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Mechanism of action of streptomycin : studies with polynucleotide phosphorylase and ribosomes Willick, Gordon Edward 1962

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MECHANISM OP ACTION OP STREPTOMYCIN: STUDIES VITH POLYNUCLEOTIDE PHOSPHORYLASE AND RIBOSOMES by GORDON EDWARD WILLICK B . S c , U n i v e r s i t y of B r i t i s h Columbia, 1960 A THESIS SUBMITTED IN PARTIAL FULFILMENT OP THE REQUIREMENTS PGR THE DEGREE OP MASTER OP SCIENCE i n the Department of BIOCHEMISTRY We accept t h i s t h e s i s as conforming to the r e q u i r e d standard THE UNIVERSITY OP BRITISH COLUMBIA J u l y , 1962 In presenting 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 of the requirements f o r an advanced degree at the U n i v e r s i t y of B r i t i s h Columbia, I agree t h a t the 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 reference and study. I f u r t h e r agree that permission f o r extensive copying of t h i s t h e s i s f o r s c h o l a r l y purposes may be granted by the Head of my Department 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 understood t h a t copying or p u b l i c a t i o n of 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 not be allowed without my w r i t t e n permission. Department of lS\ O c V y ^ T i u The U n i v e r s i t y of B r i t i s h Columbia, Vancouver 3, Canada. Date i i ABSTRACT Some o f t h e p r e v i o u s w o r k o n t h e m e c h a n i s m o f a c t i o n o f s t r e p t o m y c i n h a s i n d i c a t e d t h e g e n e r a l a r e a o f n u c l e i c a c i d m e t a b o l i s m a s b e i n g a p o s s i b l e s i t e o f a c t i o n . S p e c i f i c a l l y , a r e p o r t h a s a p p e a r e d t h a t s t r e p t o m y c i n i n h i b i t s t h e " e x c h a n g e r e a c t i o n " c a t a l y z e d b y p o l y n u c l e o t i d e p h o s p h o r y l a s e . T h e r e f o r e , t h e p o s s i b l e a c t i o n o f s t r e p t o m y c i n o n p o l y n u c l e o t i d e p h o s p h o r y l a s e f r o m a s t r e p t o m y c i n s e n s i t i v e s t r a i n o f E s c h e r i c h i a c o l i h a s b e e n r e i n v e s t i g a t e d . I n p r e p a r a t i o n f o r t h e s t u d y o n p o l y n u c l e o t i d e p h o s p h o r y l a s e , t h e n u c l e o s i d e d i p h o s p h a t e s o f a d e n o s i n e , g u a n o s i n e , c y t i d i n e , a n d u r i d i n e w e r e s y n t h e s i z e d i n s u f f i c i e n t y i e l d a n d o f a s a t i s f a c t o r y p u r i t y b y t h e r e c e n t l y d e v e l o p e d m e t h o d o f M o f f a t t a n d K h o r a n a . I t w a s n o t p o s s i b l e t o c o n f i r m t h e i n h i b i t i o n o f t h e p o l y -n u c l e o t i d e p h o s p h o r y l a s e c a t a l y z e d " e x c h a n g e r e a c t i o n " r e p o r t e d b y K o r n b e r g , u s i n g enzyme f r o m o u r s t r e p t o m y c i n s e n s i t i v e E . c o l i S A . N o r w a s i t p o s s i b l e t o d e m o n s t r a t e a n y i n h i b i t i o n o f t h e p o l y m e r i z a t i o n r e a c t i o n c a t a l y z e d b y t h i s e n z y m e , o r a n y e f f e c t o n t h e s e d i m e n t a t i o n p r o p e r t i e s o f t h e p o l y m e r so f o r m e d . H o w e v e r , t h e d i a m i n e s p u t r e s c i n e , c a d a v e r i n e , s p e r m i n e , a n d s p e r m i d i n e , w h i l e h a v i n g no e f f e c t o n t h e t i m e c o u r s e o f t h e p o l y m e r i z a t i o n r e a c t i o n , d i d l o w e r t h e s e d i m e n t a t i o n c o e f f i c i e n t o f t h e p o l y m e r f o r m e d a n d c a u s e a m o r e h e t e r o g e n e o u s p o l y m e r t o b e f o r m e d . S t r e p t o m y c i n h a s b e e n s u g g e s t e d a s a n i n h i b i t o r o f p r o t e i n i i i s y n t h e s i s . Therefore, a study of the p o s s i b l e e f f e c t s of streptomycin on ribosomee from E. c o l i was made. These sub-c e l l u l a r p a r t i c l e s have been shown to be a s i t e of p r o t e i n s y n t h e s i s . Dihydrostreptomycin(DHSM) i n t e r a c t e d s t r o n g l y w i t h the ribosomes. I t was found t h a t most of the r i b o n u c l e o -p r o t e i n p r e c i p i t a t e d when ribosomes Were d i a l y z e d overnight at 4° aga i n s t b u f f e r c o n t a i n i n g about 500 u-g./ml. of DHSM. A study of u l t r a c e n t r i f u g e p a t t e r n s of d i a l y s a t e s o f ribosomes aga i n s t lower l e v e l s of DHSM i n d i c a t e d t h a t d i s r u p t i o n , w i t h a l o s s of d i s c r e t e sedimentation c o e f f i c i e n t s , o c c u r r e d when the l e v e l of DHSM was about 350 [ig./ml. A study of e l u t i o n p a t t e r n s from DEAE-cellulose columns i n d i c a t e d o n l y a p a r t i a l change i n the p a t t e r n a f t e r breakdown. Examination of sedimentation c o e f f i c i e n t s of ribosome d i a l y s a t e s at lower DHSM l e v e l s i n d i c a t e d no s p e c i f i c e f f e c t of DHSM. The autodegradation of ribosomes by p o l y n u c l e o t i d e phosphorylase was s t u d i e d . DHSM, at low l e v e l s , had an e f f e c t on the time course o f the auto-degradation. A study of the d i s t r i b u t i o n of induced B-D-galactosidase a s s o c i a t e d w i t h the ribosomes i n d i c a t e d t h a t i t was a s s o c i a t e d w i t h t h a t p r o t e i n of the ribosomes not p r e c i p i t a t e d by DHSM. I t can be concluded t h a t streptomycin i n t e r a c t s s t r o n g l y w i t h ribosomes. T h i s g i v e s support to recent claims t h a t streptomycin i n h i b i t s * i n v i t r o ' p r o t e i n s y n t h e s i s , and t h a t the s i t e of t h i s i n h i b i t i o n i s the ribosome. ACKNOWLEDGMENT The a u t h o r w o u l d l i k e t o e x p r e s s h i s v e r y s i n c e r e t h a n k s t o D r . W . J . P o l g l a s e f o r t h e h e l p , e n c o u r a g e m e n t , a n d a d v i c e e x t e n d e d t o h i m t h r o u g h o u t t h e c o u r s e o f t h i s w o r k . i v TABLE OP CONTENTS Page A b s t r a c t i i L i s t of F i g u r e s v i L i s t of Tables v i i i Acknowledgment i x I n t r o d u c t i o n 1 Experimental 12 I. P r e p a r a t i o n of Nucleoside Diphosphates 12 A. Method 12 B. R e s u l t s 15 I I . Experiments w i t h P o l y n u c l e o t i d e Phosphorylase 17 from Dihydrostreptomycin S e n s i t i v e E s c h e r i c h i a c o l i A. A n a l y t i c a l Methods 17 B. P r e p a r a t i o n of P o l y n u c l e o t i d e Phosphorylase 20 from E s c h e r i c h i a c o l i C. E f f e c t of Dihydrostreptomycin on the Exchange 23 Rea c t i o n D. E f f e c t of Dihydrostreptomycin on Polyadenylate 26 Synt h e s i s I I I . S t u d i e s on Ribosomes from E s c h e r i c h i a c o l i 33 A. A n a l y t i c a l Methods 33 B. Growth of B a c t e r i a 34 C. P r e p a r a t i o n of Ribosomes 34 Page D. S t a b i l i t y of Ribosomes i n the Presence 35 of DHSM 1. D i s s o c i a t i o n of Ribosomes w i t h DHSM 35 2. Autodegradation of Ribosomes i n the 46 Presence of DHSM 3. P - G a l a c t o s i d a s e A c t i v i t y of Ribosomes i n 46 the Presence of DHSM D i s c u s s i o n , 51 I. P r e p a r a t i o n of Nucleoside Diphosphates 51 I I . Experiments w i t h P o l y n u c l e o t i d e Phosphorylase 52 from Dihydrostreptomycin S e n s i t i v e E s c h e r i c h i a c o l i I I I . S t u d i e s on Ribosomes from E s c h e r i c h i a c o l i 54 Summary 60 B i b l i o g r a p h y 62 v i L i s t o f F i g u r e s F i g u r e P a g e 1 S t r u c t u r e o f s t r e p t o m y c i n 1 2 T i m e s t u d y o f t h e p o l y n u c l e o t i d e p h o s p h o r y l a s e 25 c a t a l y z e d e x c h a n g e r e a c t i o n 3 T i m e c o u r s e o f p o l y a d e n y l a t e s y n t h e s i s . T e s t o f 28 p r o t a m i n e s u p e r n a t a n t ( c o n t a i n i n g n u c l e i c a c i d ) 4 L a c k o f e f f e c t o f DHSM o n t h e t i m e c o u r s e o f 29 p o l y a d e n y l a t e s y n t h e s i s 5 L a c k o f e f f e c t o f d i a m i n e s o n t h e t i m e c o u r s e 30 o f p o l y a d e n y l a t e s y n t h e s i s 6 U l t r a c e n t r i f u g e p a t t e r n s o f p o l y a d e n y l a t e 31 s y n t h e s i z e d b y p o l y n u c l e o t i d e p h o s p h o r y l a s e 7 U l t r a c e n t r i f u g e p h o t o g r a p h s o f E . c o l i SB 37 f r a c t i o n s 8 E f f e c t o n s o l u b l e RNA a n d p r o t e i n o f d i a l y s i s 38 ° ^ !!• c o l i S A r i b o s o m e s a g a i n s t DHSM i n t r i s b u f f e r , p H 7.4 9 E f f e c t o f DHSM o n u l t r a c e n t r i f u g e p a t t e r n s o f 39 r i b o s o m e s f r o m E . c o l i S B ( s e n s i t i v e ) 10 E f f e c t o f DHSM o n u l t r a c e n t r i f u g e p a t t e r n s o f 41 r i b o s o m e s f r o m E . c o l i R B ( r e s i s t a n t ) 11 R i b o s o m e s f r o m E . c o l i S B ( s e n s i t i v e ) c h r o m a t o - .42 g r a p h e d o n D E A E - c e l l u l o s e w i t h a l i n e a r g r a d i e n t o f N a C l i n 0.01 M t r i s , p H 7.4 12 R i b o s o m e s f r o m s e n s i t i v e E . c o l i SB c h r o m a t o - 43 g r a p h e d o n D E A E - c e l l u l o s e w i t h a l i n e a r g r a d i e n t o f N a C l i n TM b u f f e r 13 R i b o s o m e s f r o m r e s i s t a n t E . c o l i RB c h r o m a t o - 44 g r a p h e d o n D E A E - c e l l u l o s e w i t h a l i n e a r g r a d i e n t o f N a C l i n TM b u f f e r v i i Figure Page 14 Autodegradation of ribosomes from sensitive 47 E. c o l i SB and SA, and resistant E. c o l i RB 15 Specific a c t i v i t i e s of the 6,000 g soluble 50 portion of (3-D-galactosidase induced sensitive E. c o l i SB ribosome dialysates ^ i i i L i s t of Tables Table I I I I I I IV V VI VII V I I I Y i e l d s of Nucleoside-5' Phosphoromorpholidates Y i e l d s of Nucleoside-5' Diphosphates A c t i v i t i e s o f the F r a c t i o n s E f f e c t of DHSM(1000 jig./ml.) on the A c t i v i t i e s of the Enzyme F r a c t i o n s as Measured by the Exchange Re a c t i o n E f f e c t of Adding DHSM Before and A f t e r Incubation Sedimentation C o e f f i c i e n t s of Ribosomes from E. c o l i SB D i a l y z e d Against Increasing^ Concentrations of DHSM Sedimentation C o e f f i c i e n t s of Ribosomes from E. c o l i RB D i a l y z e d Against I n c r e a s i n g Concentrations of DHSM A c t i v i t i e s of the F r a c t i o n s of B-D-Galactosidase Induced E. c o l i SA 'age 15 16 23 23 24 45 45 49 IX A c t i v i t i e s of the PV D i a l y s a t e s 50 1 I1TR0DUCTI0BT The a n t i b i o t i c , streptomycin, was f i r s t i s o l a t e d i n 1944 from two Streptomyces g r i s e u s s t r a i n s ( l ) . I t was found to have a c h a r a c t e r i s t i c e f f e c t a g a i n s t Gram-negative m i c r o o r g a n i s m s ( l ) , and a high degree of a c t i v i t y a g a i n s t Mycobacteria, p a r t i c u l a r l y human pathogens(2) 9 The s t r u c t u r e of streptomycin i s shown i n F i g * 1. S t r e p t o -mycin i s composed of three m o i e t i e s : s t r e p t i d i n e , s t r e p t o s e , and N-»methyl-L-glucosamine. A c i d h y d r o l y s i s of streptomycin g i v e s s t r e p t i d i n e p l u s streptobiosamine. The p o i n t of attachment of streptobiosamine to s t r e p t i d i n e i s u n c e r t a i n as i n d i c a t e d . OH N-METHYL-L-GLUCOSAMINE OH \NH-C-NH II p NH * JH <j)H/ NH-C-NH " 2 NH OH STREPTIDINE F I G . 1 2 Various streptomycin d e r i v a t i v e s have been prepared; a study of such d e r i v a t i v e s has i n d i c a t e d t h a t c e r t a i n s t r u c t u r a l elements of the streptomycin molecule are probably not necessary f o r i t s b i o l o g i c a l a c t i v i t y . Mannosylstreptomycin(streptomycin B) i s b i o l o g i c a l l y a c t i v e ( 3 ) . In mannosylstreptomycin, mannose i s l i n k e d as an a 1«* 4 g l y c o s i d e with the N-methyl-L-glucosamine moiety of streptomycin(4). Reduction of the aldehyde group of the s t r e p t o s e moiety to the primary a l c o h o l group gi v e s a compound, dihydrostreptomycin, with e s s e n t i a l l y the same b i o l o g i c a l p r o p e r t i e s . Dihydrostreptomycin, the c h e m i c a l l y more s t a b l e of the two, has been used throughout t h i s work i n the form of i t s s u l f a t e . A d d i t i o n a l evidence t h a t the aldehyde group i s n o n - f u n c t i o n a l b i o l o g i c a l l y i s gi v e n by the o b s e r v a t i o n t h a t replacement of t h i s group by N - a l k y l groups with up to e i g h t s t r a i g h t c h a i n carbons gave d e r i v a t i v e s t h a t s t i l l r e t a i n e d c o n s i d e r a b l e b i o l o g i c a l a c t i v i t y ( 5 ) . Streptomycin has a b a c t e r i o s t a t i c or b a c t e r i c i d a l e f f e c t on Gram-negative b a c t e r i a , depending on i t s c o n c e n t r a t i o n ( 6 ) . From the streptomycin s e n s i t i v e s t r a i n s , r e s i s t a n t s t r a i n s can be developed, and i n some cases, n o t a b l y c e r t a i n s t r a i n s of E s c h e r i c h i a c o l i , dependent s t r a i n s a l s o . These s t r a i n s are considered to a r i s e by mutation and not by p h y s i o l o g i c a l a d a p t a t i o n of s e n s i t i v e b a c t e r i a . Although the s i t e of a c t i o n of streptomycin has been a s u b j e c t of study f o r about f i f t e e n y e a r s , i t i s s t i l l unknown. Any theory f o r the mechanism of a c t i o n of an a n t i b i o t i c should at l e a s t s a t i s f y the f o l l o w i n g c r i t e r i a ( 7 ) : 3 (1) the metabolic effects should be induced by the minimal concentrations of streptomycin effective i n bacteriostasis. ( 2 ) only the a n t i b i o t i c a l l y active derivatives of streptomycin should produce such e f f e c t s . (3) the phenomenon should be spe c i f i c to streptomycin, as distinguished from other a n t i b i o t i c s . (4) the reactions involved should be of v i t a l importance to the economy of the c e l l . Evidence has been presented that streptomycin i n h i b i t s carbohydrate and amino acid metabolism. In 1947, Geiger(8) demonstrated that the increased a b i l i t y of Escherichia c o l i c e l l s , when pretreated with fumarate or certain other carbon compounds, to oxidize amino acids was prevented by streptomycin. Umbreit(9) studied carbohydrate metabolism i n streptomycin sensitive and resistant strains of Escherichia c o l i . Umbreit could f i n d no in h i b i t i o n s of the known reactions of pyruvate and oxalacetate he studied, i . e . of the Embden—Meyerhof and tr i c a r b o x y l i c acid pathways. However, he found that the formation of a metabolite, f i r s t i s o l a t e d by Rappaport and Wagner(10) from dog l i v e r , was i n h i b i t e d by streptomycin(ll)« This compound, 2-phospho—4—hydroxy-4~carboxyadipic acid, was indicated to arise only when a dicarboxy acid and pyruvate were present. Although the mechanism put forward by Umbreit could explain G-eiger's findings, i . e . streptomycin inh i b i t e d the combination of fumarate with pyruvate from the amino acid, i t could not explain the action of the drug on organisms lacking a terminal respiration cycle for energy metabolism. For example, Rosanoff 4 and Sevag(l2) found their streptomycin sensitive s t r a i n of Escherichia c o l i , unlike the "Murray" s t r a i n used by Umbreit, had no detectable pyruvate—oxalacetate condensation mechanism. Various other papers have dealt with streptomycin i n h i b i t i o n of carbohydrate metabolism, and many indicate the i n h i b i t i o n occurs i n the v i c i n i t y of pyruvate metabolism. Barkulis(l2) studied the effect of dihydrostreptomycin on sensitive, resistant, and dependent mutants of Escherichia c o l i . I t was found that pyruvate metabolism was in h i b i t e d i n the sensitive s t r a i n , but not i n the resistant or dependent strains* However, Zebovitz and Moulder(13) found, using the same s t r a i n of E, c o l i as Barkulis, that dihydrostreptomycin inhibited pyruvate metabolism i n freshly harvested suspensions of E. c o l i only i n the presence of carbon dioxide. When c e l l s were aged at 2°, dihydrostrepto-mycin i n h i b i t i o n of pyruvate metabolism i n the absence of carbon dioxide rapidly increased to t o t a l i n h i b i t i o n i n li*2 weeks. They concluded that two pathways of pyruvate metabolism existed i n t h e i r s t r a i n of E. c o l i ; one probably dihydrostrepto-mycin sensitive, unaffected by carbon dioxide, and stable to ageing, the other insensitive to dihydrostreptomycin, i n h i b i t e d by carbon dioxide, and unstable to ageing. Rosanoff and Sevag(l4) also found anaerobic pyruvate metabolism was in h i b i t e d by streptomycin i n sensitive c e l l s , but not i n resistant or dependent. Takeuchi et a l ( l 5 ) found pyruvate metabolism blocked, t h i s time i n Mycobacterium avium. Citrate metabolism was unaffected. Hirano(l6) found pyruvate accumulated aerobically i n the presence of streptomycin, while no pyruvate accumulated 5 anaerobically with t h i s s t r a i n of E, c o l i . However, the sensitive organism was not completely in h i b i t e d by 1000 jig per ml, when under anaerobic conditions. On the other hand, his dependent s t r a i n accumulated pyruvate i n the absence of streptomycin; no accumulation occurred when grown i n the presence of streptomycin, Martin-Hernandez et a l ( l 7 ) found that pyruvic carboxylase was inh i b i t e d by streptomycin, but not at physiologically important concentrations. Possibly coupled to the i n h i b i t i o n of pyruvate metabolism i s the reported strong i n h i b i t i o n of acetoacetate metabolism by several a n t i b i o t i c s , including streptomycin(l8), The evidence thus indicates streptomycin fundamentally affects pyruvate metabolism, however the phenomenon i s complex and the various experimental results have yet to be s a t i s f a c t o r i l y explained, Davis has recently carried out studies on the mechanism of action of streptomycin i n Escherichia c o l i . Davis found that his sensitive strain(but not the resistant strain) l o s t 5'-nucleotides and amino acids to the medium, but there was l i t t l e or no r i s e i n the i n t r a - c e l l u l a r levels of these compounds(19,20). He also studied the uptake of streptomycin^U-C^ by E, c o l i . He found a rapid i n i t i a l primary uptake i n both sensitive and resistant mutants, and a slow secondary uptake i n the sensitive mutant(20). The time of the slow uptake corresponded to the time at which the nucleotides and amino acids began to be excreted. This work has been added to more recently by a study of potassium f l u x i n E. c o l i as a function of streptomycin i n h i b i t i o n ( 2 l ) . Davis interpreted these results 6 as i n d i c a t i n g t h a t streptomycin caused a breakdown of a p e r m e a b i l i t y b a r r i e r i n E . c o l i and t h a t the secondary uptake was a r e f l e c t i o n of i n c r e a s e d b i n d i n g s i t e s w i t h i n the organism which became a v a i l a b l e as streptomycin was allowed to penetrate the c e l l , presumably by a l o c a l d i s r u p t i o n of an i n i t i a l permea-. b i l i t y b a r r i e r . However, Davis r e c e n t l y r e p o r t e d t h a t a step i n 14 the procedure used f o r measuring strepjbomycin-U-C uptake had i n c r e a s e d uptake f o u r to f i v e f o l d . T h i s step was p r e - d i l u t i o n of the sample with d i s t i l l e d water(22). Therefore, the secondary uptake noted by Davis i n r e a l i t y l a r g e l y represented an uptake due to p r e - d i l u t i o n i n h i s experimental procedure before f i l t e r i n g of the c e l l s . Davis concluded t h a t the secondary phase was not due s o l e l y to b i n d i n g to s i t e s made slowly a c c e s s i b l e by s l i g h t damage to the membrane but was i n s t e a d a r e f l e c t i o n of a gradual i n c r e a s e i n f r e e l y a c c e s s i b l e b i n d i n g s i t e s . He has not y e t determined these b i n d i n g s i t e s , nor i n d i c a t e d i f t h i s has caused him to r e c o n s i d e r h i s i n t e r p r e t a t i o n of h i s e a r l i e r data. S z y b a l s k i and Mashima(23) had e a r l i e r s t u d i e d the uptake 14 of streptomycin-U-C by streptomycin s e n s i t i v e , r e s i s t a n t , and dependent mutants of E. c o l i . They found t h a t the b a c t e r i c i d a l a c t i o n of streptomycin was a s s o c i a t e d with the i r r e v e r s i b l e b i n d i n g 5 6 of 10 -10 molecules per streptomycin s e n s i t i v e E. c o l i c e l l . Under the same conditions(5-50 jig/ml.), streptomycin r e s i s t a n t c e l l s showed only low, i f any, uptake of streptomycin, and streptomycin dependent mutants e x h i b i t e d intermediate behaviour, b i n d i n g c o n s i d e r a b l y l e s s streptomycin than s e n s i t i v e 7 c e l l s , while multiplying a c t i v e l y . These results can not be compared d i r e c t l y with those of Davis since Szybalski did not take his f i r s t measurement t i l l one hour after contact of the c e l l s with streptomycin and, also, he was measuring i r r e v e r s i b l e uptake. Recently, Engelberg and Artman(24) studied the uptake 14 of streptomyc.in-U-C i n a dependent mutant of Escherichia c o l i as a function of the s a l t concentration of the medium. They came to the conclusion that streptomycin was bound by ionic linkages. No evidence has yet been presented that streptomycin i s bound by any type of covalent bond. Hancock(25) has studied streptomycin i n h i b i t i o n of Escherichia c o l i and Ba c i l l u s megaterium. He could not, by his technique, demonstrate any general breakdown of a permeability barrier i n either organism, but was able to show that the harvested organisms had, i n the presence of streptomycin, oxidative a c t i v i t i e s on glucose, lactate, and pyruvate which were between 20$ and 60$ of those of normal organisms. Using lysates from protoplasts of B a c i l l u s megaterium he found that the decrease i n oxidative a c t i v i t y with malate and succinate as substrates was due mainly to a considerable decrease i n the a c t i v i t y i n the sedimentable ghost f r a c t i o n of the lysed protoplasts. Considerable work has implicated streptomycin i n the f i e l d of nucleic acid and protein metabolism. Streptomycin has been related to protein synthesis by studies of i t s effect on induced enzyme synthesis, Roote and Polglase(26) showed that 8 the adaptive formation of enzymes for the oxidation of Imarabinose, lactose, and D—glucuronic acid was in h i b i t e d by dihydrostreptomycin i n a s t r a i n of Escherichia c o l i sensitive to t h i s a n t i b i o t i c , but not i n the resistant or dependent mutants. This work was extended to show that the formation of the same adaptive enzymes i n the dependent mutant required dihydrostreptomycin(27). Later, Peretz and Polglase(28) showed that the induction of p—galactosidase i n washed c e l l preparations was in h i b i t e d by concentrations of dihydrostrepto-mycin that i n h i b i t e d c e l l m u l t i p l i c a t i o n . Additional evidence for the effect of streptomycin on (3—galactosidase formation was obtained by Nakada(29). Using a methionine requiring s t r a i n of E. c o l i sensitive to streptomycin he found |3—galactosidase synthesis decreased to the same l e v e l i f either methionine was omitted or streptomycin added. He concluded that streptomycin inh i b i t e d the synthesis of new |3-galactosidase. Evidence i s available that streptomycin acts competitively with divalent cations; t h i s e f f e c t has been noticed i n i n h i b i t i o n reactions which appear to be connected to protein synthesis. Dihydro streptomycin i n h i b i t i o n of |3-galactosidase induction can be reversed by magnesium, but not by other divalent cations(30). In experiments designed to determine the mechanism of phytotoxicity of streptomycin, Rosen(3l) found that exposure of higher plants to streptomycin resulted i n two general phytotoxic e f f e c t s : leaf bleaching and growth i n h i b i t i o n . Manganese was found to i n h i b i t the phytotoxic effects of streptomycin competitively; calcium was the only other ion found 9 to be even p a r t i a l l y e f f e c t i v e ( 3 2 ) . The evidence thus suggests competition between streptomycin and the d i v a l e n t cation(magnesium i n b a c t e r i a , manganese i n higher p l a n t s ) f o r a common s i t e . The p r e c i p i t a t i o n of n u c l e i c a c i d s by streptomycin was f i r s t r e p o r t e d by Cohen(33)© Streptomycin has been used as a reagent f o r s e l e c t i v e l y p r e c i p i t a t i n g d e o x y r i b o n u c l e i c acid(DNA) from b a c t e r i a l e x t r a c t s ; r i b o n u c l e i c acid(RNA) i s p r e c i p i t a t e d at a higher streptomycin c o n c e n t r a t i o n ( 3 4 ) • Hamaguchi(35) st u d i e d the r e a c t i o n between streptomycin and RNA. His evidence i n d i c a t e d the guanidine groups of streptomycin combined w i t h the phosphoric a c i d r a d i c a l of the RNA„ De Deken-Grenson(36) found that streptomycin p r e c i p i t a t e d the microsomes and s o l u b l e n u c l e o p r o t e i n s from the u l t r a c e n t r i f u g e supernatants of r a t l i v e r or leaves of b a r l e y and tobacco. The p r e c i p i t a t i o n was q u a n t i t a t i v e when the s a l t c o n c e n t r a t i o n was low and the r a t i o of RNA to streptomycin was between l / l O - l / 2 0 . The p r e c i p i t a t e c o u l d be r e d i s s o l v e d on d i a l y s i s against phosphate, the phosphate presumably forming a complex w i t h the streptomycin. He also found t h a t streptomycin p r e c i p i t a t e d polymerized RNA, DNA, c e r t a i n n u c l e o p r o t e i n s , sodium polymetaphosphate, phosphoproteins, c a s e i n , l i v e t i n , and the polyglutamic a c i d s y n t h e s i z e d by B a c i l l u s s u b t i l i s , but not the s o l u b l e antigens of tobacco mosaic v i r u s e s . P o l g l a s e et a l ( 2 8 ) found t h a t while complexes of sperm DNA or thymus DNA were s u s c e p t i b l e to attack by deoxyribonuclease, and a commercial RNA-streptomycin complex by r i b o n u c l e a s e , the b a c t e r i a l n u c l e i c a c i d - s t r e p t o m y c i n complex was not s u s c e p t i b l e to a t t a c k by e i t h e r nuclease. Thus, 10 although streptomycin precipitates polyanions i n general, i t appears to form an e s p e c i a l l y stable complex with b a c t e r i a l nucleic a c i d s 9 That there i s a relationship between RNA and protein synthesis has been known for several years. Zamecnik and his colleagues(37) showed that a c e l l free system of Escherichia  c o l i . similar to those which had already been demonstrated i n plant and animal tissue, could cause the incorporation of amino acids into protein. The system required the p a r t i c u l a r ribonucleoprotein(ribosome) f r a c t i o n , the 100,000g supernatant, adenosine triphosphate(ATP), an ATP generating system, guanosine triphosphate(G-TP), magnesium, and a mixture of amino acids for maximum incorporation. Further d e f i n i t i v e work on t h i s system has recently been done by Tissieres et al(38). Erdos and Ullmann(39, 40) have reported that the amino acid transfer from soluble RNA to protein i s i n h i b i t e d by streptomycin i n sensitive c e l l s but not i n resistant c e l l s . A number of enzymes involved i n nucleic acid metabolism have been discovered i n recent years. The f i r s t enzyme found which was capable of synthesizing polynucleotides was poly-nucleotide phosphorylase, f i r s t i solated by Ochoa and his coworkers from Azotobacter v i n e l a n d i i ( 4 l ) . The enzyme required any one or a mixture of nucleoside diphosphates, plus magnesium. The reaction was reversible, i . e . the enzyme also catalyzed phosphorolysis of ribonucleic acid. In addition , the enzyme catalyzed an "exchange" reaction of the terminal phosphate of the nucleotide with inorganic phosphate, which probably 11 resulted from the reversible formation of a nucleoside monophosphate-enzyme complex(42). Littauer and Kornberg(43) described the i s o l a t i o n and p a r t i a l p u r i f i c a t i o n of t h i s enzyme from Escherichia c o l i . In t h i s paper they stated that streptomycin i n h i b i t e d the exchange reaction, under their assay conditions, by 50$ when the streptomycin concentration was 0.014^(140 n g per ml.). Thus, i t appeared that a reaction of nucleic acid metabolism was inhi b i t e d by physiologically-important concentrations of streptomycin. In view of the fact that previous evidence had indicated an i n h i b i t i o n of protein synthesis as a primary effect of streptomycin on sensitive bacteria, and since RNA synthesis appears to be a necessary precursor to protein synthesis, i t was decided to investigate Romberg's reported i n h i b i t i o n of the polynucleotide phosphorylase exchange reaction further. As part of t h i s investigation, polyadenylate synthesis was studied and also the effect of streptomycin and certain polyamines on the structure of the synthesized polymer. Following t h i s investigation a study was made on the effect of streptomycin on the structure and s t a b i l i t y of Escherichia c o l i ribosomes, p r i n c i p a l l y as re f l e c t e d i n ultracentrifuge patterns and autodegradation rates. This l a s t work was undertaken because of the involvement of ribosomes i n protein synthesis. These p a r t i c l e s consist of protein and nucleic acid s t a b i l i z e d by hydrogen bonds and by ionic bonds(44, 45). It was f e l t that streptomycin could conceivably interfere with t h i s structure with a resu l t i n g effect on protein synthesis. 12 EXPERIMENTAL I. Preparation of Nucleoside Diphosphates(46) A, Method 1. Chromatography: Descending paper chromatography of nucleoside polyphosphates was carried out on Whatman number 1 paper i n the following solvent systems: (I) isobutyric acid-1 M ammonium hydroxide-0,1 M ethylenediamine tetraacetic acid (100:60:1.6)j (II) ethanol-1 M ammonium acetate(pH 7.2)(5s2)» Chromatograms were observed under an u l t r a v i o l e t lamp for detection and i d e n t i f i c a t i o n ( b y R^ values) of products. 2, Nucleoside-5 * Phosphoromorpholidates: (a) Gytidine-5 1 Phosphoromorpholidate: A solution of dicyclohexylcarbodiimide(DCC)(1.04 g., 6,8 mmoles) i n 19 ml. of t«?butyl alcohol was added dropwise over a period of 4 hr. to a refluxing solution of sodium cytidine-5 1 phosphate(0.41 g«, 1,26 mmole) i n a mixture of water(13 ml,), t—butyl alcohol(13 ml. and morpholine(0.43 ml., 5,1 mmole). The addition was completed i n 4 hr. and the mixture was refluxed for an additional 2 hr. An additional 0.21 ml.(2.5 mmole) of morpholine was then added. The solution was maintained under reflux while DCC (0.52 g. i n 9.5 ml. of t-butyl alcohol) was added dropwise over a period of 2 hr. The mixture was refluxed for an additional 2 hr. The t—butyl alcohol was removed under reduced pressure and the aqueous solution extracted four times with ether, with f i l t r a t i o n after the f i r s t extraction. The aqueous solution 13 was then evaporated to dryness under reduced pressure, and dried i n vacuo at room temperature for 16 hr* The glassy residue was dissolved i n a minimum of methanol and concentrated c a r e f u l l y under reduced pressure to approximately 3 ml. Dry ether(35 ml.) was added; a gummy white s o l i d precipitated which changed to a dry white powder on further t r i t u r a t i o n with dry ether. After a further wash with dry ether the product (4-morpholine N,N ,-dicyclohexylcarboxamidiniura cytidine-5 1 phosphoromorpholidate) was dried i n vacuo at room temperature. (b) Other Morpholidates: The morpholidates of adenosine-5' phosphate, guanosine—5' phosphate, and uridine-5 1 phosphate were prepared i n a l i k e manner, but from the free acids. Adenosine-5* phosphate was purchased as the free acid. The commercial disbdium sal t s of guanosine-5 1 phosphate and uridine-5 1 phosphate were converted to the free acids by elution with water from 2 x 15 columns of Amberlite IR-120(H+) res i n . The eluates were concentrated to the desired starting volume. 3. Nucleoside-5 1 Diphosphates; (a) 0ytidine-5 * Diphosphate: 0«26 ml. of orthophosphoric acid was added to a solution of tri-n-butylamine(0.85 ml.) i n 12 ml. of dry pyridine(dried over anhydrous sodium sulfate) and the compound was dried by three evaporations of i t s solution i n pyridine, and by allowing to stand 2 days i n vacuo. The 4-morpholine N,N'-dicyclohexylcarboxamidinium cytidine-5 1 phosphoromorpholidate(0.41 g.) was dried by two evaporations of i t s solution i n dry pyridine and allowing to stand overnight i n vacuo. 14 The morpholidate and tri-n-butylammonium phosphate were mixed and evaporated twice more under reduced pressure from t h e i r s o l u t i o n s i n dry p y r i d i n e . A f t e r standing overnight i n vacuo, the mixture was d i s s o l v e d i n dry p y r i d i n e ( l 3 ml.) and shaken f o r one h r . A small p r e c i p i t a t e was f i l t e r e d o f f . The s o l u t i o n was allowed to stand f o r 92 h r . i n a d e s i c c a t o r over concentrated B^SO^. The s o l u t i o n was then evaporated and r e s i d u a l p y r i d i n e removed by the a d d i t i o n and evaporation of water. The r e s i d u e was d i s s o l v e d i n 5 ml. of water c o n t a i n i n g 0.23 g. of l i t h i u m a c e t a t e , and the aqueous s o l u t i o n was e x t r a c t e d with ether. The pH was adjusted to 12 with l i t h i u m hydroxide and the mixture s t o r e d a t 0° f o r 45 min. The p r e c i p i t a t e was removed by c e n t r i f u g a t i o n and washed with 0.01 N l i t h i u m hydroxide. The combined supernatant and washings were adjus t e d to pH 8.0 by treatment with Dowex 50(H +) r e s i n . The r e a c t i o n mixture(pH 8.0) was a p p l i e d to a 2 x 15 column of Dowex l ( C l ~ ) r e s i n and e l u t e d w i t h 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 i n h y d r o c h l o r i c a c i d . The mixing chamber contained 2 1. of 0.003 N h y d r o c h l o r i c a c i d and the r e s e r v o i r 2 1. of 0.05 M l i t h i u m c h l o r i d e i n 0.003 N h y d r o c h l o r i c a c i d . Two peaks were e l u t e d . The f i n a l peak was adjusted to pH 4.5 and evaporated to dryness under reduced p r e s s u r e . The r e s i d u e was w e l l s t i r r e d with a mixture of methanol(l0 ml.) and acetone (70 ml.), c o l l e c t e d by c e n t r i f u g a t i o n , and washed wi t h three s m a l l e r p o r t i o n s of raethanol-acetone(l:7), a f t e r which the supernatant was found to be f r e e of Cl""(by t e s t with s i l v e r 15 nit r a t e solution). After a further wash with ether, the product was dried i n vacuo at room temperature, 4. I d e n t i f i c a t i o n of Products: The i d e n t i t y and purity of a l l products were checked by paper chromatography with solvents I and I I . In addition, the purity of adenosine diphosphate, cytidine diphosphate, and uridine diphosphate was checked by chromatography on d i e t h y l -amino ethyl (DEAE) c e l l u l o s e . The nucleotides were eluted with a linear gradient of sodium chloride i n phosphate buffer. The mixing chamber contained 725 ml. of 0,11 M sodium phosphate buffer(pH 7.2) and the reservoir contained 675 ml, of 0.25 M sodium chloride i n 0.11 M sodium phosphate buffer. B, Results 1. Yields: The yields of expected products are given i n tables I and I I . Table I Yields of Nucleoside-5 1 Phosphoromorpholidates Nucleoside-5 1 Phosphate Weight (as the free acid) (g.) Nucleoside-5 1 Phosphoromorpholidate Adenosine-5 1 phosphate Guanosine-5* phosphate Cytidine-5' phosphate Uridine-5' phosphate 0.50 0.90 90 1.00 1.28 69 0.41 0.41 46 0.48 0.71 66 ( l ) The yields were calculated on the basis of the morpholidates being anhydrous, although i t i s known that they are hydrated to varying degrees(46). 16 G y t i d i n e - 5 ' d i p h o s p h a t e 4 0.145 22 Table I I p Y i e l d s of Nucleoside-5' Diphosphates Nucleoside-5' Diphosphate Weight $ Y i e l d (as the t r i - l i t h i u m s a l t ) (g.) ( o v e r a l l ) 3 Adenosine-5 1 diphosphate 0.26 45 ( t r i h y d r a t e ) Guanosine-5 1 diphosphate 0.24 25 ho; ( t r i h y d r a t e ) A U r i d i n e - 5 1 diphosphate 0.16 24 (mo no hydr ate) 2. P u r i t y : The R f values of a l l the nu c l e o s i d e diphosphate when t e s t e d by paper chromatography on s o l v e n t s I and I I , were the same as those recorded by M o f f a t t and Khorana(46). In a d d i t i o n , those t e s t e d on DEAE-cellulose showed no contaminatio by t h e i r r e s p e c t i v e mono- and t r i p h o s p h a t e s except i n the case of ADP. Here there was contamination by adenosine-5 1 monophosphate(4%) and adenosine-5 1 t r i p h o s p h a t e ( 1 3 $ ) • (2) The i n i t i a l weights of morpholidates were those l i s t e d i n t a b l e I . (3) The degree of h y d r a t i o n was estimated from the re p o r t e d molar e x t i n c t i o n c o e f f i c i e n t f o r ADP(54). (4) The degree of h y d r a t i o n given f o r CDP and UDP i s t h a t r e p o r t e d by M o f f a t t and Khorana(46). 17 II• Experiments with Polynucleotide Phosphorylase from  Dihydrostreptomycin Sensitive Escherichia c o l i A. Analytical Methods 1. Enzyme Assay: 32 (a) Nucleoside Diphosphate Exchange with P : This assay was based on that used by Littauer and Kornberg(43). The incubation mixture(0.5 ml.) contained 0.1 ml, of glycylglycine b u f f e r ( l M, pH 7.4), 0.10 ml, of ADP(0.004 M<), 0,05 ml. of P? 2 i n potassium phosphate buffer, pH 7.4(0.0052 M, 3.5 x 10 c#p.m, per (xmole), 0.05 ml. of MgCl 2(0.04 M), and about 0.05 unit of enzyme. The mixture was incubated for 20 min. at 37 . The reaction was stopped by immersing the tubes i n an ice bath and adding 0.5 ml. of 5 per cent perchloric acid. An amount of 0.10 ml, of an acid washed Norit A suspension(10 per cent dry weight) was added to adsorb the nucleotides and the mixture l e t stand 10 min. i n the cold. The Norit was then centrifuged and washed three times with 2.5 ml. portions of 0.003 N HCI. The precipitate was suspended i n 0.8 ml. of 50 per cent ethanol containing 3 ml. of concentrated NR^OH per l i t e r . A 0.1 ml. aliquot of the above Norit suspension(or i n some cases of the supernatant after centrifugation to remove the Norit) was dried on a planchet and the r a d i o a c t i v i t y measured(self—ablsorption correction factor 1.15). 1 unit of enzyme was defined as the 32 amount causing the incorporation of 1.0 u-mole of P into ADP per hour, and the spec i f i c activity(S.A.) was expressed as units per mg. of protein. The amount of P^ incorporated into the terminal phosphate of ADP was calculated from the equation: 18 t o t a l c.p.m. i n ADP u-moles phosphate incorporated = : i n i t i a l S.A. of P. 1 (b) Release of Orthophosphate: The following procedure, which i s a modification of that o r i g i n a l l y used by Grunberg-Manago et a l ( 4 l ) , was generally used. The reaction mixture (l.O ml.) contained 0.2 ml. of tris(hydroxymethyl)aminomethane ( t r i s ) ( l M, pH 8.0), 0.2 ml. of ADP(0.016 M), 0.1 ml. of sodium ethylenediamine tetraacetate(EDTA)(0.0016 M), 0.1 ml. of MgCl2(0.04 M), and 0.2 ml. of enzyme solution. The mixture was incubated at 37°. Orthophosphate released was determined by the method of Olmsted and Lowe(47). To 0.1 ml. of the reaction mixture was added, i n order, 1 ml. of 0.0027 M ammonium molybdate i n 1.0 N B^SO^, and 3 ml. of the N-phenyl-p-phenylenediamine solution, made up according to Dryer et al(48). After at least 10 min. o p t i c a l densities were read i n the Beckman Model B spectrophotometer at 750 mu. 2. Protein Determination: Two methods were used: 1. The method generally used was that of Lowry et al(49). Reagent A consisted of 2$ Na 2C0 3 i n 0.1 N NaOH. Reagent B was a 0.5$ solution of CuSO^.SH^G; i n 1$ aqueous sodium potassium t a r t r a t e . Reagent B ( l ml.) was mixed with 50 ml. of reagent A to give reagent C, which was discarded after one day. To 1.7 ml. or less of solution containing 150 u.g. or less of protein was added 3 ml. of reagent C, and the mixture was l e t stand at least 10 min. Folin-Ciocalteau reagent(obtained 19 commercially from the B r i t i s h Drug Houses(Canada) Ltd.)(0.3 ml.) was added with rapid mixing and the mixture l e t stand 30 min. After t h i s time, the o p t i c a l densities were read at 500 mo., and the protein concentration determined by comparison with a standard curve prepared with bovine y-globulin, f r a c t i o n Il(Calbiochem.). 2. Clear solutions were occasionally analysed by measuring the ratio of absorption at 260 mu. and 280 mp., according to the procedure of Warburg and Christian(50,51). 3. Paper Chromatography: Paper chromatography was carried out with solvent I (Sec. I . A . I . ) . 4. Ultracentrifuge Measurements: Sedimentation constants of macromolecules were measured i n a Spinco Model E ultracentrifuge. The temperature of the rotor was measured before and immediately after each run, and the average of these two temperatures was taken as the temperature of the solution during the run. The measured v i s c o s i t y of the solutions was e f f e c t i v e l y the same as d i s t i l l e d water, consequently the v i s c o s i t i e s used to standardise the sedimentation c o e f f i c i e n t s to water at 20° were the l i s t e d values for water(52). The positions of the boundaries were measured with a Gaertner microcomparator. Sedimentation c o e f f i c i e n t s were calculated from the following formulae(53): 2.303(lttg x 2 - log x x) s =s 2 • • • • • • co ( t 2 - t x ) 20 where s = sedimentation c o e f f i c i e n t X 2 , X 1 = d i s t a n c e of the boundary from the center of . r o t a t i o n at times t 2 a n d r e s p e c t i v e l y oo = angular v e l o c i t y of the r o t o r 2 ( 3 . 1 4 ) ( r e v o l u t i o n s per minute(r.p.m.)) ^ 60 <*U,w s o n = ——• x s (2) 2 0 > w T ^ C w whereff^. w » ^ 2 0 w m v i s c o s i * i e s °^ water at temperature t and 20° r e s p e c t i v e l y . B, P r e p a r a t i o n of P o l y n u c l e o t i d e Phosphorylase from E s c h e r i c h i a  c o l i ( 4 3 ) 1. Growth of B a c t e r i a : The medium(T medium) used throughout t h i s work contained the f o l l o w i n g : 1.70$ KH 2P0 4, 2.18$ K 2HP0 4, 1$ yeast e x t r a c t ( D i f c o ) , and 1$ glucose. Glucose was autoclaved s e p a r a t e l y . The f i n a l pH was 6.8-7.0, adjusted when necessary w i t h KOH. Antifoam A(Corning) was added to c u l t u r e s growing under f o r c e d a e r a t i o n . Dihydrostreptomycin s e n s i t i v e E. c o l i ( l . 3 1. of an 18 h r . c u l t u r e ) was i n n o c u l a t e d i n t o 10 1. of X medium and grown f o r 4 h r . at 37° w i t h vigorous f o r c e d a e r a t i o n . A f t e r c o l l e c t i o n by c e n t r i f u g a t i o n , the c e l l s ( 7 2 g., wet weight) were washed once wi t h f o u r volumes of c o l d 0.9$ KG1, and suspended i n c o l d 0.05 M g l y c y l g l y c i n e b u f f e r ( p H 7.4) to a t o t a l volume of 245 ml. 21 2. Preparation of Enzyme: The above suspension was treated for 10 min. i n a 10 kc. sonic o s c i l l a t o r , then centrifuged at 10,000g for 20 min. The residue was suspended i n glycylglycine buffer(0.05 M, pH 7.4) to a f i n a l volume of 245 ml. and treated an additional 30 min. i n the sonic o s c i l l a t o r at aboyt 6°. The material was centrifuged for 20 min. at 10,000g, giving a supernatant, the sonic extract of the residue. This could be, and part of i t was, stored i n the deep freeze without loss of enzyme a c t i v i t y . A l l of the following steps were carried out at 0-4°, except where otherwise indicated. To 100 ml. of the sonic extract of the residue was added 5.05 ml. of 1 M MnC^, slowly, with mechanical s t i r r i n g . The s t i r r i n g was continued for 30 min. Insoluble material was removed by centrifugation at 10,000g for 20 min. To the supernatant was added 12.0 ml. of Vfo protamine sulfate with mechanical s t i r r i n g over a period of 10 min. The precipitate was collected by centrifugation and suspended i n 48 ml. of 0.05 M phosphate buffer, pH 7.5, and recentrifuged(l0,000g, 20 min.). The supernatant f l u i d was dialysed overnight against 1 l i t r e of 0.9$ KC1. The dialysate was the protamine eluate. To 46 ml. of protamine eluate was added 0.74 ml. of 1 M potassium acetate (pH 5.5) and then 0.22 ml. of 0.5 M ZnC^ (previously adjusted to pH 5.5 with acetic acid). After standing for 10 min. the precipitate was removed by centrifuging for 3 min. at 10,000g. To the supernatant f l u i d was added 8.16 ml. of 50$ ethanol(-15°) over a 7 min. i n t e r v a l , during 22 which time the mixture was c h i l l e d to —2°. The precipitate was removed by centrifugation for 3 min. at 10,000g, and to the supernatant f l u i d was added 9.6 ml. of 50$ ethanol as before. The precipitate was collected by centrifugation and dissolved i n t r i s buffer(0.05 M, pH 8.0) to a f i n a l volume of 12.4 ml. This was the Ethano1 I f r a c t i o n . An assay of the Ethanol I f r a c t i o n showed that l i t t l e a c t i v i t y remained. Therefore, the precipitates from the zinc p r e c i p i t a t i o n and f i r s t ethanol precipitations were dissolved i n t r i s buffer(16 ml. each), dialysed against 1 l i t r e of 0.9$ KC1 at 4° overnight, and assayed(Assay 4a)). The major part of the a c t i v i t y was found i n the f i r s t ethanol p r e c i p i t a t e , which was c a l l e d Ethano 1 j r ( E i ) . Although the s p e c i f i c a c t i v i t y of the Ethanol j f r a c t i o n was less than that of the preceeding protamine eluate, i t was used for further experiments since i t was e s s e n t i a l l y free of nucleic acid contamination( as indicated by a^280 :^260 r a / t i o o f 1.82(51)). 3. A c t i v i t i e s of the Fractions: The a c t i v i t i e s of the various fractions are presented i n table I I I . 23 Table III A c t i v i t i e s of the Fractions Fraction sonic extract of the residue protamine eluate ethanol I ethanol \ Enzyme A c t i v i t y (units/ml. Of protein solution) 0.68 2.00 0.12 0.50 [Protein] (mg./ml.) 0.71 0.34 0.26 Specific A c t i v i t y (units/mg. protein) 2,82 0.34 1.92 C. Effect of Dihydrostreptomycin(DHSM) on the Exchange  Reaction(Assay (a)) 1. Eff e c t on Enzyme Fractions: The e f f e c t of 1000 fig/ml. of DHSM on the a c t i v i t i e s of the enzyme fractions i s presented i n table IV. Table IV Effect of DHSM (1000 u.g/ml. ) on the A c t i v i t i e s of the Enzyme Fractions as Measured by the Exchange Reaction(Assay (a)) Reaction conditions were as i n Assay (a). Aliquots of 0.1 ml. of the supernatant after removal of the Norit by centrifugation were counted. Since there i s considerable inaccuracy i n the absolute values of such high counts, actual incorporations 32 of P were not calculated. Counts to be compared were corrected with respect to each other for the quantity of material absorbing at 260 mp. recovered from the Norit. Enzyme Fraction control sonic extract of the residue protamine eluate ZnC^-HAc precipitate •-Counts/min. no added DHSM 1000 (ig/ml. DHSM 262 374 22,500 23,000 36,800 38,500 10,600 9100 24 2. E f f e c t of DHSM on the Ethanol \ Fraction: The e f f e c t of three concentrations of DHSM(200, 400, and 800 u.g./ml.) was studied. DHSM was added both before and after incubation. Both the ethano l-^ NH^ OH eluate and the Norit suspension were counted. The results are presented i n table V. Table V Effect of Adding DHSM Before and After Incubation Experimental conditions: glycylglycine, 100 mM, pH 7.4; 32 ADP, 0.4 mM; sodium dihydrogen phosphate, 0.52 mM with P spe c i f i c a c t i v i t y approximately 7 x 10 c .p.m./jj,mole; DHSM as indicated; t o t a l volume, 0.5 ml. Incubated 20 min. at 37°. (+) DHSM indicates addition after incubation. Experiment [DHSM] (|xg./ml. ) Incorporation (ixmoles/hr./ml. E^) Norit suspension Eluate 1 0 800 (+)800 0.21 0.24 0.24 0.15 0.10 0.10 2 0 200 (+)200 400 (+)400 0.31 0.29 0.29 0.33 0.31 0.24 0.20 0.20 0.24 0.22 3. E f f e c t of DHSM on the Time Course of the Exchange  Reaction; The time course of the exchange reaction between ADP 32 and P i n the presence of 200 |ig./ml. of DHSM and with no added DHSM i s given i n F i g . 2. The Ethanol j f r a c t i o n was used. 25 25 50 75 100 125 150 TIME(MIN) FIG. 2 Time study of the polynucleotide phosphorylase catalysed exchange reaction. Each reaction mixture contained the following: 200 mM glycylglycine, pH 7.4; 0.8 mM ADP; 0.4 mM MgCl9; sodium 32 8 dihydrogen phosphate, 0.52 mM; P , 3.4 x 10 counts; 0.35 units of Ewj. The total volume in each flask was 7.5 ml. One flask contained 200 ug./ml. of DHSM(A), the other contained no DHSM(o). The flasks were incubated at 37°. 26 D. E f f e c t of Dihydrostreptomycin on Polyadenylate Synthesis  (Assay (b)) 1. Requirement of the Reaction f o r Primer: The supernatant from the protamine p r e c i p i t a t i o n s t ep, •which contained the major p a r t of the n u c l e i c a c i d from the c e l l s , was used. Approximately 40 ml. of the protamine supernatant f r a c t i o n was d i a l y s e d a g a i n s t 1.5 1. of 0.9$ KC1 f o r 44 h r . a t 4 ° , then c e n t r i f u g e d f o r 10 min. a t 10,000g. The supernatant was r e t a i n e d : t h i s was termed P . ' s The r e s u l t s of a time study of the r e a c t i o n i n the presence and absence of P are shown i n F i g . 3. These r e s u l t s i n d i c a t e s t h a t there i s no requirement f o r a primer with t h i s enzyme preparation(E?!j) . 2. E f f e c t of DHSM on the P o l y m e r i z a t i o n R e a c t i o n : The e f f e c t of 500 p.g/ml. of DHSM on the time course of the sy n t h e s i s of polyAMP i s shown i n F i g . 4. The r e s u l t s i n d i c a t e DHSM had l i t t l e or no e f f e c t . 3. E f f e c t of Diamines on the P o l y m e r i z a t i o n R e a c t i o n : The e f f e c t of the n a t u r a l l y o c c u r r i n g diamines p u t r e s c i n e , cadaverine, spermine, and spermidine was s t u d i e d i n c o n j u n c t i o n w i t h t h i s work. The r e s u l t s ( F i g . 5) i n d i c a t e d these compounds had no e f f e c t on the time course of the r e a c t i o n . E. E f f e c t of DHSM on the Polymer Formed Re a c t i o n mixtures c o n t a i n i n g approximately the same q u a n t i t y of polyAMP, formed i n the presence and absence of 1000 u-g./ml. of DHSM, were examined i n the u l t r a c e n t r i f u g e . A c o n t r o l 27 c o n t a i n i n g enzyme, but no s u b s t r a t e , was a l s o examined. The r e s u l t s are presented i n F i g . 6. F. E f f e c t of Diamines on the Polymer Formed The u l t r a c e n t r i f u g e diagram of polyAMP s y n t h e s i z e d by p o l y n u c l e o t i d e phosphorylase i n the presence of spermidine(1.6 mM) i s shown i n F i g . 6D. The diagrams from a s i m i l a r experiment, w i t h p u t r e s c i n e , cadaverine, spermine, and spermidine added to a f i n a l c o n c e n t r a t i o n of 0.4 mM each, i s shown i n F i g . 6E. 28 T 1 1 r FIG. 3 Time course of polyadenylate s y n t h e s i s . Test of a d d i t i o n of protamine s u p e r n a t a n t ( c o n t a i n i n g n u c l e i c a c i d ) . Each r e a c t i o n mixture contained: t r i s , 200 mM, pH 8.0; ADP, 3.2 mM; MgCl~, 1 0.4 mM; E^, 0.2 ml. To one r e a c t i o n mixture was added 0.2 ml. of P g ( x ) , to the other 0.2 ml. of water(o). The t o t a l volume was 1.0 ml. The mixtures were incubated a t 37°. 29 r~ PIG, 4 Lack of effect of DHSM on the time course of polyadenylate synthesis. Each reaction mixture contained: tris, 200 mM, pH 8.0; ADP, 3.2 mM; EDTA, .1.6 mM; MgCl2, 0.4 mM; E^, 0.2 ml. To one flask was added DHSM to give a final concentration of 500 jig./ml. (•), the other contained no DHSM(o). The final volume was 1.0 ml. The mixtures were incubated at 37°. 30 FIG. 5 Lack of e f f e c t of diamines on the time course of polyadenylate s y n t h e s i s . Each r e a c t i o n mixture contained: t r i s , 200 mM, pH 8.0; ADP, 3.2 mM; EDTA, 1.6 mM;. MgCl-, 0.4 mM; E^, 0.2 ml. To one f l a s k was added p u t r e s c i n e , cadaverine, spermine, and spermidine•'to a f i n a l c o n c e n t r a t i o n of 0.5 mM each(»), while the other contained no diamines(o). The f i n a l volume was 1.0 ml. The mixtures were incubated a t 37°. 31 E FIG. 6 Ultracentrifuge patterns of polyadenylate synthesized by polynucleotide phosphorylase. Each reaction mixture contained the following: t r i s buffer, 200 mM, pH 8.0; MgC^, 4 mM; EDTA, 0.16 mM; ADP, 6.4 mM; E^, 0.2 ml. The f i n a l volume was 1.0 ml. The mixtures were incubated at 37°. 32 The sedimentation d i r e c t i o n i s from l e f t to r i g h t . A l l runs were a t 44,770 r.p.m. A. C o n t r o l , no ADP. P i c t u r e s taken 17 min. a f t e r r e a c h i n g speed. Bar angle, 60°. B. No a d d i t i o n s . P i c t u r e s taken 17 min. a f t e r r e a c h i n g speed. Bar angle, 82°. S 2 Q ^ 9 . 4 8 . C. DHSM added to a f i n a l c o n c e n t r a t i o n of 1000 ug./ml. P i c t u r e s taken 9 min. a f t e r r e a c h i n g speed. Bar angle, 70°. s20,w " 9 - 5 S -D. Spermidine added to a f i n a l c o n c e n t r a t i o n of 1.6 mM. P i c t u r e s taken 13 min. a f t e r r e a c h i n g speed. Bar angle, 70°. s20,w * 8 - 2 S ' E. P u t r e s c i n e , cadaverine, spermine, and spermidine added to a f i n a l c o n c e n t r a t i o n of 0.4 mM each. P i c t u r e s taken 9 min. a f t e r r e a c h i n g speed. Bar angle, 60°. 33 I I I . S t u d i e s on Ribosomes from E s c h e r i c h i a c o l i A. A n a l y t i c a l Methods 1. P r o t e i n Determination: P r o t e i n was determined by the method of Lowry et a l ( 4 9 ) , as i n d i c a t e d i n Sec. I I . A. 2. 2. R i b o n u c l e i c Acid(RNA) Determination: RNA was determined by a m o d i f i c a t i o n of the method of Dische and Schwarz(55). To approximately 0.5 ml. of a s o l u t i o n of RNA was added 1.5 ml. of the reagent prepared by d i s s o l v i n g 100 mg. of P e C l 3*6H 20 i n 100 ml. of concentrated HCI. Then 1.75 ml. of a 6fo s o l u t i o n of o r c i n o l ( F i s h e r reagent grade) i n ethanol was added. The r e a c t i o n mixture was heated i n a b o i l i n g water bath f o r 25 min. and cooled r a p i d l y . The volume was brought to 5.0 ml. w i t h d i s t i l l e d water. A blank without RNA was run simultaneously. The o p t i c a l d e n s i t i e s of the s o l u t i o n s were read i n a Beckman Model B spectrophotometer a t 665 mj>. A standard curve was prepared u s i n g y e a s t RNA(Matheson Coleman and B e l l ) . 3. Chromatography on D E A E - c e l l u l o s e : (a) DEAE-cellulose to be e l u t e d with a l i n e a r g r a d i e n t of NaCl i n 0.01 M t r i s b u f f e r , pH 7.4, was suspended three times i n 0.01 M t r i s , pH 7.4, and the f i n e s were decanted a f t e r a l l o w i n g the coarse m a t e r i a l to s e t t l e . (b) DEAE-cellulose to be e l u t e d with a l i n e a r g r a d i e n t of NaCl i n 0.01 M t r i s , 0.01 M MgCl 2, pH 7.4(TM b u f f e r ) was suspended three times i n TM and the f i n e s decanted a f t e r a l l o w i n g the coarse m a t e r i a l to s e t t l e . The D E A E - c e l l u l o s e was then washed on a column wi t h TM. 34 B. Growth of Bacteria Overnight cultures(10$ of the f i n a l growth medium) of E. c o l i SA or SB* i n I medium were inoculated into 3 to 10 1. KM* • of T medium and grown under vigorous forced aeration at 37° to a f i n a l o p t i c a l density at 600 mu of 1.2. The y i e l d of wet packed c e l l s was 5 to 6 g. per l i t r e . E. c o l i RB was grown i n a similar manner except that the growth media contained DHSM i n a concentration of 100 u g . / l i t r e . The c e l l s were harvested immediately and washed twice with three volumes of cold TM buffer. The c e l l s were stored at -15°. C. Preparation of Ribosomes Cell s were generally thawed overnight at 4° before use, but use of the frozen c e l l s d i r e c t l y gave a preparation with the same ch a r a c t e r i s t i c s . A l l of the following operations were carried out at 0-4°. The c e l l s ( l 0 g. wet wt.) were ground with 30 g. of levigated alumina(Alcoa Chemicals) for about 5 min. and then extracted with 30 ml. of TM. The suspension was cent-rifuged for 15 min. at 6,000 g and the precipitate(PI), consisting of alumina, whole c e l l s , and c e l l debris, was discarded. The supernatant(SI) was centrifuged again at 6,000 g and the ( l ) Two strains of Escherichia c o l i have been used i n this investigation: SA and SB. Strain SA was obtained by back-mutation from a streptomycin dependent E. coli(DA). o r i g i n a l l y obtained from Dr. Thomas F. Paine, Jr.^Massachusetts General Hospital. S t r a i n SB i s #482 of the culture c o l l e c t i o n of the National Research Council of Canada. A re s i s t a n t mutant(RB) was obtained from i t . 35 p r e c i p i t a t e ( P I I ) was d i s c a r d e d . The supernatant(SII) was c e n t r i f u g e d a t 8,000 g f o r 30 min. and the p r e c i p i t a t e ( P H I ) d i s c a r d e d . The s u p e r n a t a n t ( S i l l ) was c e n t r i f u g e d a t 100,000 g f o r 120 min. The supernatant(SIV=slOO,000 g supernatant) was u s u a l l y d i s c a r d e d . The ribosome p r e c i p i t a t e ( P I V ) was suspended i n 30 ml. of TM w i t h the a i d of an homogenizer and c e n t r i f u g e d a t 8,000 g f o r 15 min. The p r e c i p i t a t e ( P V ) was d i s c a r d e d . The supernatant(SV) c o n t a i n i n g the^ribosomes was sometimes used as such but was u s u a l l y c e n t r i f u g e d again at 100,000 g f o r 120 min. The p r e c i p i t a t e was brought up i n 15 ml. of TM and c e n t r i f u g e d a t 8,000 g f o r 15 min. to give SVI. U l t r a c e n t r i f u g e diagrams of the crude e x t r a c t ( S I I I ) and the ribosome preparation(SVI) are shown i n P i g . 7. D. S t a b i l i t y of Ribosomes i n the Presence of DHSM 1. D i s s o c i a t i o n of Ribosomes with DHSM: Ribosome s u s p e n s i o n ( S V ) ( l ml.) from E. c o l i SA was pl a c e d i n 1 cm. cellophane tubing and d i a l y z e d a g a i n s t 300 ml. of b u f f e r a t 4 ° . The b u f f e r s used were 0.01 M t r i s , pH 7.4, and TM. The b u f f e r s contained DHSM i n con c e n t r a t i o n s ranging from 0-3000 ug./ml. A f t e r 24 hr. d i a l y s i s , the d i a l y s a t e s were brought to 2.5 ml. wi t h TM and c e n t r i f u g e d a t 6,000 g f o r 15 min. A l i q u o t s of 0.1 ml. of the supernatant were used f o r p r o t e i n and RNA determinations. The p r o t e i n and RNA values were expressed as per cent of the sample d i a l y z e d a g a i n s t TM and are shown i n P i g . 8. The r e s u l t s i n d i c a t e d t h a t the ribosomes were d i s r u p t e d and r i b o n u c l e o -p r o t e i n p r e c i p i t a t e d a t 500 ug./ml. of DHSM i n the absence of 36 M g C l 2 but t h a t the l e v e l of DHSM r e q u i r e d i n c r e a s e d to about 1800 ug./ml. i n the presence of 0.01 M MgCl^. In each case about 60$ of the p r o t e i n was p r e c i p i t a t e d , and about 80$ of the RNA. Ribosomes were d i l u t e d 1:1 with TM, but were found again to p r e c i p i t a t e a t about 500 |ig./ml. of DHSM, thus i n d i c a t i n g t h a t the phenomenon was independent of ribosome c o n c e n t r a t i o n . Although ribosomes p r e c i p i t a t e d a t about 500 ug./ml. of DHSM when d i a l y z e d o v e r n i g h t , a check of d i a l y s a t e s i n the a n a l y t i c a l u l t r a c e n t r i f u g e i n d i c a t e d t h a t breakdown occurred a f t e r d i a l y s i s a g a i n s t 0.01 M t r i s , pH 7.4, c o n t a i n i n g about 350 ug./ml. of DHSM(Figs. 9, 10). Ribosomes from E. c o l i SB were d i a l y z e d a t 4° f o r 18 h r . ag a i n s t 0.01 M, t r i s , pH 7.4, c o n t a i n i n g 0 and 400 fig./ml. of DHSM. The d i a l y s a t e was placed on a 1 x 15 cm. column of DEAE-c e l l u l o s e and e l u t e d with a l i n e a r g r a d i e n t of NaCl i n 0.01 M t r i s , pH 7.4. F r a c t i o n s of 5 ml. were c o l l e c t e d and the o p t i c a l d e n s i t y a t 260 mp, determined(Fig. 11). I t was found l a t e r t h a t the use of a gr e a t e r c o n c e n t r a t i o n of Mg , coupled w i t h a longer column and s l i g h t l y shallower g r a d i e n t , gave b e t t e r r e s o l u t i o n . Ribosomes from E. c o l i SB and RB were each d i a l y z e d overnight a t 4° ag a i n s t 0.01 M t r i s , pH 7.4(250 ml.), c o n t a i n i n g 0 and 400 ug./ml. of DHSM. The d i a l y s a t e s were p l a c e d on a 0.8 x 22 cm. column of DEAE-ce l l u l o s e and e l u t e d w i t h a l i n e a r g r a d i e n t of NaCl i n TM. F r a c t i o n s of 5 ml. were c o l l e c t e d and the o p t i c a l d e n s i t y a t 260 mp, determined. The r e s u l t s are shown i n F i g s . 12 and 13. 37 B FIG* 7 Ultracentrifuge photographs of E. coli SB fractions. Sedimentation is from l e f t to right. Speed, 35,600 r.p.m. Bar angle, 60°. A. SIII. Pictures taken 10 min. after reaching speed. Interval, 8 min. B. SVI. Pictures taken 10 min. after reaching speed. Interval, 4 min. 38 1000 2 0 0 0 3 0 0 0 D I H Y D R O S T R E P T O M Y C I N ( p G / M L ) EIG. 8 Effect on soluble RNA and protein of dialysis of E. co l i SA ribosomes against DHSM in t r i s buffer, pH 7.4. After dialysis, the samples were centrifuged and the supernatant analyzed for RNA(») and. protein(o)'. A similar experiment was performed in the presence of 0.01 M UgCl^i- - - ) . Results are expressed as per cent of a control dialyzed against TM buffer. 39 FIG. 9 E f f e c t of DHSM on u l t r a c e n t r i f u g e p a t t e r n s of ribosomes from E. c o l i S B ( s e n s i t i v e ) . A l l runs were at a speed of 35,600 r.p.m. Sedimentation i s from l e f t to r i g h t . 40 A. SVI. P i c t u r e s taken 13 min. a f t e r reaching speed. Bar angle, 50°. s 2 Q > w = 53.6 S, 68.5 S, 95.6 S. B. SVI d i a l y z e d a g a i n s t 0.01 M t r i s , pH 7.4. P i c t u r e s taken 40 min. a f t e r r e a c h i n g speed. Bar angle, 45°. s 2 0 w m 2 7 , 7 S ' 4 2 , 4 S * C. SVI d i a l y z e d a g a i n s t 0.01 M t r i s , pH 7.4, c o n t a i n i n g 300 ug./ml. of DHSM. P i c t u r e s taken 14 min. a f t e r r e a c h i n g speed. Bar angle, 40°. S 2 Q v = 28.6 S, 42.5 S. D. SVI d i a l y z e d a g a i n s t 0.01 M t r i s , pH 7.4, c o n t a i n i n g 350 ug./ml. of DHSM. P i c t u r e s taken 10 min. a f t e r r e a c h i n g speed. Bar angle, 40°. E. SVI d i a l y z e d a g a i n s t 0.01 M t r i s , pH 7.4, c o n t a i n i n g 400 ug./ml. of DHSM. P i c t u r e * taken 10 min. a f t e r r e a c h i n g speed. Bar angle, 40°. 41 FIG. 10 Effect of DHSM on ultracentrifuge patterns of ribosomes from E. c o l i RB(resistant). A l l runs were at 35,600 r.p.m. Sedimentation i s from l e f t to r i g h t . A. SVI. Pictures taken 12 min. after reaching speed. Bar angle, 55°. s20,v = 5 7 , 8 S > 9 7 , 5 S ' B. SVI dialyzed against 0.01 M t r i s , pH 7.4. Pictures taken 18 min. after reaching speed. Bar angle, 55 . s20,w = 2 6 « 5 S ' 4 1 ' 4 S ' C. SVI dialyzed against 0.01 M t r i s , pH 7.4, containing 400 ug./ml. of DHSM. Pictures taken 5 min. after reaching speed. Bar angle, 55°. 42 B 4.0 " "60 '.. 80 "• FRACTION- "NUMBER 100, i2o: 140 160 FIG. 11 Ribosomes from E. c o l i S B ( s e n s i t i v e ) chromatographed on DEAE-cellulose with a l i n e a r g r a d i e n t of NaCl( ) i n 0.01 M t r i s , pH 7.4. The ribosomes were d i a l y z e d overnight a g a i n s t t r i s b u f f e r ( A ) , and t r i s b u f f e r c o n t a i n i n g 400 ug./ml. of DHSM(B). 43 -B 20 40 6 0 8 0 FRACTION NUMBER 100 120 40 60 80 FRACTION NUMBER FIG. 12 Ribosomes from s e n s i t i v e E. c o l i SB chromatographed on DEAE-cellulose w i t h &•linear g r a d i e n t of NaCl( ) i n TM b u f f e r . The ribosomes were d i a l y z e d overnight against t r i s b u f f e r (A) and t r i s b u f f e r c o n t a i n i n g 400 u-g./ml. of DHSM(B). 44 B -0-5 20 40 60 80 FRACTION NUMBER < _i o 2 FIG. 13 Ribosomes from r e s i s t a n t E. c o l i RB chromatographed on DEAE-cellulose w i t h a l i n e a r g r a d i e n t of NaCl( ) i n TM b u f f e r . The ribosomes were d i a l y z e d a g a i n s t t r i s b u f f e r ( A ) and t r i s b u f f e r c o n t a i n i n g 400 p,g./ml. of DHSM(fi); 45 Ribosomes from E. c o l i SB and RB were d i a l y z e d at 4° ag a i n s t 250 ml. of 0.01 M t r i s , pH 7.4, c o n t a i n i n g v a r y i n g amounts of DHSM below the degradation l e v e l . The sedimentation c o e f f i c i e n t s of the p a r t i c l e s i n the d i a l y s a t e s were then determined. The r e s u l t s are presented i n t a b l e s VI and V I I . Table VI Sedimentation C o e f f i c i e n t s of Ribosomes from E. c o l i SB  D i a l y z e d Against I n c r e a s i n g Concentrations o f DHSM "* E. c o l i SB ri b o s o m e s ( l ml.) were p l a c e d i n 1 cm. cellophane t u b i n g and d i a l y z e d f o r 18 hr . aga i n s t 250 ml. of 0.01 M t r i s b u f f e r , pH 7.4, c o n t a i n i n g DHSM i n the co n c e n t r a t i o n s i n d i c a t e d . The p r o t e i n c o n c e n t r a t i o n was about 1.75 mg./ml. i n each case. , s20,w (Ug./ml.) 0 33.8 39.3 19.1 32.0 34.8 38.1 29.4 38.2 76.3 30.0 41.7 152.5 28.7 45.1 Table VII Sedimentation C o e f f i c i e n t s of Ribosomes from E. c o l i RB  D i a l y z e d A g a i n s t I n c r e a s i n g Concentrations of DHSM E. c o l i RB ri b o s o m e s ( l ml.) were p l a c e d i n 1 cm. cellophane t u b i n g and d i a l y z e d f o r 18 h r . a g a i n s t 250 ml. of 0.01 M t r i s b u f f e r , pH 7.4, c o n t a i n i n g DHSM i n the co n c e n t r a t i o n s i n d i c a t e d . The p r o t e i n c o n c e n t r a t i o n was about 1.84 mg./ml. i n each case. [DHSM] s20 w (u-g./ml.) (a\ 0 26.5 41.4 15 28.2 35.0 30 28.2 36.3 60 26.4 39.5 120 28.5 40.2 46 2. Autodegradation of Ribosomes i n the Presence of DHSM; Under certa i n conditions, ribosomes from E. c o l i have been shown to undergo autodegradation with the formation of ADP, GDP, UDP, and CDP(56, 57). This has been shown, i n part, to be due to a polynucleotide phosphorylase associated with the ribosomes(57). In t h i s work, ribosomes were incubated under conditions i n which polynucleotide phosphorylase has been shown to be active i n order to determine i f the i n s t a b i l i t y of ribosomes i n the presence of DHSM could be demonstrated i n th i s way. The incubation mixture contained 0.01 M t r i s , pH 7.4, 0.01 M Na 2HP0 4, 0.15 M NaCl, and 4 to 6 mg. of ribosomes. The f i n a l volume was 10 ml. The mixture was incubated,at 37(-0.5)°. At the specified times, 0.5 ml. samples were withdrawn and added to 0.5 ml. of 5$ tr i c h l o r o a c e t i c acid at 0°. The precipitate was then removed by centrifugation and the o p t i c a l density at 260 m»x of the soluble portion was measured i n a Beckman Model DU spectrophotometer. Pig. 14 shows the course of breakdown of ribosomes from E. c o l i SA, SB, and RB c e l l s . 3. B-Galactosidase A c t i v i t y of Ribosomes i n the Presence  of Dihydrostreptomycin: The induction of B-galactosidase has been shown to be inh i b i t e d i n E. c o l i SA by DHSM(26, 28, 29). A small, but si g n i f i c a n t , f r a c t i o n of thi s enzyme has been shown to be associated with the ribosomes of E. coli(58, 59). Consequently, i t might be expected that i n the presence of DHSM, the a c t i v i t y 47 FIG. 14 Autodegradation of ribosomes from sensitive E. c o l i SB(- ), and SA(- ), and resistant E. c o l i RB( ) The reaction mixtures contained the following concentrations of DHSM: 0 u-g./ml.(o), 175 ug./ml.(•), and 350 |ig./ml.(A). 48 of B-galactosidase a s s o c i a t e d w i t h the ribosomes might i n c r e a s e due to the r e l e a s e of nascent enzyme. (a) Growth of E s c h e r i c h i a c o l i SA: I medium(l l i t r e ) was i n o c u l a t e d w i t h 2 ml. of a c u l t u r e °^ U» C ° H SA and incubated at 37° o v e r n i g h t . The c e l l s were harvested by c e n t r i f u g a t i o n and washed twice w i t h 40 ml. of c o l d b u f f e r s o l u t i o n of the f o l l o w i n g composition: 2.2$ K^HPO^, 0.39$ KH 2P0 4, 0.01 M MgCl 2, 0.1$ NaCl, f i n a l pH ad j u s t e d to 7.25(P b u f f e r ) . The y i e l d of wet packed c e l l s was 3.45 g. (b) I n d u c t i o n of 3-Galactosidase w i t h L a c t o s e : The c e l l s were suspended i n 150 ml. of P b u f f e r c o n t a i n i n g 0.01 M l a c t o s e and incubated at 37° w i t h shaking f o r 2.75 h r . The c e l l s were then harvested and washed twice w i t h c o l d TM b u f f e r . The c e l l s were ground w i t h 10 g. of alumina and e x t r a c t e d with~100 ml. of TM. The e x t r a c t was c e n t r i f u g e d at 6,000 g f o r 15 min. and the p r e c i p i t a t e ( P l ) d i s c a r d e d . The super n a t a n t ( S i ) was c e n t r i f u g e d again at 6,000 g f o r 15 min. and the p r e c i p i t a t e ( P I l ) d i s c a r d e d . The s u p e r n a t a n t ( S I l ) was c e n t r i f u g e d at 8,000 g f o r 30 min. and the p r e c i p i t a t e ( P i l l ) d i s c a r d e d . The s u p e r n a t a n t ( S i l l ) was c e n t r i f u g e d at 100,000 g f o r 120 min. to give a supernatant(SIV) and p r e c i p i t a t e ( P I V ) . PIV was brought up i n 60 ml. of TM, c e n t r i f u g e d at 8000 g f o r 15 min., and approximately 20 ml. r e t a i n e d . The remainder was c e n t r i f u g e d at 100,000 g f o r 90 min. to give a supernatant(SV) and p r e c i p i t a t e ( P V ) . PV was brought up i n 20 ml. of TM and aggregates removed by c e n t r i f u g i n g at 8,000 g f o r 15 min. 49 (c) Enzyme Assay: The assay mixture c o n s i s t e d of 2.5 ml. of P b u f f e r , 0.5 ml. of o-nitrophenyl-B-D-galactoside(ONPG), and 0.5 ml. of enzyme s o l u t i o n . At 0, 10, 20, 30, and 40 min. 0.5 ml. a l i q u o t s were withdrawn and added to 9 ml. of a s o l u t i o n of 0.10 M Na~C0 £ 3. The o p t i c a l d e n s i t y was determined at 420 imx. One u n i t o f a c t i v i t y was d e f i n e d as the h y d r o l y s i s of one fimole of 0NPG per minute at 37° i n the i n c u b a t i o n mixture used. The s p e c i f i c a c t i v i t y was d e f i n e d as u n i t s per mg. of p r o t e i n . The d i s t r i b u t i o n of a c t i v i t y i n the v a r i o u s f r a c t i o n s i s presented i n t a b l e V I I I . The PV f r a c t i o n was d i a l y z e d a g a i n s t TM b u f f e r and t r i s b u f f e r , pH 7.4, p l u s 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 DHSM overnight at 4 ° . The a c t i v i t i e s of the d i a l y s a t e s , a f t e r c e n t r i f u g a t i o n at 6,000 g f o r 15 min., are shown i n t a b l e IX and F i g . 15. A c t i v i t i e s of the F r a c t i o n s of 3-D-Galactosidase Induced E. c o l i SA Table V I I I F r a c t i o n P r o t e i n (mg./ml.) S p e c i f i c A c t i v i t y (units/mg. of p r o t e i n ) T o t a l U n i t s S I I I SIV PIV SV PV 1.35 1.27 0.31 0.08 0.26 0.74 0.75 0.61 1.80 0.063 99.5 77.5 11.4 8.65 0.27 50 Table IX A c t i v i t i e s of the PV D i a l y s a t e s D i a l y s i s S o l u t i o n S p e c i f i c A c t i v i t y (units/mg. o f p r o t e i n ) TM 0.056 t r i s + 0 [xg./ml. of DHSM 0.049 t r i s + 200 | A g./ml. of DHSM 0.050 t r i s + 400 ug./ml. of DHSM 0.055 t r i s + 600 fig./ml. of DHSM 0.074 t r i s + 1000 (ig./ml. of DHSM 0.10 Ul r-O <E 0. U. o (0 i-o < o Ul 0. V) 0 0 8 0 0 4 400 800 DIHYDROSTREPTOMYCIN (UG/ML) PIG. 15 S p e c i f i c A c t i v i t i e s of the 6,000 g s o l u b l e p o r t i o n of B-galactosidase induced s e n s i t i v e E. c o l i SB ribosome d i a l y s a t e s . 51 DISCUSSION I . P r e p a r a t i o n of Nucleoside Diphosphates The y i e l d s of n u c l e o t i d e s obtained were, i n g e n e r a l , not as h i g h as those r e p o r t e d by M o f f a t t and Khorana(46). T h e i r r e p o r t e d o v e r a l l y i e l d s were 78$ f o r CDP and 67$ f o r UDP, as compared to 22$ and 24$, r e s p e c t i v e l y , obtained here. There are two p r i n c i p a l reasons f o r the lower y i e l d s obtained here: 1. The course of the n u c l e o s i d e - 5 1 phosphoromorpholidate s y n t h e s i s was f o l l o w e d by paper e l e c t r o p h o r e s i s i n the o r i g i n a l paper. T h i s was not done here; consequently the r e a c t i o n was not always complete a t the time of workup, although r e f l u x times comparable to those i n the o r i g i n a l paper were allowed. 2. The p y r i d i n e used as s o l v e n t during the r e a c t i o n between the n u c l e o s i d e - 5 ' phosphoromorpholidate and t r i - n - b u t y l -ammonium phosphate must be as dry as p o s s i b l e i f the optimum y i e l d i s to be obtained. The p y r i d i n e used was only p a r t i a l l y d r i e d with anhydrous sodium s u l f a t e . In order to be d r i e d thoroughly, the p y r i d i n e should have been p l a c e d over calcium h y d r i d e . The c y t i d i n e diphosphate was found to be u n s t a b l e , although kept i n a dry atmosphere at 4°. A f t e r twelve months, about 20$ of the CDP had decomposed to CMP and i n o r g a n i c phosphate. The other n u c l e o t i d e s were s t a b l e . The method used i s probably the best one c u r r e n t l y a v a i l a b l e . Although the y i e l d s are reasonably good, the procedure i s 52 q u i t e lengthy. I I . Experiments w i t h P o l y n u c l e o t i d e Phosphorylase from  Dihydrostreptomycin S e n s i t i v e E s c h e r i c h i a c o l i ( E . c o l i SA) The r e p o r t e d i n h i b i t i o n , by streptomycin, of the p o l y -n u c l e o t i d e phosphorylase-catalyzed exchange r e a c t i o n ( 4 3 ) c o u l d not be d u p l i c a t e d here. None of the enzyme f r a c t i o n s showed any i n h i b i t i o n ( t a b l e IV"). Therefore, an i n h i b i t a b l e a c t i v i t y was not removed i n the course of p u r i f i c a t i o n . I f anything, DHSM appeared to sti m u l a t e the r e a c t i o n s l i g h t l y . However, s t i m u l a t i o n appeared to occur when DHSM was added a f t e r i n c u b a t i o n ( t a b l e V ) , thus t h i s s t i m u l a t i o n may have been an a r t i f a c t due to the ad s o r p t i o n of some DHSM, complexed w i t h i n o r g a n i c phosphate, onto the N o r i t . T h i s would a l s o agree w i t h the apparent i n h i b i t i o n observed when the elua n t was measured. The time course of the r e a c t i o n was examined i n order to determine i f the r e p o r t e d i n h i b i t i o n had perhaps been due to the lengthening of a l a g p e r i o d ( P i g . 2 ) . Although the r e a c t i o n had a l a g of about 30 min., t h i s was not lengthened by 200 fig./ml. of DHSM. No i n h i b i t i o n c o u l d be demonstrated of the p o l y n u c l e o t i d e phosphorylase-catalyzed p o l y m e r i z a t i o n r e a c t i o n . The r e a c t i o n has been shown to r e q u i r e an o l i g o n u c l e o t i d e primer i f a h i g h l y p u r i f i e d enzyme p r e p a r a t i o n i s u s e d ( 4 l ) . Thus, the enzyme was examined to see i f a requirement f o r a primer c o u l d be demonstrated(Fig. 3 ) . No primer requirement was found, thus the p o s s i b l e a c t i o n of DHSM on primer r e q u i r i n g enzyme c o u l d 53 not be s t u d i e d . DHSM, i n a c o n c e n t r a t i o n of 500 ug./ml., was found not to i n h i b i t the r e a c t i o n ( P i g . 4 ) . The diamines p u t r e s c i n e , cadaverine, spermine, and spermidine a l s o had no e f f e c t on the course of the r e a c t i o n . The polymer formed i n the presence of DHSM(1000 ug./ml.) and i n i t s absence had an i d e n t i c a l sedimentation c o e f f i c i e n t ( 9 S ) . That formed i n the presence of s p e r m i d i n e ( l . 6 mM) was s l i g h t l y lower(8 S ) . However, the polymer formed i n the presence of a l l of the di a m i n e s ( p u t r e s c i n e , cadaverine, spermine, and spermidine) appeared to be small e r and much more heterogeneous(Fig. 6E). T h i s was s u r p r i s i n g i n view of the re p o r t e d s t a b i l i z a t i o n of n u c l e i c a c i d s by the diamine spermine(66). Since the present work was completed, another r e p o r t of streptomycin i n h i b i t i o n of p o l y n u c l e o t i d e phosphorylase has appeared, i n which the enzyme was e x t r a c t e d from C l o s t r i d i u m p e r f i n g e n s ( 6 0 ) . In t h i s paper(60), the p o l y m e r i z a t i o n r e a c t i o n , 14 as measured by the i n c o r p o r a t i o n of 8-C -ADP i n t o p o l y a d e n y l i c a c i d , was s a i d to be i n h i b i t e d 50$ by 240 |ig./ml. of DHSM. In the course of the work repo r t e d i n t h i s t h e s i s , p r e l i m i n a r y s t u d i e s on an enzyme p r e p a r a t i o n which had been s t o r e d f o r three months i n d i c a t e d some i n h i b i t i o n of the exchange r e a c t i o n , although not as extensive as th a t r e p o r t e d by Kornberg. Since l a t e r work, where no i n h i b i t i o n c ould be shown, u t i l i z e d f r e s h l y prepared enzyme from newly harvested c e l l s , the p o s s i b i l i t y remains t h a t , w i t h ageing of e i t h e r c e l l s or e x t r a c t s , an i n h i b i t a b l e r e a c t i o n becomes apparent. Support f o r t h i s p o s s i b i l i t y e x i s t s . D o l i n (60) r e p o r t s h i s f r e s h l y harvested c e l l s were s t o r e d i n the deep f r e e z e u n t i l used. Kornberg(43) r e p o r t e d 54 t h a t the te n minute s o n i c a t e was s t o r e d up to two months a t -15° without l o s s of a c t i v i t y . I t i s not c l e a r from e i t h e r paper e x a c t l y how long e i t h e r c e l l s or e x t r a c t s were s t o r e d i n the f r e e z e r before use i n the experiments with DHSM. Since Z e b o v i t z and Moulder(13) have alr e a d y r e p o r t e d a DHSM i n h i b i t i o n of pyruvate metabolism appearing on ageing i n the c o l d , such a p o s s i b i l i t y must be taken i n t o c o n s i d e r a t i o n . I l l . S t u d i e s on Ribosomes from E s c h e r i c h i a c o l i I t i s w e l l known t h a t streptomycin w i l l p r e c i p i t a t e n u c l e i c a c i d . However, s i n c e u n p h y s i o l o g i c a l c o n c e n t r a t i o n s are r e q u i r e d , and s i n c e the n u c l e i c a c i d s of both r e s i s t a n t and dependent organisms are a l s o p r e c i p i t a b l e , such a phenomenon has been dis m i s s e d w i t h regard to the mechanism of a c t i o n of streptomycin. Nonetheless, the f a c t t h a t streptomycin can p r e c i p i t a t e n u c l e i c a c i d s a t r e l a t i v e l y low molar c o n c e n t r a t i o n s ( 3 x 10 M) i n d i c a t e s t h a t there i s a very strong i n t e r a c t i o n between streptomycin and n u c l e i c a c i d s . Using a water e x t r a c t of E. c o l i , Cohen and L i c h t e n s t e i n ( 3 4 ) demonstrated t h a t the ribosomes co u l d be p r e c i p i t a t e d by about 3800 ug./ml. of streptomycin i n the c o l d . In t h i s work, however, i t was found t h a t ribosomal RNA c o u l d be p r e c i p i t a t e d by about 500 ug./ml. of DHSM, when d i a l y z e d overnight a g a i n s t t h i s concentrationocff DHSM i n 0.01 M t r i s b u f f e r , pH 7.4(Fig. 8)). I t was found t h a t Mg*4" antagonized t h i s e f f e c t , as would be expected i n view of the known requirement of Mg f o r ribosome s t a b i l i t y . In the presence of 0.01 M MgCl_ the amount of DHSM r e q u i r e d f o r p r e c i p i t a t i o n 55 i n c r e a s e d to about 1600 ug./ml. A n a l y s i s f o r RNA and p r o t e i n i n the 6,000 g supernatant of the d i a l y s a t e s showed t h a t about 60$ of the p r o t e i n was p r e c i p i t a t e d and about 80$ of the RNA. The composition of the r e s u l t i n g p r e c i p i t a t e was then 40$ p r o t e i n , and 60$ RNA, as opposed to about 45$ p r o t e i n and 55$ RNA i n the o r i g i n a l ribosomes. A study of u l t r a c e h t r i f u g e p a t t e r n s of d i a l y s a t e s a g a i n s t i n c r e a s i n g amounts of DHSM i n d i c a t e d a ribosome breakdown at about 350 to 400 ug./ml. of DHSM, although no p r e c i p i t a t i o n o c c u r r e d ( P i g . 9, 10). When the d i a l y s a t e s of ribosomes from E. c o l i SB were chromatographed on D E A E - c e l l u l o s e ( F i g . 11) d i f f e r e n t p a t t e r n s occurred before and a f t e r breakdown. When e l u t e d with a l i n e a r g r a d i e n t of N a C l ; i n t r i s b u f f e r , pH 7.4, most of the m a t e r i a l e l u t e d a t about 1.0 M NaCl when d i a l y z e d a g a i n s t b u f f e r p l u s 400 ug./ml. of DHSM, whereas there was a major peak at 0.07 M NaCl, w i t h s m a l l e r amounts of m a t e r i a l e l u t e d a t higher s a l t c o n c e n t r a t i o n s , when d i a l y z e d a g a i n s t b u f f e r o n l y ( P i g . l l ) . I t has been shown t h a t the use of a higher Mg c o n c e n t r a t i o n prevents much of the breakdown t h a t occurs when ribosomes are chromatographed on D E A E - c e l l u l o s e ( 6 l ) . Ribosomes from E. c o l i SB d i a l y z e d a g a i n s t 0.01 M t r i s b u f f e r , pH 7.4, gave peaks at 0.07, 0.35, 0.40, 0.47, and 0.51 MqNaGl. Whwn DHSM(400 ug./ml.) was present there were only three major peaks, a t 0.07, 0.52, and 0.57 M NaCl. In a d d i t i o n there was an i n c r e a s e i n the amount of m a t e r i a l e l u t i n g at high e r s a l t c o n c e n t r a t i o n s . Ribosomes from E. c o l i RB grown i n the presence of 100 ug./ml. 56 of DHSM, a f t e r d i a l y s i s a g a i n s t t r i s b u f f e r , gave peaks at 0.07, 0.40, 0.50, and 0.52 M NaCl. When d i a l y z e d i n the presence of 400 jig./ml. of DHSM, there were peaks at 0.07, 0.56, and 0.62 M NaCl, and a great i n c r e a s e i n m a t e r i a l e l u t i n g at a higher s a l t c o n c e n t r a t i o n . S e v e r a l p o i n t s concerning t h i s chromatography i n the presence of Mg can be made. F i r s t l y , the presence of Mg (0.01 M) i n the e l u t i n g f l u i d r e s u l t s i n l e s s breakdown of r i b o n u c l e o p r o t e i n on the column. Secondly, the peak at 0.07 M NaCl, common to a l l chromatograms, r e s u l t s from ribosomal breakdown on the DEAE- c e l l u l o s e , and c o n s i s t s of p r o t e i n ( 4 5 ) . T h i r d l y , although d i a l y s i s of ribosomes a g a i n s t 400 u-g./ml. of DHSM r e s u l t s i n the breakdown of ribosomes as r e v e a l e d i n the u l t r a c e n t r i f u g e , i t does not seem to r e s u l t i n complete breakdown as measured by DEAE-cellulose chromatography. F i n a l l y , there appears to be a d i f f e r e n c e i n the ribosomes from E. c o l i SB and from E. c o l i RB(grown i n the presence of 100 (xg./ml. of DHSM) i n t h a t there i s no peak at 0.35 M NaCl i n the chromatogram from E. c o l i RB. With regard to the t h i r d p o i n t , o n l y p a r t o f the ribosomal RNA, as r e v e a l e d by DEAE-cellulose column chromatography i n the presence of Mg , appears to be l a b i l e to DHSM. A l a t e n t ribonuclease(RNase) has been r e p o r t e d to be present i n E. c o l i ribosomes(62). A recent r e p o r t has appeared i n which the s o l u b i l i z a t i o n of ribosomal p r o t e i n was e f f e c t e d by d i a l y s i s a g a i n s t 1 M t r i s b u f f e r , pH 7.4, at 24°(63). T h i s r e s u l t e d 57 from the activation of ribosomal RNase. RNase would be expected to be active when the ribosomes were broken down, but might not cause much breakdown i n 18 hr. at 4°. Another possible explanation i s that DHSM caused the uncoiling of an RNA aggregate. This would explain the increase i n material absorbing at 260 mu. being eluted at higher s a l t concentrations, since the res u l t i n g subunits would be expected to have more available charge to the column. Further work i s therefore necessary i n order to establish the nature of the compounds included i n the various peaks. The results of the autodegradation experiments tend to support the hypothesis that DHSM interacts strongly with the ribosomes to effect some change i n structure, the nature of which i s unknown. At high enough concentrations of DHSM the result i s a total.breakdown of structure. However, even at low concentrations of DHSM, changes i n the autodegradation curves indicate an effect on ribosome structure. The E. c o l i SA has been found to be considerably less sensitive than E. c o l i SB. E. c o l i SA i s sensitive to about 1000 |xg./ml., whereas E.. c o l i SB i s sensitive to about 30 ^ g./ml. This i s based on.the concentration of DHSM required to prevent overnight growth of an inoculum i n 5 ml. of growth medium. The pr i n c i p l e difference i n the autodegradation rates °f M' c o l i SA, SB, and RB.is i n the divergence of rate when i n the presence of DHSM(l75 p,g./ml. and 350 (ig./ml.). This divergence occurs with E. c o l i SB after about 15 min., whereas i t occurs with E. c o l i SA after about 80 min. (Fig. 14). 58 With E. c o l i EB there i s no a c t u a l p o i n t at which the degradation r a t e s d i v e r g e , but the r a t e s do d i f f e r s l i g h t l y . Another d i f f e r e n c e i s t h a t the a c t i v i t y of p o l y n u c l e o t i d e phosphorylase i s much gr e a t e r i n the h i g h l y s e n s i t i v e E. c o l i SB then i n e i t h e r E. c o l i SA or RB. Davis has r e c e n t l y r e i n v e s t i g a t e d the e x t r a c e l l u l a r p r o d u c t i o n of n u c l e o t i d e s by E. c o l i when i n the presence of DHSM, u s i n g c e l l s l a b e l l e d w i t h P 3 2 and S 3 5 ( 6 4 ) . As a r e s u l t of these experiments, he was l e d to hypothesize t h a t DHSM t r i g g e r e d a dep o l y m e r i z a t i o n of E. c o l i ribosomal RNA, r e s u l t i n g i n 5 ' - n u c l e o t i d e s . The r e s u l t s here with E. c o l i ribosomes do not support such an hyp o t h e s i s . The experiments w i t h ribosomes from 6-D-galactosidase induced E. c o l i SA i n d i c a t e t h a t the 40$ of the ribosomal p r o t e i n l e f t i n s o l u t i o n a f t e r p r e c i p i t a t i o n with DHSM(Pig. 8) contained e s s e n t i a l l y a l l of the B-D-galactosidase a c t i v i t y a s s o c i a t e d w i t h the ribosomes, s i n c e the s p e c i f i c a c t i v i t y of the 6,000 g supernatant doubled a f t e r p r e c i p i t a t i o n ( t a b l e IX and P i g . 15). Proct e r ( 6 5 ) has shown t h a t new ribosomes are sy n t h e s i z e d when o-nitr o p h e n y l - 8 - D - g a l a c t o s i d a s e i s induced i n E. c o l i . u s i n g m e l l i b i o s e as the induc e r . C e l l s were pre-incubated 14 14 14 with C -guanine, C - u r a c i l , or C -adenine. Only the ribosomes of c e l l s which were then induced contained any r a d i o a c t i v i t y . He also found t h a t i n c u b a t i o n of ribosomes w i t h protamine s u l f a t e or r i b o n u c l e a s e f o r 30 min. p r i o r to enzyme assay i n c r e a s e d the a c t i v i t y s i x - f o l d , thus i n d i c a t i n g the presence 59 of 'nascent' enzyme on the ribosomes. No nascent enzyme was observed i n these experiments. If such nascent enzyme was present then i t must have been part of the nucleoprotein precipitated by the DHSM, since only existing a c t i v i t y was apparently eliminated from the ribosomes. 60 SUMMARY -1. Nucleoside diphosphates(adenosine diphosphate, guanosine diphosphate, cytidine diphosphate, and uridine diphosphate) of s atisfactory purity were synthesized from the corresponding monophosphates by the method of Khorana and Moffatt. 2. The formation of polynucleotides from adenosine diphosphate by p u r i f i e d polynucleotide phosphorylase from a dihydrostreptomycin (DHSM) sensitive s t r a i n of Escherichia c o l i was studied. 3. A reported i n h i b i t i o n by streptomycin of the polynucleotide phosphorylase catalyzed "exchange reaction" could not be confirmed. 4. DHSM had no effect on the time course of the polymerization reaction, nor on the sedimentation properties of the polymer formed(polyadenylic acid). 5. The diamines putrescine, cadaverine, spermine, and spermidine were found to have no effect on the course of the polymerization reaction, but did affect the sedimentation properties of the polymer formed. 6. 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