e a r to h a v e b e n e f i t t e d by s t o r a g e at - 1 8 ° i n 4 0 % g l y c e r o l w e r e the g l y c y l - a n d m e t h i o n y l - s - R N A s y n t h e t a s e s . T e n of the 16 a m i n o a c i d s s t u d i e d w e r e f o u n d t o be e n r i c h e d i n F r a c t i o n A as c o m p a r e d to the d e s a l t e d F r a c t i o n III (see T a b l e V I ) . H o w e v e r c o m p a r i s o n of the v a l u e s l i s t e d f o r F r a c t i o n A i n T a b l e V I w i t h t h o s e f o r F r a c t i o n III i n T a b l e V s h o w that o n l y 8 of the 16 a m i n o a c i d s s t u d i e d have e n r i c h e d s y n t h e t a s e a c t i v i t i e s i n F r a c t i o n A . A l s o a p p a r e n t f r o m t h i s c o m p a r i s o n i s the s u g g e s t i o n that the i s o l e u c y l - , l e u c y l - , p h e n y l a l a n y l - , s e r y l , t r y p t o p h a n y l - a n d v a l y l - s - R N A s y n t h e t a s e s w e r e the m o s t l a b i l e of the 16 s t u d i e d . H o w e v e r , i t w a s l a t e r d i s c o v e r e d that the f i r s t f o u r m e n t i o n e d s y n t h e t a s e s w e r e e l u t e d by the p h o s p h a t e g r a d i e n t ( F i g s 5 a n d 6). A l t h o u g h the a r g i n y l - s - R N A s y n t h e t a s e a c t i v i t y w a s e n r i c h e d i n F r a c t i o n A , c o n s i d e r a b l e s y n t h e t a s e a c t i v i t y f o r t h i s a m i n o a c i d w a s a l s o e l u t e d i n the l a t t e r p a r t of the p h o s p h a t e g r a d i e n t (see F i g . 3, f r a c t i o n B ) . - 2 4 9 -T h e s p e c i f i c a c t i v i t i e s of s e v e r a l of the a m i n o a c y l - s - R N A s y n t h e t a s e s p r e s e n t a s e n r i c h e d a c t i v i t i e s i n F r a c t i o n A w e r e d e t e r m i n e d ( T a b l e V I I ) . T h e s y n t h e t a s e a c t i v i t y f o r a r g i n i n e e l u t e d by the p h o s p h a t e g r a d i e n t ( f r a c t i o n B ) w a s a l s o d e t e r m i n e d . T h e r e c o v e r i e s of the e n z y m e a c t i v i t i e s i n f r a c t i o n A w e r e p o o r a n d the o v e r a l l p u r i f i c a t i o n s w e r e not e n c o u r a g i n g ( w i t h the p o s s i b l e e x c e p t i o n of the a r g i n y l - s - R N A s y n t h e t a s e a c t i v i t y i n f r a c t i o n B w h i c h w a s 15 f o l d p u r i f i e d ) . F r o m the a m o u n t of p r o t e i n e l u t e d b y the p h o s p h a t e g r a d i e n t the e n z y m e a c t i v i t i e s e l u t e d by the a m m o n i u m s u l f a t e w a s h s h o u l d be m u c h h i g h e r t h a n o b s e r v e d . E v e n t a k i n g i n t o a c c o u n t the s k e p t i c i s m w i t h w h i c h one m u s t v i e w the d e t e r m i n a t i o n s of s p e c i f i c a c t i v i t y v a l u e s f o r t h e s e e n z y m e s , i t i s o b v i o u s that the c h r o m a t o g r a p h y m u s t be c a r r i e d out i n the p r e s e n c e of g l y c e r o l a n d that the c o l u m n f r a c t i o n s m u s t be s t o r e d at - 1 8 ° as s o o n as p o s s i b l e a f t e r b e i n g c o l l e c t e d . A l t h o u g h e n r i c h e d f r a c t i o n s m a y be o b t a i n e d , i t i s a p p a r e n t f r o m T a b l e V that f r a c t i o n a t i o n of a c e l l - f r e e e x t r a c t w i t h a m m o n i u m s u l f a t e does not c l e a n l y s e p a r a t e any of the s y n t h e t a s e s , one f r o m the o t h e r . It w a s t h e r e f o r e d e c i d e d to p r e c i p i t a t e the p r o t e i n f r o m the c e l l -f r e e e x t r a c t i n one s t e p a n d s u b s e q u e n t l y c h r o m a t o g r a p h the a m m o n i u m s u l f a t e p r e c i p i t a t e d p r o t e i n on h y d r o x y a p a t i t e (see C h a r t II f o r s u m m a r y of e x p e r i m e n t a l p r o c e d u r e s ) . T h i s t i m e g l y c e r o l w a s a d d e d to 4 0 % (v/v) f o l l o w i n g a m m o n i u m s u l f a t e p r e c i p i t a t i o n . A l s o i n c o r p o r a t e d i n t o -250-CHART II Summary of Experimental (Method 3, modified) 2 lb bakers' yeast - toluene, dry ice (1 hr) - incubation at 3-5° (72 hr) - centrifugation Cell-free extract - ammonium sulfate precipitation 0 - 80% protein fraction - G-25 Sephadex (40% glycerol) assay with 20 amino acids (Table VIII) Hydr oxyapatite chromatog raphy (40% glycerol)(Fig. 4) V Position of elution of the activities for 20 amino acids in Figs 5 and 6 - 2 5 1 -_4 the e l u t i n g s y s t e m w a s 10 M E D T A . T h e l a t t e r c o m p o u n d h a s b e e n s h o w n to have a s t a b i l i z i n g ef fect o n the s e r y l - s - R N A s y n t h e t a s e e n z y m e ( M a k m a n a n d C a n t o n i , 1965). T h e c r u d e p r o t e i n p r e p a r a t i o n w a s a s s a y e d f o r s y n t h e t a s e a c t i v i t y t o w a r d 20 a m i n o a c i d s ( T a b l e V I I I ) . C o m p a r i s o n of the v a l u e s l i s t e d i n T a b l e V w i t h t h o s e i n T a b l e V I I I ( k e e p i n g i n m i n d that the r e s u l t s i n T a b l e V I I I w e r e d e r i v e d f r o m p r o t e i n o b t a i n e d b y a s i n g l e a m m o n i u m s u l f a t e p r e c i p i t a t i o n ) s u g g e s t that t h e r e i s l i t t l e to be g a i n e d b y e x t e n d e d i n c u b a t i o n at 3 - 5 ° . O n the o t h e r h a n d the 72 h r i n c u b a t i o n p e r i o d d i d not r e s u l t i n a s i g n i f i c a n t l o s s of s y n t h e t a s e a c t i v i t y . W h e n the c r u d e , w h o l e - p r o t e i n e x t r a c t w a s c h r o m a t o g r a p h e d on h y d r o x y a p a t i t e , the u s u a l e l u t i o n p a t t e r n w a s o b t a i n e d ( F i g . 4 ) . T h e c o l u m n e l u a t e w a s a s s a y e d i n d e t a i l f o r a l l 20 a m i n o a c y l - s - R N A s y n t h e t a s e a c t i v i t i e s ( F i g s 5 a n d 6). T h e a c t i v i t i e s d e t e c t e d o n l y o r l a r g e l y i n the a m m o n i u m s u l f a t e e l u a t e w e r e a l a n y l - , a s p a r t y l - , a s p a r a g i n y l - , c y s t e i n y l - , g l u t a m y l - , g l u t a m i n y l - , l y s y l - , h i s t i d y l - , m e t h i o n y l - , p r o l y l - , t r y p t o p h a n y l - , t h r e o n y l - , t y r o s y l - a n d v a l y l - s - R N A s y n t h e t a s e s . A c t i v i t i e s d e t e c t e d o n l y o r l a r g e l y i n the e l u a t e f r o m the p h o s p h a t e g r a d i e n t w e r e a r g i n y l - , i s o l e u c y l - , l e u c y l r , p h e n y l a l a n y l - a n d s e r y l - s - R N A s y n t h e t a s e s . T w o p e a k s of a c t i v i t y w e r e d e t e c t e d f o r the g l y c y l - s - R N A s y n t h e t a s e . T h e f i r s t p e a k w a s e l u t e d t o w a r d s the end of the p h o s p h a t e g r a d i e n t a n d the s e c o n d p e a k « a s e l u t e d by the a m m o n i u m - 2 5 2 -s u l f a t e w a s h ( F i g . 5) . T h e s i g n i f i c a n c e of the s h o u l d e r s on the act iv i ty-p r o f i l e s f o r s e v e r a l a m i n o a c i d s (see F i g s 5 a n d 6) i s u n k n o w n a l t h o u g h a r e c e n t r e p o r t by B a r n e t t a n d B r o w n (1967) has s h o w n that m i t o c h o n d r i a m i g h t h a v e t h e i r o w n u n i q u e s - R N A ' s a n d a m i n o a c y l - s - R N A s y n t h e t a s e s . The p o s s i b l e s i g n i f i c a n c e of the t w o p e a k s of g l y c y l - s - R N A s y n t h e t a s e a c t i v i t y i s d i s c u s s e d i n m o r e d e t a i l i n P a r t I I - D , S e c t i o n 3 - B . T h e l y s i s of y e a s t c e l l s w i t h t o l u e n e at l o w t e m p e r a t u r e has p r o v i d e d a m e t h o d f o r the p r e p a r a t i o n of m o s t of the a m i n o a c y l - s - R N A s y n t h e t a s e s i n q u a n t i t i e s s u f f i c i e n t f o r c h a r g i n g l a r g e q u a n t i t i e s of m i x e d s - R N A w i t h a g i v e n a m i n o a c i d . B a s e d o n the w o r k d e s c r i b e d i n P a r t I I - D , S e c t i o n 3 - B , i t i s e s t i m a t e d that t w o p o u n d s of p r e s s e d b a k e r s ' y e a s t c a n p r o v i d e e n o u g h e n z y m e to c h a r g e at l e a s t 10 g of y e a s t s - R N A w i t h g l y c i n e . C h r o m a t o g r a p h y of the c r u d e p r o t e i n p r e p a r a t i o n s on h y d r o x y -a p a t i t e s e p a r a t e s m o s t of the s y n t h e t a s e e n z y m e s f r o m the r e s t of the c e l l u l a r p r o t e i n . I n a d d i t i o n , l o w t e m p e r a t u r e l y s i s f o l l o w e d by h y d r o x y a p a t i t e c h r o m a t o g r a p h y of the c e l l - f r e e e x t r a c t a p p e a r s to be a s u i t a b l e a p p r o a c h f o r the p u r i f i c a t i o n of i n d i v i d u a l a m i n o a c y l - s - R N A s y n t h e t a s e s by f u r t h e r c h r o m a t o g r a p h i e s . S h o r t e r i n c u b a t i o n p e r i o d s at 3 - 5 ° a r e s u g g e s t e d . It m i g h t a l s o be a d v i s a b l e to a d d g l y c e r o l a n d / o r n e u t r a l b u f f e r a f t e r the y e a s t p a s t e w a r m s to 3 - 5 ° . H o w e v e r , i t s h o u l d be k e p t i n m i n d that the a d d i t i o n of g l y c e r o l at t h i s s tage c a n s - 2 5 3 -c o m p l i c a t e the f r a c t i o n a t i o n of the c e l l - f r e e p r e p a r a t i o n w i t h a m m o n i u m s u l f a t e . T h e m a j o r l i m i t i n g f a c t o r f o r the p r e p a r a t i o n of e v e n l a r g e r q u a n t i t i e s of the s y n t h e t a s e e n z y m e s i s the r e l a t i v e l y l o w c a p a c i t y of the h y d r o x y a p a t i t e c o l u m n s . M o r e p r o t e i n c o u l d p r o b a b l y be a p p l i e d to t h e s e c o l u m n s i f the n u c l e i c a c i d c o n t e n t of the c e l l - f r e e e x t r a c t w e r e d i m i n i s h e d . T h i s c o u l d be a c h i e v e d by p a s s i n g the c e l l - f r e e e x t r a c t t h r o u g h a D E A E - c e l l u l o s e c o l u m n u n d e r c o n d i t i o n s w h i c h r e t a r d n u c l e i c a c i d s but not the s y n t h e t a s e e n z y m e s ( H o l l e y , a n d G o l d s t e i n , 1959) a n d i n the p r e s e n c e of g l y c e r o l . 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