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Purine synthesis in suspensions of mucosa from the small intestine of the rat Paterson, Alan Robb Phillips 1956

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PURINE SYNTHESIS IN SUSPENSIONS OP MUCOSA PROM THE SMALL INTESTINE OF THE RAT by A l a n Robb P h i l l i p s Paterson B.A., U n i v e r s i t y of B r i t i s h Columbia, 19^0 M.A., U n i v e r s i t y of B r i t i s h Columbia, 1952 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OP DOCTOR OF PHILOSOPHY i n the Department of B i o c h e m i s t r y We accept t h i s t h e s i s as conforming t o the standard r e q u i r e d from candidates f o r the degree of DOCTOR OP PHILOSOPHY. Members of the Department of B i o c h e m i s t r y . The U n i v e r s i t y of B r i t i s h Columbia October, 19£6 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 a t the U n i v e r s i t y of B r i t i s h Columbia, I agree that 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 qr by h i s r e p r e s e n t a t i v e . I t i s under-stood that 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 The U n i v e r s i t y of B r i t i s h Columbia, Vancouver 8, Canada. Date Wc\z pmiiersti]} of ^rtttsij (EolumHa Faculty of Graduate Studies P R O G R A M M E O F T H E F I N A L O R A L E X A M I N A T I O N 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 */ ALAN ROBB PHILLIPS PATERSON B.A. (British Columbia) M.A. (British Columbia) W E D N E S D A Y , OCTOBER 10, 195<5, at 3:00 p.m. IN R O O M 300, PHYSICS B U I L D I N G C O M M I T T E E IN C H A R G E D E A N G. M . S H R U M , Chairman M . D A R R A C H D. H . C O P P W . J. POLGLASE I. McTAGGART COWAN S. H . Z B A R S K Y J. J. R. C A M P B E L L R. B. K E R R L. D. H A Y W A R D External Examiner: G . C. B U T L E R University of Toronto PURINE SYNTHESIS IN SUSPENSIONS OF MUCOSA FROM THE SMALL INTESTINE OF THE RAT ABSTRACT Purine biosynthesis has been studied in vitro using surviving, whole cell suspensions of the mucosa from the small intestine of the rat. The de novo synthesis of purines by this tissue system was demonstrated by measuring the incorporation of C14-labelled purine precursors into adenine and guanine of the acid-soluble (AS) nucleotides and the nucleic acids. Essential parts of this demonstration consisted of establishing the radiochemical purity of the isolated purines, and of showing that the isotope incorporation was not attributable to bacteria present in the tissue suspension. Upon incubation of mucosal suspen-sions with formate-C14, adenine and guanine of the AS nucleotides displayed a rapid renewal, being approximately 40 and 14 times as radioactive, respectively, as the adenine and guanine of the mixed nucleic acids. Incorporation of formate-C14 by exchange reactions (as opposed to de novo synthetics) appeared to be excluded as a major process by the demonstra-tion that two other purine precursors, glycine-1-C14 and bicarbonate-C4, also became incorporated into the purines. The theoretical incorporation of 2 mole-cules of formate for each glycine molecule used in the process of de novo purine synthesis was approached in these experiments by the observed incorporation of 2- 4 molecules of formate per molecule of glycine. This is sufficiently close to the theoretical value to exclude a major incorporation of formate-C14 by known exchange reactions. Time studies indicated that the renewal of AS adenine and guanine pro-ceeded at an approximately constant rate with a decline in rate beginning at 3- 4 hours of incubation. In several experiments an initial lag in the rate of synthesis of nucleic acid purines was apparent. The rate of purine synthesis as measured by the incorporation of formate-C 1 4 was not significantly affected lay the addition of non-isotopic glycine or glutamine to the incubation medium. Similarly, glycinamide and 4-amino-5-imidazolecarboxamidine, both of which resemble known intermediates in purine biosynthesis, did not alter the incorporation of formate-C14 into the purines. Renewal of either AS or nucleic acid purines from formate-C14 did not take place in homogenates of intestinal mucosa. The incorporation of formate-C14 by the purines of the intestinal mucosa of the intact rat was measured 24 hours after administration of the isotope. In contrast to the in vitro experiments, the large differences between the specific activities of the AS and nucleic acid purines were absent in the intact animal. A comparison of purine synthesis in the intact animal with that of the in vitro system suggested that guanine synthesis was suppressed in the latter. PUBLICATIONS "The Preparation of 2-C"-Adenine." A. R. P. Paterson and S. H . Zbarsky. Journal of the American Chemical Society, 75, 5753 (1953). "In vitro Synthesis of Purines by Rat Intestinal Mucosa." A. R. P. Paterson and S. H . Zbarsky. Biochimica et Biophysica Acta, 18, 441 (1955). "Ribonucleic Acid Synthesis in Tumor Humogenates." A. R. P. Paterson and G. A. LePage. Federation Proceedings, 15, 324 (1956). "The Metabolism of 2-C"-Adenine in the Rat." S. H . Zbarsky and A. R. P. Pat-erson. Accepted for publication in the Canadian Journal of Biochemistry and Physiology. GRADUATE STUDIES Field of Study: Biochemistry Intermediary Metabolism S. H. Zbarsky Enzymology W. J. Polglase Biochemistry of Antibiotics W. J. Polglase Biochemistry of Cancer M. Darrach Biochemistry of Steroids M. Darrach Seminar in Biochemistry Other Studies: Bacteriology L. E. Ranta Carbohydrates E. V. White Organic Analysis A. E. Werner Physical Inorganic Chemistry C. Reid Stereochemistry L. D. Hayward Biometry .V. C. Brink Human Physiology {^ E. C Black Bacterial Metabolism J. j . R. Campbell ACKNOWLEDGEMENTS In the course of t h i s work, the author was i n r e c e i p t of U n i v e r s i t y of B r i t i s h Columbia Teaching F e l l o w -ships d u r i n g the 1952-53 and 1953~51i s e s s i o n s . A N a t i o n a l Research C o u n c i l of Canada F e l l o w s h i p was h e l d i n the 55 s e s s i o n . T h i s support i s acknowledged w i t h g r a t i t u d e . The author wishes' a l s o t o thank Dr. S. H. Zbarsky f o r h i s ad v i c e and many h e l p f u l d i s c u s s i o n s i n t h i s r e s e a r c h p r o j e c t . ABSTRACT Purine b i o s y n t h e s i s has been s t u d i e d i n v i t r o u s i n g s u r v i v i n g , whole c e l l suspensions of the mucosa from the s m a l l i n t e s t i n e of the r a t . The p r e p a r a t i o n and use of such suspen-s i o n s have been d e s c r i b e d as have been c e r t a i n f e a t u r e s of t h e i r m e t a b o l i c behaviour. The techniques used t o i s o l a t e and measure the r a d i o a c t i v i t y of the s e v e r a l p u r i n e f r a c t i o n s of mucosal suspensions c o n t a i n i n g 1^0-200 mg. of f r e s h t i s s u e have been o u t l i n e d . The de novo s y n t h e s i s of purines by t h i s t i s s u e system was demonstrated by measuring the i n c o r p o r a t i o n of s e v e r a l C 1 ^ - l a b e l l e d p u r i n e p r e c u r s o r s i n t o adenine and guanine of the a c i d - s o l u b l e (AS) n u c l e o t i d e s and the n u c l e i c a c i d s . E s s e n t i a l p a r t s of t h i s demonstration c o n s i s t e d of e s t a b l i s h -i n g the r a d i o c h e m i c a l p u r i t y of the i s o l a t e d , p u r i n e s , and of showing t h a t the i s o t o p e i n c o r p o r a t i o n was not a t t r i b u t a b l e to b a c t e r i a present i n the t i s s u e suspension. Upon i n c u b a t i o n of mucosal suspensions w i t h f o r m a t e - C ^ , adenine and guanine of the AS n u c l e o t i d e s d i s p l a y e d a r a p i d renewal, b e i n g approx-im a t e l y liO and llj. times as r a d i o a c t i v e , r e s p e c t i v e l y , as the adenine and guanine of the mixed n u c l e i c a c i d s . The s p e c i f i c a c t i v i t y of AS adenine was approximately 6 times g r e a t e r than t h a t of AS guanine, and n u c l e i c a c i d (NA) adenine was 2 - 3 times as a c t i v e as NA guanine. F o l l o w i n g i n c u b a t i o n of the mucosal p r e p a r a t i o n , the suspending medium contained adenine, guanine and u r i c a c i d , but s i n c e these purines were only s l i g h t l y r a d i o a c t i v e they were regarded as breakdown p r o d u c t s . I n c o r p o r a t i o n of format e - C ^ by exchange r e a c t i o n s (as opposed t o de novo s y n t h e s i s ) appeared to be excluded as a major process by t h e demonstration t h a t two other p u r i n e p r e c u r s o r s , • g l y c i n e - l - C ^ and b i carbonate-C"^, a l s o became i n c o r p o r a t e d i n t o the p u r i n e s . The t h e o r e t i c a l i n c o r p o r a t i o n of 2 molecules of formate f o r each g l y c i n e molecule used i n the process of de novo puri n e s y n t h e s i s was approached i n these experiments by the observed i n c o r p o r a t i o n o f 2 - I4. mole-cules of formate per molecule of g l y c i n e . T h i s i s s u f f i c i e n t l y c l o s e to the t h e o r e t i c a l v a l u e to exclude a major i n c o r p o r a t i o n by known exchange r e a c t i o n s . Time s t u d i e s i n d i c a t e d t h a t the renewal of AS adenine and guanine proceeded at an approximately constant r a t e w i t h a d e c l i n e i n r a t e s t a r t i n g a t 3 - I4. hours, of i n c u b a t i o n . I n s e v e r a l experiments an i n i t i a l l a g i n the r a t e of s y n t h e s i s of NA purines was apparent. The r a t e of p u r i n e s y n t h e s i s as measured by the i n c o r p o r a t i o n of f o r m a t e - C 1 ^ was not s i g n i f i c a n t l y a f f e c t e d by the a d d i t i o n of n o n - i s o t o p i c g l y c i n e or glutamine t o the i n c u b a t i o n medium. S i m i l a r l y , glycinamide and [|.-amino-5-imidazolecarboxamidine, b o t h of which resemble known i n t e r -mediates i n p u r i n e b i o s y n t h e s i s , d i d not a l t e r t h e i n c o r p o r -a t i o n of formate-C i n t o the p u r i n e s . Renewal of e i t h e r AS or NA purines from formate-d i d not t ake p l a c e i n homogenates of i n t e s t i n a l mucosa. i i i The i n c o r p o r a t i o n of f o r m a t e - C 1 ^ by the purines of the i n t e s t i n a l mucosa of the i n t a c t r a t was measured 2I4. hours a f t e r a d m i n i s t r a t i o n of the i s o t o p e . I n c o n t r a s t t o the i n  v i t r o experiments, the l a r g e d i f f e r e n c e s between the s p e c i f i c a c t i v i t i e s of the a c i d - s o l u b l e and NA p u r i n e s were absent i n the i n t a c t animal, the l e v e l l i n g e f f e c t very probably b e i n g due t o the 2[L hour i n t e r v a l . A comparison of p u r i n e s y n t h e s i s i n the i n t a c t animal w i t h t h a t of the i n v i t r o system suggested t h a t guanine s y n t h e s i s was suppressed i n the l a t t e r . The use of i n t e s t i n a l mucosa f o r i n v i t r o s t u d i e s of p u r i n e metabolism has not h i t h e r t o been r e p o r t e d . I n view of the^demonstrated r a p i d s y n t h e s i s of s o l u b l e p u r i n e n u c l e o t i d e s i n t h i s system, p a r t i c u l a r l y of adenine n u c l e o t i d e s , i t i s f e l t t h a t suspensions of mucosa may have u s e f u l a p p l i c a t i o n s i n i n v i t r o s t u d i e s of p u r i n e b i o s y n t h e s i s . I i v TABLE OF CONTENTS Page INTRODUCTION 1 1. PURINE NUCLEOTIDE SYNTHESIS 2 A. E a r l y p r e c u r s o r s of the pu r i n e carbon atoms 3 B. E a r l y p r e c u r s o r s of the purin e n i t r o g e n atoms 5 C. Intermediary compounds 7 2. POLYNUCLEOTIDE SYNTHESIS 17 A. D e s o x y r i b o n u c l e i c a c i d s y n t h e s i s . . . . . . . 17 B. R i b o n u c l e i c a c i d s y n t h e s i s 19 3. PURINE SYNTHESIS IN INTESTINAL MUCOSA 22 EXPERIMENTAL 280 1. ANALYTICAL METHODS 28 A. Ion exchange chromatography 28 ( i ) The s e p a r a t i o n of adenine, guanine, hypoxanthine, xanthine and u r i c a c i d . 28 ( i i ) I s o l a t i o n o f adenine and guanine from i n t e s t i n a l mucosa .30 B. Paper chromatography 31 ( i ) S olvent systems 31 ( i i ) V i s u a l i z i n g p u r i n e areas 32 ( i i i ) C a p i l l a r y s i p h o n a p p l i c a t o r 3I4-C. Spectrophotometry 36 ( i ) U l t r a v i o l e t a b s o r p t i o n s p e c t r a . . . . 36 ( i i ) D e t e r m i n a t i o n of purines i l l ( i i i ) Accuracy of pu r i n e a n a l y s i s lj.2 D. R a d i o a c t i v i t y measurements I4.3 ( i ) I n s t r u m e n t a t i o n ij.3 ( i i ) Methods of mounting samples Ijlj. (a) " I n f i n i t e l y - t h i n " samples . . . . I4.I4. (b) Samples mounted on f i l t e r paper d i s c s 45 (c) " I n f i n i t e l y f ; t h i c k " barium carbon-ate samples I4.7 ( i v ) E s t i m a t i o n of e r r o r s E. Respirometry  2. THE PREPARATION OF SUSPENSIONS OF INTESTINAL MUCOSAI 3. THE PURINES OF THE INTESTINAL MUCOSA SUSPENSION. ]\.m USE OF THE MUCOSA SUSPENSION A. Media  B. Procedure f o r a t y p i c a l experiment . . . . ( i ) I n c u b a t i o n . ' ( i i ) I s o l a t i o n and a n a l y s i s of pu r i n e s . . ( i i i ) A n a l y s i s of centre w e l l c o n t e n t s . . . . 5 . ISOTOPICALLY LABELLED SUBSTRATED A. Sodium f o r m a t e - C 1 ^  B. G l y c i n e - l - C 1 ^  C. Sodium b i c a r b o n a t e . . 6. NON-ISOTOPIC SUBSTRATE A. Glycinamide h y d r o c h l o r i d e . _,. B. lj.-Amino -5-imidazolecarboxamidine d i h y d r o - c h l o r i d e  RESULTS  1. CHARACTERISTICS OF THE INTESTINAL MUCOSA SUSPENSION A. P h y s i c a l c h a r a c t e r i s t i c s ; . . B. R e s p i r a t o r y a c t i v i t y  C. B a c t e r i a l contamination  D. The purines of the mucosa suspension . . . ( i ) Whole suspension ( i i ) Medium ( i i i ) A c i d - s o l u b l e f r a c t i o n ( i v ) P o l y n u c l e o t i d e f r a c t i o n Page E. The o x i d a t i o n of g l y c i n e - l - C 1 ^ and f o r m a t e - C 1 ^ 62 2 . PURINE SYNTHESIS IN MUCOSA SUSPENSIONS 61i A. Radiochemical p u r i t y 65 B. I n c o r p o r a t i o n of formate, g l y c i n e , and  carbonate 68 3 . SOME CHARACTERISTICS OP PURINE SYNTHESIS IN INTESTINAL MUCOSA SUSPENSIONS. . 72 A. U n i f o r m i t y o f susp e n s i o n 72 B. A comparison of the s p e c i f i c a c t i v i t i e s of the s e v e r a l p u r i n e f r a c t i o n s . 7^ ( i ) A c i d - s o l u b l e and n u c l e i c a c i d p u r i n e s . . *7LL. ( i i ) Purines i n the medium 7 l i C. Formate i n c o r p o r a t i o n w i t h r e s p e c t t o time. . . 76 1L. THE EFFECT OP ADDITIVES ON PURINES SYNTHESIS . . . . 83 5 . PURINE SYNTHESIS IN HOMOGENATES 88 6 . INCORPORATION OF FORMATE-C 1^ BY THE PURINES OF INTESTINAL MUCOSA IN THE INTACT RAT 89 DISCUSSION 92 SUMMARY 101 BIBLIOGRAPHY 105 v i i TABLES Page '.. I. Rate of new c e l l formation i n rat tissue . . . 23 I I . U l t r a v i o l e t absorption data used i n the determination of purines lj.2 I I I . The oxidation of formate-C 1^ to carbon dioxide-C 1^ 63 IV. Radiochemical purity of.AS and NA purines derived from formate-C 1^. A comparison of s p e c i f i c a c t i v i t i e s before and a f t e r re chromatography 67 V. A comparison of formate-C^ and g l y c i n e - l - C " ^ incorporation by the AS purines 69 VI. A comparison of formate- glycine-C and bicarbonate-CI 1^ incorporation by adenine . . . 70 VII. A comparison of r e p l i c a t e samples 73 VIII. S p e c i f i c a c t i v i t y of u r i c acid of the medium r e l a t i v e to AS and NA purines. . . 76 IX. A comparison of the s p e c i f i c a c t i v i t i e s of acid-soluble and nucleic acid purines and the purines of the medium 77 X. The effects of glycine, glutamine and glycina:-.' mide on purine synthesis 85 XI. The effect of glycinamide on purine synthesis. 86 XII. The effect of I i-amino-^-imidazolecarboxamidine on purine synthesis 87 XIII. Incorporation of formate-C 1^ by the purines of i n t e s t i n a l mucosa i n the intact r a t . . . . . . 91 v i i i FIGURES Page 1. The p r i n c i p a l precursors of the carbon atoms of u r i c acid ii 2 . The p r i n c i p a l precursors of the nitrogen atoms of u r i c acid 7 3. Intermediates i n u r i c acid biosynthesis 9 i i . Scheme for purine biosynthesis l l i 5. Ion exchange column with s e l f - f i l l i n g , pressur-i z i n g reservoir 30 6. C a p i l l a r y siphon applicator f o r preparing chroma to grams 35 7. Absorption spectrum of adenine i n 0.1N HC1 . . . . 38 8. Absorption spectrum of guanine i n 0.1N HC1 . . . . 39 9. Absorption spectrum of u r i c acid i n 0.1N HC1 . . . lj.0 10. A comparison between " i n f i n i t e l y - t h i n " and f i l t e r paper-mounted samples.of adenine-C 1^ I4.6 11. Intestine s p l i t t e r 50 12. Apparatus f o r the preparation of sodium bicarbon-ate-C 1^ . 57 13. Photomicrographs of a t y p i c a l preparation of rat i n t e s t i n a l mucosa. . . . 60 l l i . Photomicrographs of a t y p i c a l preparation of rat i n t e s t i n a l mucosa . 60 15. The oxidation of c ^ -formate to C^Oo by a suspension of i n t e s t i n a l mucosa 62 16. T y p i c a l s p e c i f i c a c t i v i t i e s of the purine fractions 75 17. A time study of the incorporation of formate-into adenine and guanine 78 i x 1 8 . A time study of the i n c o r p o r a t i o n of f o r m a t e - C ^ i n t o adenine and guanine 79 1 9 . A time study o f the i n c o r p o r a t i o n o f f o r m a t e - C 1 ^ . Into adenine and guanine 80 2 0 . A time study of the i n c o r p o r a t i o n o f f o r m a t e - C ^ i n t o adenine and guanine 8 l 2 1 . A time study o f the uptake of f o r m a t e - C 1 ^ i n t o adenine and guanine 82 X THE FOLLOWING LIST DEFINES THE ABBREVIATIONS USED IN THIS.THESIS R-NA r i b o n u c l e i c a c i d DNA d e s o x y r i b o n u c l e i c a c i d AS a c i d - s o l u b l e NA n u c l e i c a c i d AMP adenosine monophosphate ATP adenosine t r i p h o s p h a t e DPN di p h o s p h o p y r i d i n e n u c l e o t i d e TPN t r i p h o s p h o p y r i d i n e n u c l e o t i d e carboxamide J^-amino-^-imidazolecarbbxamide PRPP 5-phosphoribosy.l pyrophosphate GAR glycinamide r i b o t i d e FGAR oc N-f ormylglycinamidine r i b o t i d e FGAM oc N-f ormylglycinamidine r i b o t i d e AIR 5-a.minoimidazole r i b o t i d e AICAR 5-amino-l|.-imidazolecarboxamide r i b o t i d e ("carboxamide" r i b o t i d e ) INTRODUCTION I n e a r l y i n v e s t i g a t i o n s of the b i o s y n t h e s i s o f n u c l e i c a c i d s , c l a s s i c a l f e e d i n g techniques and n u t r i t i o n s t u d i e s were employed. These s t u d i e s p r o v i d e d c l e a r c u t evidence t h a t the h i g h e r forms of l i f e are a b l e t o s y n t h e s i z e n u c l e i c a c i d s from simple p r e c u r s o r s , but gave l i t t l e s p e c i f i c I n f o r m a t i o n about the i d e n t i t y o f the u l t i m a t e p r e c u r s o r s . W i t h i n the l a s t f i f t e e n y e a r s , the advent of the i s o t o p i c t r a c e r technique and the a v a i l a b i l i t y of i s o t o p e s i n q u a n t i t i e s l a r g e enough f o r chemical and b i o l o g i c a l e x p e r i m e n t a t i o n have p r o v i d e d the b a s i c methodology f o r what has proven to be a very f r u i t f u l a t t a c k on the problem of n u c l e i c a c i d b i o s y n t h e s i s . Phosphate and ammonium ions were the f i r s t primary substances shown by i s o t o p i c techniques t o be i n c o r p o r a t e d i n t o n u c l e i c a c i d s . Subsequently, more s p e c i f i c experiments, employ-i n g m e t a b o l i t e s l a b e l l e d w i t h i s o t o p i c carbon, r e v e a l e d the i d e n t i t y o f the compounds from which the carbon atoms o f the p u r i n e and p y r i m i d i n e bases o r i g i n a t e . There i s much to suggest t h a t the n u c l e i c a c i d s are not b u i l t up by the stepwise i n t e -g r a t i o n of these simple compounds, but r a t h e r t h a t they are polymers of more complex s u b - u n i t s , the p u r i n e and p y r i m i d i n e r i b o t i d e s . These sub-units are p r e s e n t l y c o n s i d e r e d t o be d e r i v e d from the f r e e r i b o t i d e s found i n the " a c i d - s o l u b l e f r a c t i o n " of t i s s u e s , which Is an e x t r a c t u s u a l l y made w i t h c o l d , d i l u t e p e r c h l o r i c or t r i c h l o r a c e t i c a c i d . The composi-t i o n of the a c i d - s o l u b l e f r a c t i o n , which i n c l u d e s a complex mixture of the p u r i n e and p y r i m i d i n e r iboside-^ 1-mono-, d i - , and t r i p h o s p h a t e s , has been s t u d i e d by H u r l b e r t et a l . ( 1,2). T h i s f r a c t i o n i s c u r r e n t l y r e c e i v i n g much a t t e n t i o n because of i t s key p o s i t i o n i n the b i o s y n t h e s i s of n u c l e i c a c i d s . I s o t o p i c p r e c u r s o r s o f the purines ( 3 - 6 ) and p y r i m i d i n e s (7,8) of the n u c l e i c a c i d s appear r a p i d l y i n t h i s f r a c t i o n which seems to be i n t e r m e d i a t e between the simple or e a r l y p r e c u r s o r s and the n u c l e i c a c i d s (9)» F o r t h i s d i s c u s s i o n , n u c l e i c a c i d b i o s y n t h e s i s i s considered under two p r i n c i p a l t o p i c s , n u c l e o -t i d e ( r i b o t i d e ) s y n t h e s i s and p o l y n u c l e o t i d e s y n t h e s i s . As t h i s i n v e s t i g a t i o n i s concerned p r i m a r i l y w i t h p u r i n e metabol-ism, the b i o s y n t h e s i s of the p y r i m i d i n e s w i l l not be d i s c u s s e d . The l a t t e r t o p i c has been reviewed r e c e n t l y by R e i c h a r d ( 1 0 ) . 1 . PURINE NUCLEOTIDE SYNTHESIS P r i o r t o 1 9 5 0 , i t was i m p l i c i t i n most d i s c u s s i o n s of n u c l e i c a c i d b i o s y n t h e s i s t h a t the bases were e l a b o r a t e d as separate e n t i t i e s , and experimental work was undertaken from t h i s p o i n t of view. However, i t has become apparent i n r e c e n t years that n e i t h e r the f r e e bases nor t h e i r r i b o s e d e r i v a t i v e s , the r i b o s i d e s , a r e i n t e r m e d i a t e s i n e i t h e r r i b o n u c l e i c a c i d or i n r i b o t i d e s y n t h e s i s . The p u r i n e r i n g i s assembled v i a r e a c t i o n sequences i n which the i n t e r m e d i a t e compounds a r e r i b o t i d e s , and the f i r s t complete pur i n e s t r u c t u r e on the s y n t h e t i c pathway i s a r i b o s i d e monophosphate. Thus, the process of p u r i n e b i o s y n t h e s i s i s a c t u a l l y one of p u r i n e n u c l e o t i d e s y n t h e s i s . The f o l l o w i n g scheme i l l u s t r a t e s , i n g e n e r a l terms, the r e l a t i o n s h i p s between the e a r l y p r e c u r s o r s , the n u c l e o -t i d e s and the p o l y n u c l e o t i d e s and demonstrates t h a t p u r i n e b i o s y n t h e s i s i s a c t u a l l y p u r i n e n u c l e o t i d e b i o s y n t h e s i s . • INTERMEDIATES SIMPLE PRECURSORS r ^ i n t e r - ^.purine r i b o t _ _ ^ . t i s s u e ^.POLYNUC mediate s i d e mono- n u c l e o t i d e r i b o t i d e s phosphates p o o l LEOTIDE PURINES c a t a b o l i s m n u c l e o s i d e s FREE PURINES ( e x c r e t o r y f u n c -t i o n i n b i r d s ) A. E a r l y p r e c u r s o r s of the p u r i n e carbon atoms. The b a s i c experiments on the p r e c u r s o r s o f u r i c a c i d carbon atoms have been adequately reviewed by s e v e r a l authors (10 - lij.) and a r e summarized i n F i g u r e 1, which shows the r e l a t i o n s h i p s between the p r i n c i p a l p r e c u r s o r s and their, u l t i m a t e l o c a t i o n s i n the u r i c a c i d molecule. The b a s i c experiments on t h i s t o p i c were performed by Buchanan and h i s c o l l e a g u e s who a d m i n i s t e r e d c a r b o n - l a b e l l e d compounds t o pigeons and determined the d i s t r i b u t i o n of the i s o t o p e among the carbon atoms of the e x c r e t e d u r i c a c i d . Inherent i n the a p p l i c a t i o n o f these s t u d i e s t o pu r i n e metabolism i n g e n e r a l , was the assumption t h a t u r i c a c i d , i n a d d i t i o n t o b e i n g the c h i e f e x c r e t o r y product of n i t r o g e n metabolism i n the b i r d , was a l s o the end product o f p u r i n e c a t a b o l i s m . The f u r t h e r assumption was made t h a t u r i c a c i d , s y n t h e s i z e d f o r t h e purpose o f n i t r o g e n e l i m i n a t i o n , was el a b o r a t e d i n the same manner as the p u r i n e s , adenine and guanine, employed i n f u n c t i o n a l r o l e s as coenzyme and n u c l e i c a c i d components. These a p p a r e n t l y reasonable assumptions have been s u b s t a n t i a t e d t o the extent t h a t i s o t o p i c p r e c u r s o r s were found t o l a b e l the p o l y n u c l e o t i d e p u r i n e s o f the r a t , the pigeon and y e a s t c e l l s i n the same p a t t e r n as found i n u r i c a c i d (10, l l i . ) . FIGURE 1. THE PRINCIPAL PRECURSORS OF THE CARBON ATOMS OF URIC ACID. f o r m i c a c i d u r i c a c i d The p u r i n e p r e c u r s o r s shown i n F i g u r e i a r e merely e a r l y p r e c u r s o r s and do not r e p r e s e n t the a c t u a l compounds which p a r t i c i p a t e I n puri n e r i n g s y n t h e s i s . I n t a c t animals were employed i n the b a s i c s t u d i e s , but more r e c e n t l y , i n v i t r o systems have been used t o great advantage, p a r t i c u l a r l y i n s t u d i e s o f the i n t e r m e d i a r y processes i n pu r i n e s y n t h e s i s . Pigeon l i v e r p r e p a r a t i o n s have been favoured because of the prominence of hypoxanthine s y n t h e s i s i n t h i s t i s s u e . I n the b i r d , the l i v e r i s the p r i n c i p a l s i t e of u r i c a c i d s y n t h e s i s . T h i s p r o c e s s , i n i t s f i n a l stages,, passes through hypoxanthine, xanthine and has as the f i n a l product, u r i c a c i d . As pi g e o n l i v e r l a c k s the enzyme xanthine oxidase, p u r i n e s y n t h e s i s stops at the hypoxanthine stage. Hypoxanthine s y n t h e s i s was shown to occur I n v i t r o I n s l i c e s and homogenates of pigeon l i v e r , but a most important s t e p i n the development of these s t u d i e s was the d i s c o v e r y t h a t the enzymes r e s p o n s i b l e f o r t h i s s y n t h e s i s are s o l u b l e ( l £ , l 6 ) . Schulman et a l . ( 1 7 ) showed th a t i n the s y n t h e s i s o f hypoxanthine by pigeon l i v e r e x t r a c t s , g l y c i n e , formate and carbon d i o x i d e are used i n the r a t i o 1 : 2 : 1 , p r o v i d i n g e x c e l l e n t c o n f i r m a t i o n o f the r e s u l t s of the i n v i v o experiments. B. E a r l y p r e c u r s o r s of the p u r i n e n i t r o g e n atoms. T h i s t o p i c has been reviewed by R e i c h a r d ( 1 0 ) . The e a r l y o b s e r v a t i o n s t h a t ammonium s a l t s c o n t r i b u t e d t o the p u r i n e n i t r o g e n s were made more meaningful by the r e c e n t experiments of L a g e r k v i s t (18) who degraded u r i c a c i d by a method which permit-ted the measurement o f the i s o t o p e content of each n i t r o g e n atom. L a g e r k v i s t showed t h a t i n pigeons, ammonium s a l t s c o n t r i b u t e d p r e f e r e n t i a l l y t o p o s i t i o n s 3 and 9 °f e x c r e t e d u r i c a c i d . T h i s d i s t r i b u t i o n was very l i k e l y e f f e c t e d by c o n v e r s i o n of the ammonia t o the amide n i t r o g e n of glutamine. Pigeon l i v e r e x t r a c t s were employed i n the important experiments of Sonne et a l . ( 1 9 , 2 0 ) who demonstrated t h a t i n the s y n t h e s i s of hypoxanthine, g l y c i n e and glutamic (or a s p a r t i c ) a c i d each c o n t r i b u t e d one n i t r o g e n atom per molecule of hypo-xanthine formed, w h i l e the amide group of glutamine c o n t r i b u t e d 2 molecules of n i t r o g e n . I n these experiments d e g r a d a t i o n methods f o r u r i c a c i d were not a v a i l a b l e by which n i t r o g e n atoms 1 and 3 c o u l d be separated f o r i s o t o p i c a n a l y s i s , however i t appeared t h a t the n i t r o g e n atoms at p o s i t i o n s 3 and 9 were d e r i v e d from t h e amide group of glutamine, while a s p a r t i c and glutamic amino n i t r o g e n s c o n t r i b u t e d t o p o s i t i o n 1 . The g l y c -in e amino group formed p u r i n e n i t r o g e n 7. ^lie c o n t r i b u t i o n of the glutamine amide group t o p o s i t i o n s 3 and 9 w a s f u r t h e r s u b s t a n t i a t e d by the r e p o r t t h a t glutamine i s r e q u i r e d f o r the s y n t h e s i s o f glycinamide r i b o t i d e i n p i g e o n l i v e r e x t r a c t s ( 2 1 ) . R e c e n t l y Leveriberg and h i s c o l l a b o r a t o r s have employed newly developed d e g r a d a t i o n techniques which p e r m i t t e d the separate i s o l a t i o n of p u r i n e n i t r o g e n s 3 and 9 I n experiments which d i r e c t l y confirmed the above i n f e r e n c e s r e g a r d i n g the m e t a b o l i c o r i g i n s of these n i t r o g e n s ( 2 2 ) . 1-his t o p i c i s summarized i n F i g u r e 2 . G l y c i n e and ammonium s a l t s appear t o be the only purine p r e c u r s o r s t h a t have been s t u d i e d as sources of t h e n i t r o g e n atoms i n p o l y n u c l e o t i d e p u r i n e s ( 1 0 ) . 7 FIGURE 2 . THE PRINCIPAL PRECURSORS OF THE NITROGEN ATOMS OF THE PURINE RING. ammonium s a l t s C. Intermediary compounds. The i s o l a t i o n and c h a r a c t e r i z a t i o n of the incompleted p u r i n e , l4.-amino-5-imidazolecarboxamide ( "carboxamide" ), from s u l f o n a m i d e - i n h i b i t e d c u l t u r e s of E. c o l i was an Important s t e p i n the i s o l a t i o n of in t e r m e d i a t e s i n puri n e n u c l e o t i d e s y n t h e s i s ( 2 3 ) . The s t r u c t u r e of carboxamide suggested a r o l e i n p u r i n e b i o s y n t h e s i s , as d i d the u t i l i z a t i o n o f t h i s compound i n the p l a c e o f purines by c e r t a i n p u r i n e - r e q u i r i n g b a c t e r i a (10, see r e f . 5 1 - 5 3 ) . L a b e l l e d carboxamide was i n c o r p o r a t e d i n t o n u c l e i c a c i d p u r i n e s o f the r a t (2li) and y e a s t ( 2 5 ) . Broken-c e l l p r e p a r a t i o n s o f pige o n l i v e r (26) and y e a s t (27) i n c o r p o r -ated -carboxamide i n t o hypoxanthine and i n o s i n i c a c i d , r e s p e c t i v e l y . Thus, the p a r t i c i p a t i o n of carboxamide i n p u r i n e s y n t h e s i s was e s t a b l i s h e d , but t r a p p i n g and d i l u t i o n experiments designed t o d e t e c t the f o r m a t i o n of l a b e l l e d carboxamide from i s o t o p i c p r e c u r s o r s o f hypoxanthine demonstrated t h a t carboxamide per se was not an i n t e r m e d i a t e (27 ,28) . 8 A balance study of formate i n c o r p o r a t i o n i n t o hypoxanthine i n pigeon l i v e r e x t r a c t s , r e p o r t e d at t h a t time by Greeriberg (28),showed that i n o s i n i c a c i d was the p r e c u r s o r of i n o s i n e which, i n t u r n , formed nypoxanthine. T h i s work i n d i c a t e d t h a t the f i r s t complete p u r i n e s t r u c t u r e formed de novo i n t h i s system was a r i b o t i d e . The e x c l u s i o n of carboxamide as an i n t e r m e d i a t e was e n t i r e l y compatible w i t h the i d e a t h a t r i b o t i d e f o r m a t i o n was an i n t e g r a l p a r t of p u r i n e s y n t h e s i s and prompted the s u g g e s t i o n t h a t carboxamide r i b o t i d e was the immediate p r e c u r s o r of i n o s i n i c a c i d i n the p i g e o n l i v e r system. The carboxamide r i b o s i d e has been i s o l a t e d from s u l f a d i a z i n e - i n h i b i t e d E. c o l i c u l t u r e s ( 2 9 ) . P h o s p h o r y l a t i o n of the r i b o s i d e to form the proposed i n t e r m e d i a t e r i b o t i d e has been demonstrated i n pigeon l i v e r e x t r a c t s and y e a s t a u t o l y s -ates ( 3 0 ) . The o b s e r v a t i o n s of Buchanan and h i s c o l l e a g u e s (31) on the enzymatic exchange r e a c t i o n of formate w i t h pos-i t i o n 2 of i n o s i n i c a c i d agreed w e l l w i t h the p a r t i c i p a t i o n of the carboxamide r i b o t i d e i n p u r i n e s y n t h e s i s . F u r t h e r , these authors have presented evidence f o r the f o r m a t i o n of carboxamide r i b o t i d e from hypoxanthine i n f r a c t i o n a t e d p i g e o n l i v e r e x t r a c t s ( 3 1 ) . Thus f a r , the known i n t e r m e d i a t e s i n u r i c a c i d s y n t h e s i s i n the p i g e o n l i v e r system were as f o l l o w s : 9 e a r l y p r e c u r s o r s u r i c a c i d FIGURE 3 . INTERMEDIATES IN URIC ACID BIOSYNTHESIS I n 1953 Greeriberg proposed t h a t a c y c l i c r i b o t i d e d e r i v a t i v e s of g l y c i n e were l i k e l y p r e c u r s o r s o f carboxamide r i b o t i d e (32) and r e c e n t l y , such i n t e r m e d i a t e s have been i s o l a t e d and c h a r a c t e r i z e d . The i s o l a t i o n o f glycinamide r i b o t i d e (GAR) and i t s N-formyl d e r i v a t i v e (FGAR) from pigeon l i v e r e x t r a c t s was f i r s t r e p o r t e d by Greeriberg and h i s coworkers (33) • T h i s was confirmed by Hartman et ' a l . who showed subsequently t h a t these compounds accumulate i n the presence of L - a z a s e r i n e ( 2 1 ) . •L'hese authors a l s o demonstrated t h a t 5>-phosphoribosylpyrophosphate (PRPP) was the donor o f the r i b o s y l p h o s p h a t e p o r t i o n o f the GAR and FGAR, r e p l a c i n g r i b o s e -5-phosphate and adenosine t r i p h o s p h a t e (ATP) I n t h e i r system. 10 Korriberg et a l . nave shown w i t h p u r i f i e d y e a s t enzymes th a t PRPP i s a key Intermediate i n the s y n t h e s i s of n u c l e o t i d e s from the f r e e p u r i n e and p y r i m i d i n e bases (3^)« R e c e n t l y Buchanan and h i s coworkers have p a r t i a l l y p u r i f i e d enzymes from pigeon l i v e r which w i l l condense PRPP and the f r e e p u r i n e s , adenine and hypoxanthine, t o form a d e n y l i c and I n o s i n i c . a c i d s . A separate f r a c t i o n c ontains the enzyme r e s p o n s i b l e f o r the f o r m a t i o n of PRPP from ribose - 5-phosphate and ATP (35,36). Hartman et a l . showed t h a t the amide n i t r o g e n of GAR and FGAR was d e r i v e d from the amide n i t r o g e n of glutamine (21). From t h i s work L - a z a s e r i n e would appear t o b l o c k the i n c o r p o r a -t i o n of the glutamine amide n i t r o g e n d e s i g n a t e d f o r p o s i t i o n 9 of the p u r i n e r i n g and thus, t o prevent f o r m a t i o n of the i m i d a z o l e r i n g . GAR and FGAR ar e e f f i c i e n t p r e c u r s o r s of i n o s i n i c a c i d i n the p i g e o n l i v e r system. The demonstration t h a t g l y c i n e was i n c o r p o r a t e d by way of a p h o s p h o r i b o s y l d e r i v a t i v e of glycinamide r a i s e d the q u e s t i o n of the p a r t i c i p a t i o n of the amide per se as an i n t e r -mediate i n p u r i n e s y n t h e s i s . I n the present i n v e s t i g a t i o n , glycinamide was shown to have no s i g n i f i c a n t e f f e c t on p u r i n e s y n t h e s i s i n suspensions o f i n t e s t i n a l mucosa, although, p e r m e a b i l i t y e f f e c t s were not excluded. A r e c e n t r e p o r t by Goldthwait et a l . (37) makes i t u n l i k e l y t h a t glycinamide p a r t i c i p a t e s i n p u r i n e s y n t h e s i s d i r e c t l y s i n c e these authors demonstrate t h a t the amide n i t r o g e n of GAR formed i n p i g e o n l i v e r e x t r a c t s was i n t r o d u c e d from 5-phosphoribosylamine. T h i s compound was formed from glutamine and PRPP. 11 A number of r i b o t i d e s which r e p r e s e n t i n t e r m e d i a t e stages i n i n o s i n i c a c i d s y n t h e s i s have r e c e n t l y been i s o l a t e d by Buchanan and h i s coworkers. They have employed f r a c t i o n a t e d pigeon l i v e r e x t r a c t s which, I n e f f e c t , were p o r t i o n s of the enzyme system which s y n t h e s i z e d i n o s i n i c a c i d , ^h.e enzyme p r e p a r a t i o n s c a r r i e d out only c e r t a i n steps i n t h i s process and i n t h i s way per m i t t e d the I s o l a t i o n of i n t e r m e d i a t e s . Levenberg and Buchanan (38) i s o l a t e d a f r a c t i o n from pigeon l i v e r e x t r a c t s which converted FGAR t o 5-aminoimidazolecarboxamide r i b o t i d e (AIR) i n the presence o f glutamine and ATP. F u r t h e r f r a c t i o n -a t i o n o f the AIR-forming enzyme system y i e l d e d two p r e p a r a t i o n s , one of which c a r r i e d the r e a c t i o n of FGAR w i t h glutamine and ATP t o another i n t e r m e d i a t e stage, forming N-f o r m y l g l y c i n a m i -d i n e r i b o t i d e (FGAM). The oth e r enzyme f r a c t i o n converted FGAM to AIR without f u r t h e r a d d i t i o n s . The new i n t e r m e d i a t e s were c h a r a c t e r i z e d c h e m i c a l l y and both were r e a d i l y converted t o i n o s i n i c a c i d by pigeon l i v e r enzymes. I n t h i s work the s i t e of a c t i o n of L - a z a s e r i n e was. l o c a l i z e d to the i n h i b i t i o n o f t h e c o n v e r s i o n o f FGAR t o FGAM. The a d d i t i o n of the carboxamide group t o AIR, forming 5-amino-I|.-imidazolecarboxamide r i b o t i d e (AICAK)"*", was shown by Lukens and Buchanan (39) t o take p l a c e i n a f r a c t i o n a t e d p i g e o n l i v e r e x t r a c t and r e q u i r e d ATP, aspar-t i c a c i d , and b i c a r b o n a t e . T h i s enzyme system was a l s o f r a c t i o n a t e d Into two p r e p a r a t i o n s , one of which accumulated 1 F o l l o w i n g the a b b r e v i a t i o n used i n the l i t e r a t u r e c i t e d , t h i s compound has been r e f e r r e d t o above as "carboxamide r i b o t i d e " 12 a new i n t e r m e d i a t e . The second enzyme p r e p a r a t i o n converted t h i s i n t e r m e d i a t e t o AICAR without f u r t h e r a d d i t i o n s . Proof of s t r u c t u r e Is l a c k i n g , but the a n a l y t i c a l data suggest t h a t the i n t e r m e d i a t e i s £ - a m i n o - l i - i m i d a z o l e - (N-suecinoylcarboxa-mide) r i b o t i d e . Warren and F l a k s (I4.0) have s t u d i e d f o r m y l a t i o n r e a c t i o n s i n t h i s s e r i e s of i n t e r m e d i a t e s and have shown t h a t pigeon l i v e r enzymes i n the presence of s e r i n e , t r i p h o s p h o -p y r i d i n e n u c l e o t i d e (TPN), and l e u c o v o r i n w i l l convert AICAR to i n o s i n i c a c i d . The formyl d e r i v a t i v e of AICAR ( 5-formyl-amino - l i-imidazolecarboxamide r i b o t i d e ) has been prepared c h e m i c a l l y , and p i g e o n l i v e r enzyme p r e p a r a t i o n s c a t a l y s e i t s c o n v e r s i o n t o i n o s i n i c a c i d without f u r t h e r a d d i t i o n s . Our present understanding of the i n t e r m e d i a t e compounds of p u r i n e s y n t h e s i s may be summarized i n the scheme shown i n F i g u r e "4. The processes of de novo s y n t h e s i s of the p u r i n e n u c l e o t i d e s l e a d t o the t i s s u e p o o l of s o l u b l e n u c l e o t i d e s (the a c i d - s o l u b l e f r a c t i o n ) . T h i s f r a c t i o n i s a very complex mixture, c o n t a i n i n g the 5-mono, d i - , and t r i p h o s p h a t e s of the r i b o s i d e s of adenine, guanine, c y t o s i n e and u r a c i l . The pur i n e r i b o s i d e phosphates of t h i s f r a c t i o n a re known t o have f u n c t i o n a l r o l e s as coenzymes i n i n t e r m e d i a r y m e t a b o l i c p r o c e s s e s , l a r g e l y those of energy t r a n s f e r through p h o s p h o r y l a t i o n r e a c t i o n s . The polyphosphates t u r n over r a p i d l y and are maintained by g l y c o l y s -i s and o x i d a t i v e p h o s p h o r y l a t i o n ( l i l ) . I s o t o p i c n u c l e i c a c i d p r e c u r s o r s have been shown r e p e a t e d l y t o cause a r a p i d l a b e l l i n g 13 of the a c i d - s o l u b l e p o o l . E a r l y In the i n c o r p o r a t i o n p r o c e s s , i s o t e p e c o n c e n t r a t i o n s i n the n u c l e i c a c i d s are i n v a r i a b l y subordinate to those of the f r e e n u c l e o t i d e s , and f o r t h i s r e a s o n the p o l y n u c l e o t i d e s a r e b e l i e v e d to be d e r i v e d from the f r e e n u c l e o t i d e p o o l . The above scheme ( F i g u r e I4.) i s based l a r g e l y on i n v i t r o experiments w i t h p i g e o n l i v e r p r e p a r a t i o n s , but no experimental evidence has been r e p o r t e d to e s t a b l i s h the r e l a t i o n s h i p s between the b i o s y n t h e t i c pathway and the p u r i n e n u c l e o s i d e phosphate p o o l ( a c i d - s o l u b l e f r a c t i o n ) In t h i s t i s s u e system. I t i s not known, f o r example, i f carboxamide r i b o t i d e (AICAR) or i n o s i n i c a c i d are o b l i g a t o r y Intermediates i n a d e n y l i c or g u a n y l i c a c i d b i o s y n t h e s i s . Abrams and B e n t l e y have d e s c r i b e d the t r a n s f o r m a t i o n of i n o s i n i c a c i d t o a d e n y l i c and g u a n y l i c a c i d s i n a s o l u b l e enzyme e x t r a c t from r a b b i t bone marrow (1^ 5>). T h i s work e s t a b l i s h e s the c o n n e c t i o n between i n o s i n i c a c i d and the adenine and guanine n u c l e o s i d e phosphates, but i t does not i n d i c a t e any s e q u e n t i a l r e l a t i o n s h i p s between them i n b i o s y n t h e s i s . There has been a r e c e n t s u g g e s t i o n t h a t a d e n y l i c a c i d may be formed by way of a carboxamidine d e r i v a t i v e of aminoimidazole r i b o t i d e (lj.6). I f such were the case, i n o s i n i c a c i d would be excluded as an o b l i g a t o r y i n t e r m e d i a t e i n p u r i n e s y n t h e s i s . At present t h e r e i s l i t t l e t o suggest t h a t the f a c t s of p u r i n e b i o s y n t h e s i s t h a t apply s p e c i f i c a l l y t o p i g e o n l i v e r systems, are not of g e n e r a l a p p l i c a b i l i t y t o mammalian systems. Indeed, the p a t t e r n of l a b e l l i n g i n the p u r i n e s from p r e c u r s o r s such as formate, g l y c i n e , and COg, b e i n g the same i n y e a s t , Ribose-5>-phosphate ATP Glutamic a c i d ATP FIGURE 1L. SCHEME FOR PURINE BIOSYNTHESIS (l|2 f35) 5-Phosphoribosyl-pyrophos phat e (PRPP) (l|3) Glutamine (H 2P0 3)0CH2 /0 NNH2 OH OH 5-Phospb.oribosylamine G l y c i n e (37) Formate L e u c o v o r i n H 2 c / ^ 2 (ILO) ^ r i b o s e - 5 -phosphate ,TH Glycinamide r i b o t i d e (GAR) Hp ^ N c. H OH -H «r • Tibose-5>-phos-phate H H 2 ^ -N \ H N< c=o-H N-Formyl glycinamide r i b o t i d e (FGAR) rib o s e -5-pnos-phate N-Formyl-g l y c i n a m i d i n e r i b o t i d e (FGAM) FIGURE ij.. (continued) H R2 HOOC—C-C-COOH N H FGAM-H2N (39) N > l b o s e - 5 ' - H 2 N / ^ N \ phosphate 5-Aminoimidaz o1e r i b o t i d e (Am) r i b o s e - 5 ' -phosphate 5 - Ami no -1].- i m i d -a z o l e - ( N - S u c c i n o y l -carboxamide) r i b o -t i d e Nucleic^ A c i d s T i s s u e p o o l of s o l u b l e - nucleoside" phosphates i b o s e - 5 ' -phosphate I n o s i n i c A c i d (IMP) r i b o s e - 5 1 -phosphate 5-Amino-ij.-imid-a z o l e carboxamide r i b o t i d e (''carboxamide r i b o -t i d e " or AICAR) s e r i n e TPN l e u c o -v o r i n U r i c a c i d ^ r i b o s e - S ' -phosphate 5 -F ormy lami no - Ij.- imida z o 1 e • carboxamide r i b o t i d e (FAICAR) 16 b a c t e r i a , b i r d s , and mammals, would suggest t h a t the same b a s i c mechanisms apply f o r p u r i n e b i o s y n t h e s i s . However, i n a v i a n l i v e r the s y n t h e s i s of i n o s i n a t e i s a major pathway d i r e c t e d a t the p r o d u c t i o n of u r a t e , the e x c r e t o r y form of n i t r o g e n i n the b i r d . The s y n t h e s i s of p u r i n e molecules, h a v i n g as t h e i r f a t e n u c l e i c a c i d components, n u c l e o t i d e s and coenzymes, c o n s t i t u t e s the major p u r i n e s y n t h e t i c f u n c t i o n i n other a v i a n t i s s u e s and i n other animals which do not excrete p u r i n e s as t h e i r p r i n c i p a l nitrogenous waste p r o d u c t s . T h e r e f o r e , the q u e s t i o n a r i s e s : are the f i n d i n g s from pigeon l i v e r experiments, where the u r a t e p r o d u c t i o n mechanism i s b e i n g e x p l o i t e d , a p p l i c a b l e t o the b i o s y n t h e s i s of n u c l e i c a c i d and coenzyme p u r i n e s ? The b a s i c steps of r i n g s y n t h e s i s are probably s i m i l a r , but the p o i n t s at which pur i n e components a r e drawn o f f f o r n u c l e i c a c i d , n u c l e o t i d e , and coenzyme s y n t h e s i s remain und i s c o v e r e d . Edmonds and LePage have observed i n s h o r t term experiments w i t h whole animals t h a t i n o s i n a t e , adenylate, and guanylate appear t o have separate o r i g i n s •(£)• They conclude, t h a t the n u c l e o t i d e s a r i s e from a common p r e c u r s o r a t some e a r l y stage of s y n t h e s i s and are probably not m e t a b o l i z e d a l o n g a common pathway. T h i s p o i n t i s i n t r o d u c e d to emphasize t h a t a v i a n l i v e r p r e p a r a t i o n s are adequate f o r s t u d i e s of i n o s i n a t e s y n t h e s i s , but the b a s i c p u r i n e p r e c u r s o r may not be i n o s i n i c a c i d which, w i t h subsequent o x i d a t i o n and amination, would form a d e n y l i c and g u a n y l i c a c i d s These f u n c t i o n a l groups may be i n t r o d u c e d p r i o r t o t h e complete c y c l i z a t i o n of the p u r i n e r i n g (I4.6). Thus, t h e r e appears t o be a d i s t i n c t need f o r p a r a l l e l , comparative s t u d i e s of p u r i n e b i o s y n t h e s i s i n mammalian systems. 17 2. POLYNUCLEOTIDE SYNTHESIS. N u c l e i c a c i d s y n t h e s i s i s c o n s i d e r e d t o take p l a c e by the u n i o n of s u b - u n i t s , the n u c l e o t i d e s . T h i s concept i s d e r i v e d I n p a r t from s t r u c t u r a l and enzymatic s t u d i e s which have shown t h a t n u c l e i c a c i d s can be h y d r o l y s e d c h e m i c a l l y and e n z y m a t i c a l l y i n t o n u c l e o t i d e s . I d e n t i f i c a t i o n of n u c l e o s i d e -5«-phosphates as the proximal or immediate p r e c u r s o r s of the n u c l e i c a c i d polymer ( r a t h e r t h a n s t i l l s m a l l e r sub-units such as f r e e bases, r i b o s e , and phosphate) has come about through s t u d i e s of the composition of the s o l u b l e n u c l e o t i d e f r a c t i o n of t i s s u e s and the m e t a b o l i c r e l a t i o n s h i p s between t h i s p o o l and the n u c l e i c a c i d s . S t r i c t l y , t h i s mechanism can be a p p l i e d only t o RNA s y n t h e s i s ; however, i n view of the s i m i l a r i t i e s i n DNA and RNA s t r u c t u r e , i t s e x t e n s i o n t o DNA s y n t h e s i s seems re a s o n a b l e . S t u d i e s i n DNA s y n t h e s i s are not y e t advanced as f a r as those i n the RNA f i e l d , p a r t i c u l a r l y I n regard t o the i s o l a t i o n of p r e c u r s o r s . S t u d i e s of n u c l e i c a c i d s y n t h e s i s u s i n g t r a c e r techniques have employed two p r i n c i p a l approaches, (a) through the use of l a b e l l e d p r e c u r s o r s which g a i n access to the a c i d -s o l u b l e p o o l by the de novo pathways, (b) through the use of l a b e l l e d p u r i n e s , p y r i m i d i n e s and t h e i r d e r i v a t i v e s , the n u c l e o s i d e s and n u c l e o t i d e s . The e x t e n s i v e l i t e r a t u r e on t h i s l a t t e r t o p i c has been reviewed by s e v e r a l authors (11 - ii}.), most completely by Brown and R o l l (\\.7)» A. D e s o x y r i b o n u c l e l c A c i d S y n t h e s i s Small molecule p r e c u r s o r s of the p u r i n e s of DNA a r e , i n g e n e r a l , i n c o r p o r a t e d t o a s e v e r a l f o l d s m a l l e r extent t h a n 18 i n t o the purines of RNA. DNA s y n t h e s i s , as measured by the I n c o r p o r a t i o n o f l a b e l l e d p r e c u r s o r molecules, occurs a t h i g h e r r a t e s i n p r o l i f e r a t i n g t i s s u e s than i n non-growing t i s s u e s . DNA purines a r e d e r i v e d from adenine t o an extent which Is d i r e c t l y r e l a t e d t o the r a t e o f c e l l d i v i s i o n i n the t i s s u e under c o n s i d e r a t i o n , the I n c o r p o r a t i o n b e i n g s l i g h t i n non-p r o l i f e r a t i n g t i s s u e s such as l i v e r and kidney, but approaching t h a t o f RNA i n i n t e s t i n e , bone marrow, and r e g e n e r a t i n g l i v e r which a r e p r o l i f e r a t i n g t i s s u e s . I n comparisons of the renewal of DNA purines i n non-growing and p r o l i f e r a t i n g til s sues, adenine i n c o r p o r a t i o n Is enhanced i n the l a t t e r case t o a much g r e a t e r extent t h a n i s seen w i t h "de novo" p r e c u r s o r s , formate and g l y c i n e . Adenosine and the Isomers of a d e n y l i c a c i d are poor p r e c u r s o r s o f RNA adenine r e l a t i v e t o the f r e e base and a r e very l i k e l y degraded t o t h i s form b e f o r e I n c o r p o r a t i o n ( I L 8 ) . However, adenosine has been shown t o be i n c o r p o r a t e d i n t o DNA w i t h the N - g l y c o s y l bond i n t a c t ( I 1 8 ) . Guanine i s u t i l i z e d only s l i g h t l y f o r n u c l e i c a c i d s y n t h e s i s by the r a t , but, I n c o n t r a s t t o adenine and i t s d e r i v a t i v e s , the n u c l e o t i d e , g u a n y l i c a c i d , i s a s u p e r i o r RNA p r e c u r s o r . G u a n y l i c a c i d Is used to a s m a l l extent i n DNA renewal, and i n t h i s p r o c e s s , the N - g l y c o s y l bond i s broken (1L8) . I n g e n e r a l , t h e r e i s l i t t l e known of the immediate p r e c u r s o r s o f the DNA p u r i n e s . S t u d i e s o f DNA s y n t h e s i s , i n which p y r i m i d i n e d e r i v a t i v e s are u t i l i z e d , o f f e r more d e f i n i t e evidence o f n u c l e o t i d e p a r t i c i p a t i o n . The known p r e c u r s o r s o f DNA p y r i m i -dines d i s p l a y p a t t e r n s o f u t i l i z a t i o n d i f f e r e n t from t h o s e . o f 19 adenine and i t s d e r i v a t i v e s i n the r e s p e c t t h a t the f r e e bases of the p o l y n u c l e o t i d e s a r e i n e f f e c t i v e p r e c u r s o r s , w h i l e the n u c l e o s i d e s and n u c l e o t i d e s a r e w e l l i n c o r p o r a t e d . O r o t i c a c i d i s r e a d i l y converted i n t o the p y r i m i d i n e bases of RNA and DNA and must pass through the a c i d - s o l u b l e n u c l e o t i d e p o o l as oro-t o d y l i c and u r i d y l i c a c i d s i n t h i s p r o c e s s . D e s o x y c y t i d i n e and thymidine a r e unique i n t h a t they are s p e c i f i c DNA p r e c u r s o r s , showing no c o n v e r s i o n to RNA p y r i m i d i n e s i n the r a t (l| .9»50). The p h o s p h o r y l a t i o n of d e s o x y r i b o n u c l e o s i d e s i n an i n v i t r o system has been demonstrated ( 5 l ) > and s m a l l amounts of f r e e d e s o x y r i b o n u c l e o s i d e s and d e s o x y r i b b n u c l e o t i d e s have been shown t o be present i n l i v e r ( 5 2 ) . Rose and Schweigert have shown t h a t t h e N - g l y c o s y l bond of c y t i d i n e remained i n t a c t i n i t s i n c o r p o r a t i o n i n t o d e s o x y c y t i d i n e and thymidine of DNA ( 5 3 ) . The knowledge t h a t thymidine i s a s p e c i f i c p r e c u r s o r f o r DNA, and t h e r e c e n t development of i n v i t r o systems i n which DNA s y n t h e s i s take p l a c e (50) promise advances i n the f i e l d of DNA s y n t h e s i s . The immediate p r e c u r s o r s o f t h e d e s o x y r i b o p o l y -n u c l e o t i d e s are not y e t known, but the evidence c i t e d above, and the analogy w i t h RNA s y n t h e s i s suggest t h a t desoxyribonuc-l e o t i d e s are l i k e l y p r e c u r s o r s . B. R i b o n u c l e i c A c i d S y n t h e s i s The renewal of RNA bases from i s o t o p i c a l l y l a b e l l e d bases has an e x t e n s i v e l i t e r a t u r e and w i l l be t r e a t e d only summarily i n t h i s d i s c u s s i o n , w i t h the o b j e c t i v e of p o i n t i n g out evidence t h a t bears on the i d e n t i t y of the immediate p r e c u r s o r s of the r i b o n u c l e i c a c i d macromolecule. I n g e n e r a l , 20 whole animal experiments have shown t h a t adenine Is w e l l i i n c o r p o r a t e d i n t o RNA p u r i n e s , whereas guanine and hypoxanthine are p o o r l y taken up. The p u r i n e r i b o s i d e s and r i b o t i d e s have proven to be i n f e r i o r t o the f r e e bases as RNA p r e c u r s o r s i n whole animal experiments. Such i s not the case w i t h the p y r i m i d i n e bases, where the n u c l e o s i d e s and n u c l e o t i d e s o f c y t o s i n e and u r a c i l are b e t t e r i n c o r p o r a t e d i n t o RNA t h a n a r e the f r e e bases. E a r l y s t u d i e s i n t h i s area produced l i t t l e i n f o r m a t i o n on the nature o f the i n t e r m e d i a t e n u c l e i c a c i d p r e c u r s o r s because the importance of the a c i d - s o l u b l e f r a c t i o n i n t h i s r e s p e c t was t h e n unrecognized. I n r e c e n t i n v i v o s t u d i e s , the i s o t o p e contents of the a c i d - s o l u b l e n u c l e o t i d e s d e r i v e d from l a b e l l e d p urines ( 5 , 9 ) a n d p y r i m i d i n e s ( 1 , 2 ) have been measured and compared, showing, i n g e n e r a l , t h a t t u r n o v e r was r a p i d i n the n u c l e o t i d e s and slower i n the n u c l e i c a c i d s . Thus, the pathways of de novo s y n t h e s i s and the anabolism of the preformed bases appear t o have a confluence i n t h e a c i d - s o l u b l e f r a c t i o n , '^he f r e e p u r i n e s are probably converted t o the n u c l e o t i d e form by r e a c t i o n w i t h PRPP (3I4. - 3 6 ) . The r e l a t i v e Importance of the de novo and preformed pathways has been compared i n l i v e r and i n t e s t i n e , w i t h a s u r p r i s i n g d i f f e r e n c e b e i n g noted. I n the r a t , the s y n t h e s i s of RNA i n the l i v e r from adenine Is favoured over the de novo process, whereas the r e v e r s e i s t r u e i n i n t e s t i n e . Developments i n the technique of Ion-exchange chromatography have been a p p l i e d w i t h g r e a t success t o the r e s o l u t i o n of the complex a c i d - s o l u b l e f r a c t i o n o f t i s s u e s . 21 The n u c l e o t i d e composition of t h i s f r a c t i o n has been s t u d i e d by the use of g r a d i e n t e l u t i o n chromatography ( 1 , 2 ) . The adenosine phosphates a r e p a r t i c u l a r l y prominent i n t h i s complex mixture o f n u c l e o s i d e phosphates. As Brown and R o l l p o i n t out, "the abundance of the adenosine phosphates l e a d t o the t a c i t assumption t h a t t i s s u e adenosine monophosphate (AMP) or i t s polyphosphates probably serve as a source of p u r i n e s , a t l e a s t of adenine, f o r p o l y n u c l e o t i d e s y n t h e s i s 11 (I4.7). Adenine - 8-C has been found t o be i n c o r p o r a t e d by the s o l u b l e adenosine phosphates at much h i g h e r r a t e s t h a n by p o l y n u c l e o t i d e adenine i n the mouse (9) and the r a t (Ij.). H u r l b e r t and P o t t e r i n t h e i r study of the i n c o r p o r a t i o n of - o r o t i c a c i d i n t o RNA, showed t h a t t h i s compound i s r a p i d l y I n c o r p o r a t e d i n t o u r i d i n e phosphates of the a c i d - s o l u b l e n u c l e o t i d e p o o l and thence Into RNA ( 7 , 8 ) . Nuclear RNA was more r a p i d l y renewed than c y t o p l a s m i c RNA. The suthors concluded t h a t s o l u b l e u r i d i n e phosphates were the immediate p r e c u r s o r s of t h e u r a c i l of n u c l e a r RNA. A time study of adenine i n c o r p o r -a t i o n Into a c i d - s o l u b l e adenine and n u c l e a r RNA adenine by F r e s c o and Marshak (51+) i n d i c a t e d t h a t a g e n e r a l p r e c u r s o r -product r e l a t i o n s h i p may e x i s t between the two. R e c e n t l y , s t u d i e s of RNA s y n t h e s i s w i t h c e l l - f r e e systems have shown t h a t i t may be p o s s i b l e t o study polynuc-l e o t i d e s y n t h e s i s at the enzyme l e v e l . Goldwasser has demon-s t r a t e d t h a t RNA adenine can be d e r i v e d from AMP l a b e l l e d w i t h P-" or i n homogenates of pigeon l i v e r (55,56). H e i d e l b e r g e r et a l . have shown a s i m i l a r i n c o r p o r a t i o n of P ^ ^ - i a b e l l e d AMP 22 i n homogenates of r e g e n e r a t i n g l i v e r (57)• I n c o r p o r a t i o n of -AMP i n t o the RNA of g l y c o l y s i n g tumour homogenates has been r e p o r t e d by P a t e r s o n and LePage (58)• I n general, the homogenate systems show a g r e a t e r a b i l i t y t o i n c o r p o r a t e the r i b o t i d e s t han f r e e bases or e a r l y p r e c u r s o r s . T i s s u e p r e p a r a t i o n s , i n which high-energy phosphate compounds ar e maintained by energyrproducing processes such as g l y c o l y s i s , w i l l phosphorylate l a b e l l e d n u c l e o t i d e s u b s t r a t e s , making I t d i f f i c u l t t o a s c e r t a i n a t what l e v e l of p h o s p h o r y l a t i o n the s u b s t r a t e Is i n c o r p o r a t e d i n t o the n u c l e i c a c i d polymer. The very important c o n t r i b u t i o n s of Ochoa and h i s coworkers suggest t h a t RNA i s s y n t h e s i z e d from n u c l e o s i d e diphosphates (59)• These workers have shown t h a t a p a r t l y p u r i f i e d b a c t e r i a l enzyme p r e p a r a t i o n w i l l form RNA-like polymers from the 5 '-diphosphates of adenosine, guanosine, In o s i n e and c y t i d i n e . T h i s important d i s c o v e r y has p r o v i d e d a system I n which p o l y n u c l e o t i d e synthe-s i s can be s t u d i e d at the enzyme l e v e l . I t remains t o be e s t a b l i s h e d whether these RNA-like polymers t r u l y resemble the n a t i v e p o l y n u c l e o t i d e s , or whether the s i m i l a r i t y i s s u p e r f i c i a l , s p e c i f i c arrangements of the component n u c l e o t i d e s perhaps b e i n g absent. 3 . PURINE SYNTHESIS IN INTESTINAL MUCOSA. A c t i v e l y p r o l i f e r a t i n g t i s s u e s such as r e g e n e r a t i n g l i v e r ( 6 0 ) , bone marrow ( 6 l , 6 2 ) , and tumour (3) have been employed f o r i n v i t r o s t u d i e s of n u c l e i c a c i d p u r i n e s y n t h e s i s . I n the whole animal, such t i s s u e s d i s p l a y a r a p i d t u r n o v e r o f n u c l e i c a c i d components r e l a t i v e t o t i s s u e s w i t h low r a t e s of 23 c e l l d i v i s i o n . A v i a n l i v e r has been favoured f o r the i n v i t r o i n v e s t i g a t i o n of p u r i n e s y n t h e s i s , s i n c e t h i s t i s s u e has as a p a r t i c u l a r l y prominent m e t a b o l i c f u n c t i o n , the s y n t h e s i s of u r i c a c i d . The r a t e of n u c l e i c a c i d s y n t h e s i s i n i n t e s t i n a l mucosa i s h i g h i n the i n t a c t animal, v e r y probably, because of the h i g h r a t e of c e l l d i v i s i o n i n t h i s t i s s u e . T h i s suggests t h a t i n t e s t i n a l mucosa would be a s u i t a b l e t i s s u e f o r i n v i t r o s t u d i e s of p u r i n e metabolism. Such, a use has not been r e p o r t e d p r i o r t o the present work. Hevesy and h i s coworkers (63) f i r s t r e p o r t e d t h a t a c o r r e l a t i o n e x i s t s between the r a t e of c e l l d i v i s i o n i n a t i s s u e and the uptake of P 32 -phosphate by DNA. T h e i r d a t a show t h a t i n t e s t i n e had the h i g h e s t phosphate renewal of the p r i n c i p a l t i s s u e s of the r a t . T h i s i s not merely a f e a t u r e of phosphate metabolism, as other DNA p r e c u r s o r s such as formate and g l y c i n e have s i n c e been shown t o be r a p i d l y i n c o r p o r a t e d i n t o the DNA of I n t e s t i n e and other p r o l i f e r a t i n g t i s s u e s (lj.7, 61}. - 6 6 ) . Stevens et a l . have measured the c e l l renewal r a t e s i n s e v e r a l t i s s u e s of the r a t , as shown i n the f o l l o w i n g t a b l e : TABLE I RATE OF NEW CELL FORMATION IN RAT TISSUES (61}.) T i s s u e Per cent d a i l y f o r m a t i o n of c e l l s l i v e r l u ng i n t e s t i n a l mucosa 0.71 51*. 3 21* T h e i r work demonstrated t h a t p a r a l l e l t o t h i s very r a p i d p r o l i f e r a t i o n of the mucosal e p i t h e l i u m of the i n t e s t i n e , t h e r e was a c o r r e s p o n d i n g l y h i g h r a t e of i n c o r p o r a t i o n o f i n o r g a n i c phosphate i n t o DNA.. The RNA of i n t e s t i n a l mucosa a l s o d i s p l a y s a vigourous renewal. The I n c o r p o r a t i o n o f i s o t o p i c p r e c u r s o r s (ammonia, phosphate, formate, g l y c i n e , and adenine) by the RNA of i n t e s -t i n e , when compared w i t h t h a t of l i v e r and other t i s s u e s , shows t h a t i n t e s t i n e has one of the most r a p i d renewal r a t e s of RNA I n the body (see Tables I I and I I I of the review by Brown and R o l l (Ij.7) ). T h i s f e a t u r e may be r e l a t e d t o the h i g h r a t e of c e l l d i v i s i o n , a l t h o u g h t i s s u e f u n c t i o n must a l s o be considered i n t h i s r e s p e c t . Comparisons of formate and adenine I n c o r p o r a t i o n i n t o i n t e s t i n e and l i v e r RNA ( l i 7 , 6 7 ) have r e v e a l e d t h a t i n the l a t t e r t i s s u e , adenine i s u t i l i z e d i n p r e f e r e n c e t o the i n c o r p o r a t i o n of simple p r e c u r s o r s v i a the de novo pathways of pu r i n e s y n t h e s i s . The converse i s t r u e f o r i n t e s t i n e where the de novo pathway Is favoured. I n the metabolism o f carbohydrate i n t e s t i n a l mucosa has an unusual f e a t u r e i n which r e s p e c t i t bears a resemblance to tumour t i s s u e . I n most normal t i s s u e s g l y c o l y s i s i s supressed under a e r o b i c c o n d i t i o n s . A s i g n i f i c a n t p r o d u c t i o n of l a c t a t e from pyruvate occurs i n most t i s s u e s only i n t h e absence of oxygen. The s u p r e s s i o n of g l y c o l y s i s , as measured by l a c t a t e f o r m a t i o n , when r e s p i r a t i o n takes p l a c e i s w e l l known as the Pasteur e f f e c t . A h i g h g l y c o l y t i c r a t e under b o t h 2$ a e r o b i c and a n a e r o b i c c o n d i t i o n s i s considered t o be c h a r a c t e r i s t i c of tumours, w i t h c e r t a i n normal t i s s u e s d i s p l a y i n g t h i s f e a t u r e t o a s i m i l a r extent ( 6 8 ) . D i c k e n s and Weil-Malherbe have r e p o r t e d a complete l a c k of the Pasteur e f f e c t i n I n t e s t i n a l mucosa ( 6 9 ) . R o s e n t h a l has subsequently r e p o r t e d t h a t the h i g h r a t e of a e r o b i c g l y c o l y s i s may be, i n p a r t , a t t r i b u t a b l e t o damage i n c u r r e d d u r i n g p r e p a r a t i o n of the t i s s u e ( 7 0 ) . Dickens and Weil-Malherbe s t u d i e d the r e s p i r a t i o n and g l y c o l y s i s of s t r i p s o f i n t e s t i n a l mucosa and t h e i r data p l a c e j e j u n a l mucosa as one of the most a c t i v e l y r e s p i r i n g t i s s u e s of the body ( 6 9 ) . I n t h e i r experiments, r e s p i r a t o r y r a t e s f o r mucosa from v a r i o u s p a r t s of the gut showed a marked d e c l i n e w i t h progress down the i n t e s t i n e . With r e s p e c t t o time, Dickens and Weil-Malherbe found t h a t the r e s p i r a t i o n of t h i s t i s s u e was more s t a b l e t han g l y c o l y s i s . The i n s t a b i l i t y of g l y c o l y s i s may be r e l a t e d to the abundance of phosphatases i n t h i s t i s s u e . Lowenstein and Cohen have assayed v a r i o u s t i s s u e s of the r a t f o r the enzyme carbamylphosphate-aspartate t r a n s c a r b a m y l -ase, and r e p o r t t h a t i n t e s t i n a l mucosa has the h i g h e s t a c t i v i t y ( 7 1 ) . T h i s enzyme c a t a l y s e s the f o r m a t i o n of carbamylaspartate ( u r e i d o s u c c i n a t e ) , an important i n t e r m e d i a t e i n p y r i m i d i n e b i o s y n t h e s i s . The data of A l l f r e y et a l . (72) show t h a t I n t e s t i n a l mucosa i s p a r t i c u l a r l y r i c h i n c e r t a i n enzymes which are i n v o l v e d i n the c a t a b o l i s m of n u c l e i c a c i d s , namely: adenosine deaminase ATPase a l k a l i n e phosphatase adenosine - 5 1-phosphatase DNAase I I adenosine - 3 1-phosphatase 26 These enzymes a r e mainly c y t o p l a s m i c i n d i s t r i b u t i o n . Xanthine oxidase i s a l s o abundant i n mucosa ( 7 3 ) • T k e prominence o f these c a t a b o l i c enzymes may be r e l a t e d t o the f a i l u r e t o d e t e c t p u r i n e s y n t h e s i s i n homogenates of i n t e s t i n a l mucosa i n the experiments d e s c r i b e d subsequently. While t h i s manuscript was i n p r e p a r a t i o n s e v e r a l r e p o r t s appeared i n which the i n v i t r o use of i n t e s t i n a l mucosa has been d e s c r i b e d . H e i d e l b e r g e r and Harbers have r e p o r t e d the i n c o r p o r a t i o n of o r o t i c a c i d i n t o the u r i d i n e n u c l e o t i d e s o f the h i g h speed supernatant f r a c t i o n o f homo-genates of s e v e r a l r a t t i s s u e s , i n c l u d i n g i n t e s t i n a l mucosa ( 7 J 1 ) . The e f f e c t of a m i n o p t e r i n on i n t e s t i n a l mucosa i n the I n t a c t r a t and on the r e s p i r a t i o n o f t h i s t i s s u e i n v i t r o has been d e s c r i b e d by V i t a l e et a l . ( 7 5 » 7 * b ) » I n i n i t i a t i n g the present s t u d i e s , r e c o g n i t i o n was made of the need f o r a study of the mechanisms of de novo adenine and guanine s y n t h e s i s i n mammalian t i s s u e s . More p a r t i c u l a r l y , i t Is important t o know whether or not the known pathways f o r i n o s i n i c a c i d b i o s y n t h e s i s i n a v i a n l i v e r a re a l s o p r i m a r i l y i n v o l v e d i n a d e n y l i c and g u a n y l i c a c i d b i o s y n t h e s i s I n mammalian t i s s u e s . The use of i n v i t r o systems and t i s s u e e x t r a c t s i n such s t u d i e s i s i n d i c a t e d s i n c e t h i s approach has been f r u i t f u l i n the i s o l a t i o n o f Intermediates i n i n o s i n i c a c i d b i o s y n t h e s i s . C e r t a i n o f the f e a t u r e s d i s c u s s e d p r e v i o u s l y recommended i n t e s t i n a l mucosa as a l i k e l y mammalian t i s s u e f o r i n v i t r o s t u d i e s of p u r i n e metabolism: (a) the very h i g h r a t e of c e l l d i v i s i o n , (b) the r a p i d renewal of DNA and RNA, (c) the 27 prominence of de novo pathways of p u r i n e renewal, (d) the h i g h r a t e o f r e s p i r a t i o n and g l y c o l y s i s i n v i t r o . I n the present s t u d i e s a method was developed f o r p r e p a r i n g the mucosa of the s m a l l i n t e s t i n e of the r a t as a suspension i n Krebs-Ringer b u f f e r s . I n t h i s form the t i s s u e s u r v i v e d f o r s e v e r a l hours of i n c u b a t i o n , r e s p i r i n g a c t i v e l y . The de novo s y n t h e s i s of a c i d - s o l u b l e purines was shown t o occur i n such suspensions by measuring the i n c o r p o r a t i o n o f I s o t o p i c formate, g l y c i n e , and carbon d i o x i d e i n t o t h i s f r a c t i o n . The renewal o f s o l u b l e adenine n u c l e o t i d e s was prominent, b e i n g approximately 6 times t h a t o f the guanine n u c l e o t i d e s . The s y n t h e s i s o f p o l y n u c l e o t i d e purines a l s o occured d u r i n g i n c u b a t i o n . The s p e c i f i c a c t i v i t y r e l a t i o n s h i p s between t h e i s o t o p i c p r e c u r s o r s and the products suggested t h a t the a c i d -s o l u b l e n u c l e o t i d e s were in t e r m e d i a t e s i n n u c l e i c a c i d s y n t h e s i s . The demonstration t h a t r a t : i n t e s t i n a l mucosa i s capable of a r a p i d s y n t h e s i s o f adenine and guanine i n v i t r o suggests t h a t t h i s system may be a p p l i e d p r o f i t a b l y t o a study of the i n t e r -mediates i n v o l v e d i n puri n e b i o s y n t h e s i s as i t occurs i n mammalian t i s s u e . 28 EXPERIMENTAL 1. ANALYTICAL METHODS In t h i s s e c t i o n a n a l y t i c a l methods are d i s c u s s e d from the t e c h n i c a l p o i n t of view. T h e i r a p p l i c a t i o n t o b i o l o g i c a l m a t e r i a l and the r e s u l t i n g problems are d i s c u s s e d i n subsequent s e c t i o n s . A. Ion exchange chromatography ( i ) The s e p a r a t i o n of adenine, guanine, hypoxanthine.  xanthine, xanthine and u r i c a c i d S o l u t i o n s of these p u r i n e s were r e s o l v e d i n t o t h e i r components on columns of Dowex-50 i o n exchange r e s i n by f i r s t e l u t i n g w i t h 0.1N HC1 t o remove the oxy-purines, which emerged i n the f o l l o w i n g sequence: u r i c a c i d (not r e t a i n e d ) , xanthine, and hypoxanthine. By changing the eluent t o 1.5>N HC1, guanine was removed, f o l l o w e d by adenine. A s i m i l a r scheme was r e c e n t l y d e s c r i b e d by Abrams and Bentley ( 7 7 ) . Procedure Dowex-50 c a t i o n exchange r e s i n (20O-I4.OO mesh) was prepared f o r use by f i r s t removing t h e " f i n e s " by d e c a n t a t i o n 29 from a water suspension of the r e s i n . Removal of the " f i n e s " i n c r e a s e d the r a t e of flow through the r e s i n . S e v e r a l volumes of 6N HC1 were used t o wash the r e s i n and the excess a c i d was removed by s e v e r a l r i n s e s w i t h water. The r e s i n was t h e n washed w i t h 6N NaOH f o l l o w e d by s e v e r a l r i n s e s w i t h water. F i n a l l y , the r e s i n was washed a g a i n w i t h 6N HC1, f o l l o w e d by washes w i t h water which were continued u n t i l the washings were n e u t r a l . T h i s washing procedure f r e e d the r e s i n o f a brownish, s o l u b l e i m p u r i t y . A s o l u t i o n of the above purines i n 0.1N HC1 was poured onto a 1 x 7 cm* Dowex-50 column (200-lj.OO mesh) and the e f f l u e n t was c o l l e c t e d i n 5 ml. f r a c t i o n s u s i n g a T e c h n i -con automatic f r a c t i o n c o l l e c t o r . The purines were l o c a t e d i n the e f f l u e n t by measuring the o p t i c a l d e n s i t y of each f r a c t i o n at 260 mu i n t h e Beckman Model DTJ Spectrophotometer and were i d e n t i f i e d by t h e i r a b s o r p t i o n s p e c t r a . The column was f i r s t e l u t e d w i t h 0.1N HC1, f l o w i n g a t 0 . 5 ml. per min., u n t i l hypoxanthine was removed. The eluent was t h e n changed to 1 . 5N HC1. The flow r a t e was achieved by a t t a c h i n g t o the column an eluent r e s e r v o i r made from g l a s s t u b i n g h i g h enough to g i v e a h y d r o s t a t i c head o f 1 . 7 m. The eluent l e v e l i n the r e s e r v o i r was kept approximately constant by the f i l l i n g d e v i c e shown i n F i g u r e 5« 30 FIGURE $. ION EXCHANGE COLUMN WITH SELF-FILLING PRESSURIZING RESERVOIR 7V H E A D OF 1.7 M. ION EXCHANGE COLUMN ( I I ) I s o l a t i o n of adenine and guanine from i n t e s -t i n a l mucosa Adenine and guanine were r o u t i n e l y i s o l a t e d from p e r c h l o r i c a c i d e x t r a c t s of i n t e s t i n a l mucosa u s i n g the i o n exchange technique d e s c r i b e d by LePage (3)« Procedure The p u r i n e - c o n t a i n i n g e x t r a c t s were passed through s m a l l columns (6 x l£ mm.) of Dowex-^0 r e s i n (200-1*00 mesh) which r e t a i n the p u r i n e s . A f t e r washing the r e s i n w i t h 2.0 ml. of 0.1N HC1, f o l l o w e d by 2.0 ml. of water, adenine and guanine were recovered t o g e t h e r by e l u t i n g w i t h I4..O ml. of 6N HC1. The 6N HC1 f r a c t i o n s were evaporated t o dryness i n vacuo over s u l f u r i c a c i d and sodium hydroxide. I 31 B. Paper chromatography Descending paper chromatography on Whatman No. 1 f i l t e r paper was used throughout these experiments. (I) Solvent systems The choice of solvent systems for chromatography was influenced by two f a c t o r s : the behaviour of guanine, and the contamination of the i s o l a t e d purines by amino acids, especially glycine. Strongly a c i d i c systems such as the tertiary-butanol-KCl solvent of Smith and Markham (78) and the iso-propanol-HCl system of Wyatt (79) have proven to be among the most s a t i s f a c t o r y solvents f o r the chromatography of guanine, having the capacity f o r moving larger amounts of guanine without streaking than neutral or basic systems (80). The Wyatt system was used routinely as the f i r s t solvent i n the chromatography of the acid-soluble f r a c t i o n . The overlap-ping of glycine and adenine spots introduced the problem of radiochemical purity since g l y c i n e - l - C ^ was used as a substrate In c e r t a i n experiments. The iso-amyl alcohol-disodium phos-phate solvent of Carter (81) separated glycine (R f 0 . 7 0 ) and adenine (Rf O.ljii) but gave poor r e s u l t s with guanine. In t h i s solvent guanine formed double spots and streaked r e a d i l y i f overloaded on the paper. Guanine has a low s o l u b i l i t y i n t h i s solvent and accordingly, does not move from the point of app l i c a t i o n . I t i s usually not d i s t r i b u t e d uniformly through-out the area of application, more accumulating on the side of the paper to which i t was applied than on the side opposite 2. 2 LePage, G.A., personal communication. 32 T h i s behaviour o f guanine has obvious disadvantages i f p u r i n e areas of chromatograms are t o be assayed f o r r a d i o a c t i v i t y d i r e c t l y . The c o i n c i d e n c e of n i n h y d r i n - p o s i t i v e areas w i t h p u r i n e areas was observed on chromatograms of the a c i d - s o l u b l e purines which were r u n i n Wyatt's s o l v e n t . T h i s r a i s e d the p o s s i b i l i t y of r a d i o a c t i v e contamination. The n - b u t a n o l - e t h a n o l s o l v e n t o f Berry et a l . ( 8 2 ) was found t o l e a v e near the o r i g i n the amino a c i d s which accompany adenine and guanine i n the i s o - p r o p a n o l s o l v e n t . F o r these reasons, i n the m a j o r i t y o f the experiments d e s c r i b e d h e r e i n , the a c i d - s o l u b l e p u r i n e s were f i r s t chromato-graphed i n Wyatt's iso-propanol-HC1 s o l v e n t . The p u r i n e s from these chromatograms were recovered by e x t r a c t i o n w i t h 0.1N KC1 and rechromatographed i n n - b u t a n o l - e t h a n o l . T h i s procedure separated t h e purines from accompanying amino a c i d s and i n c r e a s e d the s p e c i f i c a c t i v i t y of adenine. Unless otherwise noted, adenine and guanine were p u r i f i e d i n t h i s manner. Other systems were t r i e d but w i t h l e s s s a t i s f a c t o r y r e s u l t s . Chromatograms r u n f i r s t i n butanol-ammonia (83) showed c o n s i d e r a b l e l o s s of a c t i v i t y I n the r e g i o n of guanine when rechromatographed i n i s o - p r o p a n o l - H C l . ( i i ) V i s u a l i z i n g p u r i n e areas The n u c l e i c a c i d bases were l o c a t e d on paper chromatograms by the c o n v e n t i o n a l u l t r a v i o l e t techniques which employ the h i g h absorbancy d i s p l a y e d by these compounds i n the r e g i o n of 260 mu. I n order to o u t l i n e the p u r i n e areas i n p e n c i l , chromatograms were examined i n t r a n s m i t t e d l i g h t from an u l t r a v i o l e t p r i n t i n g box 33 which had a g e r m i c i d a l lamp^ aa a l i g h t source. The u l t r a -v i o l e t l i g h t was f i l t e r e d through a l a r g e g l a s s f i l t e r ^ " . T h i s apparatus was f a r s u p e r i o r t o the w i d e l y recommended Minera-l i g h t lamp f o r s u r v e y i n g chromatograms. The d e f i n i t i o n of the purine spots, which appear as dark areas a g a i n s t the f a i n t f l u o r e s c e n c e of the paper, was e s p e c i a l l y improved by the use of t r a n s m i t t e d l i g h t . T ^ Q o u t l i n i n g of t h e spots was f a c i l i -t a t e d by u s i n g the f i l t e r as a s u r f a c e a g a i n s t which t o draw. Contact p r i n t s of p u r i n e chromatograms, made w i t h u n f i l t e r e d l i g h t from the g e r m i c i d a l lamp, were prepared r o u t i n e l y f o r r e c o r d purposes and to a i d i n c u t t i n g out the p u r i n e areas f o r r a d i o c a r b o n assay and spectrophotometry. F o r the d e t e c t i o n of u l t r a v i o l e t a b s o r b i n g areas, the photographic p r i n t was much more s e n s i t i v e t h a n the v i s u a l examination. Since p e n c i l marks were recorded c l e a r l y on such p r i n t s , p u r i n e areas t h a t had been o u t l i n e d i n p e n c i l were l o c a t e d and cut out w i t h p r e c i s i o n . With t h i s technique adenine and guanine i n amounts of 1 #, as chromatogram spots of o r d i n a r y s i z e ( 2 - 3 cm. ), were manipulated w i t h ease. U r i c a c i d spots were only f a i n t l y r ecorded w i t h t h i s technique because t h i s p u r i n e does not absorb s t r o n g l y i n the s p e c t r a l r e g i o n i n which the g e r m i c i d a l lamp has i t s p r i n c i p a l emmision. To c o n f i r m the i d e n t i t y of 3 A General E l e c t r i c 1$ watt g e r m i c i d a l lamp, which emits 90 per cent of i t s r a d i a t i o n at 253-.7 rou., was found t o be a s a t i s f a c t o r y u l t r a v i o l e t source f o r scanning and p r i n t i n g paper chromatograms. [[. C orning No. 9863 g l a s s f i l t e r 31* the f a i n t l y a b s o r b i n g u r i c a c i d s p o t s , samples of a u t h e n t i c u r i c a c i d were chromatographed on the same paper. The spots were l o c a t e d by c u t t i n g out the l a ne and s p r a y i n g i t w i t h ammoniacal s i l v e r n i t r a t e . The s t r i k i n g b l u e f l u o r e s c e n c e of guanine h y d r o c h l o r i d e i n the l i g h t from the g e r m i c i d a l lamp enabled t h i s p u r i n e to be d i s t i n g u i s h e d from others on the chromatogram. The f l u o r e s c e n t spot i s more d i f f i c u l t t o d e l i n e a t e than the dark spot obtained when the same amount of guanine i s present as the f r e e base. ( i i i ) C a p i l l a r y s i p h o n a p p l i c a t o r P r i o r t o p u r i f i c a t i o n by chromatography, adenine and guanine were obtained as a r e s i d u e l e f t by e v a p o r a t i o n o f the 6N HC1 e l u a t e i n the i o n exchange st e p of t h e i r i s o l a t i o n procedure. As t h e q u a n t i t a t i v e t r a n s f e r o f t h i s m a t e r i a l t o chromatograms proved to be a t e d i o u s o p e r a t i o n , an apparatus u t i l i z i n g c a p i l l a r y siphons was c o n s t r u c t e d which would a u t o m a t i c a l l y apply the p u r i n e s o l u t i o n s t o the chromatograms. The d e v i c e , which i s i l l u s t r a t e d i n F i g u r e 6 employed a j e t of warm a i r d i r e c t e d a g a i n s t the chromatograms, immediately below the p o i n t at which each s i p h o n was a p p l i e d . By c o n t r o l l i n g the flow o f a i r and I t s temperature, the p u r i n e s o l u t i o n was evaporated as f a s t as i t was a p p l i e d and the area of a p p l i c a -t i o n kept t o approximately 1 cm. i n diameter. An apparatus c o n s i s t i n g of f o u r of the a p p l i c a t o r u n i t s shown i n F i g u r e 6 was used i n t h i s i n v e s t i g a t i o n . I n use, the c a p i l l a r y siphons were f i r s t "primed" by f i l l i n g them w i t h water and were h e l d i n p o s i t i o n a g a i n s t the paper by 35 FIGURE 6. CAPILLARY SIPHON APPLICATOR FOR PREPARING CHROMATOGRAMS. The c a p i l l a r y siphons were made from heavy-walled pyrex t u b i n g (7 mm. O.D. x 2 mm. l.D) drawn out t o 0.5 mm. O.D. The s i p h o n h o l d e r was cut from heavy rubber sheet, li mm. t h i c k , and was s t a p l e d to a p i e c e of heavy s o l i d cardboard. T h i s u n i t was a t t a c h e d t o the ramp, as i l l u s t r a t e d , by a hinge of cellophane tape. The s u r f a c e of the ramp was p a i n t e d w i t h rubber-base paper adhesive t o prevent the sample beakers from s l i d i n g out of p o s i t i o n . The a i r j e t s were made from 7 nmu O.D. g l a s s t u b i n g w i t h a 2 mm. h o l e and were p o s i t i o n e d i n the apparatus w i t h the h o l e a l i g n e d d i r e c t l y below the p o i n t a t which the c a p i l l a r y s i p h o n was a p p l i e d t o the papergrams. A i r s u p p l i e d t o the j e t s was warmed by passage through a pyrex tube heated by a bunsen flame. A wire gauze p r o t e c t e d the p o r t i o n o f t h e tube a g a i n s t which the bunsen flame was d i r e c t e d . 36 the rubber f i n g e r s of the s i p h o n h o l d e r assembly. The sample t o be chromatographed was contained i n a 5 or 10 ml. beaker. With the a i r temperature between 1*0 and 5 0°C. the apparatus a p p l i e d aqueous s o l u t i o n s at approximately 2 ml. per hour. P i l l i n g and c l e a n i n g the siphons was g r e a t l y f a c i l i t a t e d by use of a p i e c e of s m a l l diameter p o l y e t h y l e n e t u b i n g , which, w i t h c a r e f u l h e a t i n g , was drawn out t o make a "neck" near one end, w i t h a diameter of l e s s than 0 . 5 mm. The siphons were i n s e r t e d i n t o the tube and g e n t l y f o r c e d i n t o the "neck", making a s e a l . By a p p l y i n g s u c t i o n t o the p l a s t i c tube, water was drawn i n t o the tube f o r f i l l i n g or c l e a n i n g . C. Spectrophotometry (I) U l t r a v i o l e t a b s o r p t i o n s p e c t r a The h i g h absorbancy i n the u l t r a v i o l e t r e g i o n which i s c h a r a c t e r i s t i c of the n u c l e i c a c i d bases and the development of the photo-e l e c t r i c spectrophotometer have p r o v i d e d the b a s i s f o r very s e n s i t i v e methods of a n a l y s i s f o r these compounds. The a b s o r p t i o n s p e c t r a , b e i n g c h a r a c t e r i s t i c f o r each base, are w i d e l y used t o i d e n t i f y p u r i n e s , p y r i m i d i n e s and t h e i r com-pounds. T h e i r o p t i c a l p r o p e r t i e s have been r e c e n t l y compiled i n a comprehensive review by Beavan, H o l i d a y and Johnson (81*). The Beckman Model DU q u a r t z spectrophotometer was used f o r the measurement of o p t i c a l d e n s i t i e s throughout t h i s work. The a b s o r p t i o n s p e c t r a of adenine, guanine and u r i c a c i d were determined i n 0.1N HC1 and are shown i n F i g u r e s 7, 8, and 9. The p u r i n e s used f o r t h i s purpose were commercial products and were p u r i f i e d i n the f o l l o w i n g manner: 37 Adenine Adenine ( N u t r i t i o n a l B iochemicals Corp.), shown t o be c h r o m a t o g r a p h i c a l l y pure, was twice c r y s t a l l i z e d as the s u l f a t e from water. The f r e e base was prepared from a s o l u t i o n of the s u l f a t e by the a d d i t i o n of excess sodium b i c a r b o n a t e . The p r e c i p i t a t e d adenine was washed w i t h water u n t i l the washings were f r e e of carbonate and s u l f a t e . Guanine Guanine ( N u t r i t i o n a l Biochemicals Corp.), known t o c o n t a i n adenine, was d i s s o l v e d i n d i l u t e HC1. Guanine wa3 s e l e c t i v e l y p r e c i p i t a t e d by a d j u s t i n g the a c i d i t y of t h i s s o l u t i o n w i t h sodium hydroxide u n t i l i t was a c i d t o l i t m u s yet a l k a l i n e t o congo r e d (85) and was t h e n r e c r y s t a l l i z e d t hree times as the s u l f a t e . The f r e e base was prepared from a s o l u t i o n of the s u l f a t e by the a d d i t i o n of excess ammonium hydroxide and the guanine p r e c i p i t a t e was washed u n t i l f r e e of s u l f a t e . U r i c a c i d U r i c a c i d (Kodak) was d i s s o l v e d i n warm concen-t r a t e d s u l f u r i c a c i d and re c o v e r e d from t h i s s o l u t i o n by d i l -u t i n g w i t h water. A f t e r r e p e a t i n g t h i s o p e r a t i o n , the u r i c a c i d was washed f r e e o f s u l f a t e and r e c r y s t a l l i z e d from water. P r i o r t o use, the p u r i f i e d p urines were d r i e d f o r 2 hours, i n vacuo, a t 100° C. over P2°5« Known amounts of the purines were d i s s o l v e d i n 0.1N HC1 and the o p t i c a l d e n s i t i e s of t h e s o l u t i o n s measured a t v a r i o u s wavelengths. Prom these data, molar e x t i n c t i o n c o e f f i c i e n t s were c a l c u l a t e d and are p l o t t e d i n F i g u r e s 7, 8 and 9» There i s c l o s e agreement between these curves and p u b l i s h e d data (81i,86). 1.2 2 0.8 l-u 0.6 0.2 230 FIGURE 7. i 250 270 290 W A V E L E N G T H ( M JJ ) ABSORPTION SPECTRUM OF ADENINE IN 0.1N HC1. . . CO 230 250 270 290 W A V E L E N G T H ( M JJ ) FIGURE 9. ABSORPTION SPECTRUM OF URIC ^ ACID IN 0.1N HC1. o I IP-( i i ) D e t e r m i n a t i o n of p u r i n e s The p u r i n e content os d i s c s punched from chromatograms was estimated s p e e t r o -p h o t o m e t r i c a l l y a f t e r the measurements of r a d i o a c t i v i t y . T h i s widely used method of a n a l y s i s ( 8 0 ) c o n s i s t s of e x t r a c t i n g the p u r i n e - c o n t a i n i n g d i s c of paper w i t h 0 . 1 N H C 1 and measuring the u l t r a v i o l e t a b s o r p t i o n o f the e x t r a c t at c e r t a i n a p p r o p r i -ate wavelengths. The paper d i s c was cut i n t o s m a l l p i e c e s , p l a c e d i n a t e s t tube w i t h I I . 0 ml. of 0 . 1 N H C 1 , allowed t o stand over-n i g h t a t room temperature, and then shaken f o r a t l e a s t one h a l f hour i n a mechanical shaker. The e x t r a c t i o n of p u r i n e was v i r t u a l l y q u a n t i t a t i v e by t h i s method, as t r i a l r e c o v e r y experiments showed r e c o v e r i e s of 91* - 1 0 3 per cent f o r adenine and 9^ - 1 0 5 per cent f o r guanine. F o r e s t i m a t i o n of the p u r i n e content of the e x t r a c t the d i f f e r e n c e was measured between the o p t i e a l d e n s i t i e s at c e r t a i n wavelengths which are i n d i c a t e d i n Tab l e I I . By a p p l y i n g the a p p r o p r i a t e molar e x t i n c t i o n v a l u e , € A , the purin e content of the e x t r a c t was c a l c u l a t e d . A l l o p t i c a l d e n s i t y measurements were made a g a i n s t blanks which c o n s i s t e d of e x t r a c t s o f d i s c s cut from blank lanes of the same chroma-togram. The use of e x t i n c t i o n s measured between the s p e c t r a l maximum and a r e f e r e n c e wavelength where the a b s o r p t i o n i s low (as opposed t o measurements made only a t the maximum) was intended t o compensate f o r the e r r o r i n t r o d u c e d by the presence of non-purine u l t r a v i o l e t - a b s o r b i n g i m p u r i t i e s which occur t o I ij.2 TABLE I I ULTRAVIOLET ABSORPTION DATA USED IN THE DETERMINATION OP PURINES Purine ^ M * R Adenine Guanine U r i c a c i d 2 6 2 . 5 mu 2l±8 " 28ii " 290 mu 300 mu 310 mu 1-25 x 10^ 1*02 1 1 • 1*13 " 1 . A M i s the wavelength of maximum o p t i c a l d e n s i t y . 2 . A R i s the reference wavelength, a r b i t r a r i l y chosen at a point where the e x t i n c t i o n i s low, yet d e f i n i t e . 3 . i s the d i f f e r e n c e between the molar e x t i n c t i o n c o e f f i c i e n t s measured at A J J a n d A ^ . v a r y i n g extents i n the f i l t e r paper e x t r a c t s . With t h i s method the d i f f e r e n c e i n e x t i n c t i o n between the two wavelengths Is a s p e c i f i c p r o p e r t y of the purines and i s u n a f f e c t e d by other a b s o r p t i o n I n t h i s r e g i o n p r o v i d i n g the l a t t e r Is equal a t b o t h wavelengths. I n p r e l i m i n a r y experiments w i t h e x t r a c t s of r e p l i c a t e adenine spots on chromatograms, t h e v a r i a t i o n between analyses based on £ ^ w a s reduced t o one h a l f of t h a t when (: 2 6 2 . 5 w a s used. I n a d d i t i o n , p u r i n e contents c a l c u l a t e d w i t h € 2 6 2 . 5 w e r e k- ~ % P e r cent h i g h e r t h a n those c a l c u l a t e d w i t h £ A . ( i i i ) Accuracy of p u r i n e a n a l y s i s The e r r o r i n h e r e n t i n t h i s a n a l y t i c a l method was estimated by comparing analyses of 10 r e p l i c a t e p u r i n e spots from the same chromato-gram. At the l e v e l of 2 , 2 S o f adenine per spot, the extreme 1*3 values d i f f e r e d from the mean by 3 and 5. per cent. W i t h guanine, a t the l e v e l of ^ . l S p e r spot, the analagous experiment i n d i c a t e d a l a r g e r v a r i a t i o n , the extremes of the analyses b e i n g 11 per cent from the means. I n a s i m i l a r experiment, a lower v a r i a t i o n was noted when ll}..l{.tfof adenine per spot was used, the extremes d i f f e r i n g from t h e mean of 17 analyses by l e s s t han 2 per cent. Thus, i t would appear that when t h i s method i s a p p l i e d t o f i l t e r paper d i s c s c o n t a i n i n g 2 - j? of p u r i n e , d u p l i c a t e analyses could be expected t o vary approximately 5 per cent from t h e i r mean i n the case of adenine and t o the l a r g e r extent of approximately 10 per cent w i t h guanine. D. R a d i o a c t i v i t y measurements ( i ) I n s t r u m e n t a t i o n C a r b o n 1 ^ r a d i o a c t i v i t y was assayed i n e i t h e r of two gas flow counters^ connected w i t h commercial s c a l i n g u n i t s 0 . The counters were operated i n the p r o p o r t i o n a l r e g i o n and used a gas mixture of argon and methane ( 9 0 : 1 0 ) . D i f f e r e n c e s between the two counting u n i t s and i n d i v i d u a l d a i l y v a r i a t i o n s i n counter and s c a l e r performance were compensated f o r by the use of a standard r a d i o c a r b o n source. Each count was c o r r e c t e d by a f a c t o r c o n s i s t i n g o f the counting r a t e o f the standard source at some a r b i t r a r i l y chosen time ( t h i s term i s constant) d i v i d e d by the cou n t i n g r a t e of t h e standard source at the time of the observed count,, ( 8 7 ) . 5 Counter Model C F I S - 2 , manufactured by Atomic Energy of Canada L i m i t e d . 6 Nuclear Instrument and Chemical C o r p o r a t i o n s c a l e r , Model l 6 3 ; T r a c e r l a b Incorporated, Model SC - 3 2 A m p l i s c a l e r . ( i i ) Methods of mounting samples Three types of sample mounting were employed i n C"^ assay, " i n f i n i t e l y - t h i n " samples mounted on aluminum p l a n c h e t s , samples contained i n f i l t e r paper d i s c s ( u s u a l l y cut from chromatograms), and " i n f i n i t e l y - t h i c k " samples of barium carbonate. Purine samples were r o u t i n e l y counted on f i l t e r paper d i s c s and carbonates were counted i n the form of barium carbonate. The only except-ions t o t h i s procedure are noted i n the t e x t , (a) " I n f i n i t e l y - t h i n " samples T h i s type of mounting was used i n s e v e r a l p r e l i m i n a r y experiments but was subsequently r e p l a c e d by the more convenient f i l t e r . p a p e r d i s c method. S o l u t i o n s of the r a d i o a c t i v e sample, c o n t a i n i n g s u b m i l l i g r a m amounts of m a t e r i a l , were p l a c e d i n f l a t aluminum p l a n c h e t s which were pr o v i d e d w i t h a f i n e r i n g of wax t o c o n f i n e the l i q u i d t o a d e f i n i t e a r e a . A s p r e a d i n g agent 100 A of a 1:1000 d i l u t i o n of T r i t o n X-100? (8) was used to d i s t r i b u t e the s o l u t i o n u n i f o r m l y over the wax-rimmed area and promoted the d e p o s i t i o n of the sample as a uniform l a y e r upon d r y i n g . An attempt t o r e l a t e a c t i v i t i e s of samples mounted i n t h i s f a s h i o n w i t h a c t i v i t i e s of i d e n t i c a l samples mounted on f i l t e r paper d i s c s i n d i c a t e d t h a t the t h i n - f i l m samples were s u b j e c t e d to a space charge e f f e c t . T h i s e f f e c t was manifested as an i n a b i l i t y t o get a c c e p t a b l e agreement between r e p l i c a t e counts on the same sample, the d i s c r e p a n c y u s u a l l y b e i n g i n the form of a d i m i n i s h i n g a c t i v i t y w i t h s u c c e s s i v e counts. The s c a t t e r 7 A product of the Rohm and Haas Company. 1*5 of the p o i n t s i n the upper curve i n F i g u r e 10 i l l u s t r a t e s the poor r e p r o d u c a b i l i t y w i t h t h i s type of sample mount when used i n the above mentioned counter. The departure from a l i n e a r r e l a t i o n s h i p between counting r a t e and amount of sample was i n t e r p r e t e d as b e i n g due to a space charge. I n the case of the f i l t e r paper-mounted samples, prepared from the same s o l -u t i o n of adenine-C 1^, the e x c e l l e n t l i n e a r i t y shown i n the lower curve of F i g u r e 10 I n d i c a t e s t h a t t h i s e f f e c t was absent. The a p p l i c a t i o n of c o l l o i d a l g r a p h i t e t o thin-sample p l a n c h e t s , as suggested'by R e i d and Robbins (88), may p o s s i b l y overcome t h i s d i f f i c u l t y . i (b) Samples mounted on f i l t e r paper d i s c s T h i s method of sample mounting has an obvious, a p p l i c a t i o n to the assay of r a d i o a c t i v e substances on chromatograms and was examined w i t h t h i s use i n mind. In the experiment i l l u s t r a t e d I n F i g u r e 10, e x c e l l e n t l i n e a r i t y was obtained between the observed r a d i o -a c t i v i t y and the amount of r a d i o a c t i v e m a t e r i a l p r e s e n t on the paper d i s c , i n d i c a t i n g the s u i t a b i l i t y of t h i s type of sample mounting f o r the assay of r a d i o c a r b o n . The d i f f e r e n c e i n the sl o p e s of the two curves i n F i g u r e 10 shows t h a t a b s o r p t i o n by the f i l t e r paper reduces the r a d i o a c t i v i t y by a f a c t o r of approximately 1*. T h i s f a c t o r c ould not be e v a l u a t e d a c c u r a t e l y from these data, but the t r e n d towards l i n e a r i t y i n the t h i n sample curve suggests t h a t i t would be constant i n the absence of the space charge e f f e c t . Experiments of the same type w i t h b i o l o g i c a l l y l a b e l l e d guanine gave s i m i l a r r e s u l t s . 1+6 FIGURE 10. A COMPARISON BETWEEN " INFINITELY-THIN" AND FILTER PAPER-MOUNTED SAMPLES OF ADENINE-C 1^. Samples o f l a b e l l e d adenine (obtained from i n t e s t i n a l mucosa which had been incubated i n v i t r o w i t h C ^-formate) were prepared as " i n f i n i t e l y - t h i n " samples mounted on aluminum p l a n c h e t s . D u p l i c a t e samples were p l a c e d on d i s c s o f Whatman no. 1 f i l t e r paper, 26 mm. i n diameter. D u r i n g <bounting, the paper d i s c s were mounted on aluminum p l a n c h e t s which were pro v i d e d w i t h tabs designed t o h o l d t h e d i s c by the edges. The same p r e p a r a t i o n o f adenine was used f o r a l l data shown. R e i d and Robbina (88) s t a t e t h a t a conducting s u r f a c e on the r a d i o a c t i v e sample improves c o u n t i n g reproduc-a b i l i t y i n the gas flow type of counter. F o r t h i s r e a s o n the apparent absence of the charge e f f e c t i n f i l t e r paper-mounted samples may be due t o c o n d u c t i v i t y of the paper, a t t r i b u t a b l e perhaps t o i t s water content®. Purine areas from chromatograms were r o u t i n e l y assayed f o r r a d i o a c t i v i t y i n the form o f paper d i s c s and unless otherwise mentioned, i t may be assumed t h a t t h i s procedure was f o l l o w e d . T h i s type of sample mounting has been employed by LePage (3) and H u r l b e r t ( 8 ) . A s i m i l a r type of sample mounting technique has been d e s c r i b e d r e c e n t l y ( 8 9 , 9 0 ) i n which l e n s paper was used t o f a c i l i t a t e uniform d i s t r i b u t i o n o f the sample on the p l a n c h e t . To prevent a p o s s i b l e l o s s of a c t i v i t y through v o l a t i l i t y caused by a c i d vapours i n the l a b o r a t o r y a i r , sodium f o r m a t e - C ^ samples were assayed on paper d i s c s punched from f i l t e r paper t h a t had been p r e v i o u s l y sprayed w i t h 0.5 M K 2HP0^ ( 6 9 ) . (c) I n f i n i t e l y - t h i c k barium carbonate samples T h i s method of sample mounting was used f o r r a d i o c a r b o n assay of the b i c a r b o n a t e s u b s t r a t e , carbonate trapped i n the centre w e l l s of Warburg v e s s e l s and carbonate produced from formate by o x i d a t i o n w i t h m e r c u r i c s u l f a t e ( 9 1 ) . ^b.e two techniques d e s c r i b e d p r e v i o u s l y cannot be used f o r carbonates s i n c e they 8 P o l g l a s e , W.J., p e r s o n a l communication. undergo a r a p i d exchange w i t h the carbon d i o x i d e of the a i r ( 8 7 ). T h e carbonate sample f o r assay was p r e c i p i t a t e d as barium carbonate and prepared f o r counting by a m o d i f i c a t i o n of the procedure p r e v i o u s l y f o l l o w e d i n t h i s l a b o r a t o r y ( 9 2 , 9 3 ) which c o n s i s t s e s s e n t i a l l y i n c o l l e c t i n g the sample by f i l t r a -t i o n i n p a p e r - l i n e d , brass f i l t e r i n g d i s h e s . I n the m o d i f i c a -t i o n employed here, the barium carbonate samples were c o l l e c t e d on s i n g l e d i s c s of f i l t e r paper p l a c e d on the p e r f o r a t e d bottom of the brass d i s h e s . T h i s e s t a b l i s h e d d i r e c t contact of the carbonate sample w i t h the brass d i s h (the l i n i n g of f i l t e r paper used p r e v i o u s l y prevented t h i s ) . T h i s m o d i f i c a t i o n and the use of f i l t e r paper d i s c s which had been p r e v i o u s l y o impregnated w i t h c o l l o i d a l g r a p h i t e 7 improved the r e p r o d u c t -i b i l i t y of counting r a t e s when compared w i t h samples prepared by the e a r l i e r t e c h n i q u e 1 ^ . ( i i i ) S p e c i f i c a c t i v i t y 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 counts per minute per m i l l i g r a m of sample (c.p.m. per mg.). ( i v ) E s t i m a t i o n of e r r o r s The s t a t i s t i c a l e r r o r s i n h e r e n t i n the c o u n t i n g procedure were estimated w i t h the f o l l o w i n g formula f o r the percentage e r r o r of a net a c t i v i t y ( 8 7 ) : TC = 100 K V (Nh + N a ) N S + N b 9 F i l t e r paper d i s c s were dipped i n a 1 : 2 £ d i l u t i o n of concentrated Aquadag (Acheson C o l l o i d s Company, Port Huron, Michigan, U.S.A.) and d r i e d , w i t h the process b e i n g repeated u n t i l they had a uniform g r e y - b l a c k appearance. Two d i p s were u s u a l l y r e q u i r e d . 10 S. H. Zbarsky, unpublished i n f o r m a t i o n . 1*9 where, E = e r r o r of net a c t i v i t y N-^  s background counts N s = t o t a l o f background and sample counts K = p r o b a b i l i t y constant (K = 1, standard e r r o r , was used i n these experiments). F o r use i n t h i s formula, background and the unknown a c t i v i t y must be counted f o r equal p e r i o d s . Unless otherwise noted counting e r r o r s are l e s s than $ per cent. E. Respirometry Oxygen consumption was measured by the c o n v e n t i o n a l Warburg technique (9l+) and r e s u l t s are expressed i n terms of Q02 ( m i c r o l i t r e s of oxygen consumed per mg. of t i s s u e , dry weight per h o u r ) . 2. THE PREPARATION OF SUSPENSIONS OF INTESTINAL MUCOSA The i n v i t r o experiments of Dickens and Weil-Malherbe (69) w i t h s t r i p s of i n t e s t i n a l mucosa showed t h a t the m e t a b o l i c a c t i v i t y of t h i s t i s s u e decreases p r o g r e s s i v e l y a l o n g the •length of the s m a l l i n t e s t i n e . Consequently, i n s t u d i e s of the metabolism of mucosa i n v i t r o t h a t r e q u i r e comparisons between r e p l i c a t e samples of t h i s t i s s u e , i t becomes e s s e n t i a l t h a t some way be found t o make these samples i d e n t i c a l . I t was found t h a t r e l a t i v e l y uniform, w h o l e ^ c e l l suspensions of mucosa could be obtained by g e n t l y s y r i n g i n g a s u s p e n s i o n of mucosal s t r i p s . Of course, homogenates w i l l p r o v i d e completely i d e n t i c a l aliquots-, but such p r e p a r a t i o n s were found t o be i n a c t i v e i n r e s p e c t t o p u r i n e s y n t h e s i s . 5 0 I n o b t a i n i n g the s t r i p s of i n t e s t i n a l mucosa, the technique suggested by Dickens and Weil-Malherbe (69) was f o l l o w e d . Male W i s t e r r a t s were k i l l e d by a blow on the head and d e c a p i t a t e d . The s m a l l i n t e s t i n e was removed immediately, c h i l l e d and cut i n t o 10 cm. segments. These were f l u s h e d f r e e of contents w i t h the Krebs-Ringer medium d e s c r i b e d below. The segments were next s p l i t open and a p p l i e d t o a c h i l l e d g l a s s s l a b w i t h the mucosa s u r f a c e upwards. The s p l i t t i n g d e v i c e i l l u s t r a t e d In F i g u r e 11 g r e a t l y f a c i l i t a t e d t h i s step, s p l i t t i n g the segment and a p p l y i n g i t to. the p l a t e i n one o p e r a t i o n . FIGURE 11. INTESTINE SPLITTER BARD PARKER No.l I SCALPEL BLADE GUIDE PIECE,CARVED | FROM POLYETHYLENE , SHEET ,2MM THICK •While p i n n i n g the m u s c u l a r i s down w i t h a microscope s l i d e , the mucosa was s t r i p p e d o f f w i t h another s l i d e , u s i n g a s c r a p i n g motion. I f the s t r i p p i n g s l i d e had a s t r a i g h t , sharp edge, such as t h a t obtained on a f r e s h l y broken s l i d e , the mucosa was e a s i l y removed as a continuous s t r i p , l e a v i n g the m u s c u l a r i s a p p l i e d t o the p l a t e . 5 1 The s t r i p s o f mucosa were p l a c e d i n 10-15 ml. of i c e - c o l d medium and f r e e d by g e n t l y mixing w i t h a s t i r r i n g r o d . The t i s s u e was washed s e v e r a l times by c e n t r i f u g i n g the p r e p a r a t i o n at low speed, f o l l o w e d by r e s u s p e n s i o n i n f r e s h medium. The s t r i p s were then fragmented by g e n t l y and r e p e a t e d l y drawing t h e suspension i n t o , and e x p e l l i n g i t from, a 10 ml. s y r i n g e . The r e s u l t i n g suspension was t h e n s t r a i n e d through one t h i c k n e s s of gauze, the t i s s u 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 and resuspended i n the c o l d medium, making the f i n a l volume 25 ml. With t h i s procedure the s m a l l i n t e s t i n e of a 200 g. r a t p r o v i d e d a s u s p e n s i o n c o n t a i n i n g approximately 10 mg. dry weight of t i s s u e per ml. 3 . THE PURINES OP THE INTESTINAL MUCOSA SUSPENSION I n p r e l i m i n a r y experiments the p u r i n e composition of the mucosa suspension was examined by the i o n exchange procedure d e s c r i b e d above. The t o t a l p u r i n e content of the suspension was i s o l a t e d by means of the hot p e r c h l o r i c a c i d e x t r a c t i o n method of S c h e i d e r ( 9 5 ) , f o l l o w e d by p r e c i p i t a t i o n as s i l v e r s a l t s . Procedure A suspension of r a t i n t e s t i n a l mucosa was incubated at 3 7 ° C. i n Krebs-Ringer b i c a r b o n a t e medium (9l|.) f o r 2 . 5 hours, at which time i t was made 1* per cent i n p e r c h l o r i c a c i d and heated at 90° C. f o r 30 minutes ( 9 5 )• T h e t i s s u e r e s i d u e was separated from the e x t r a c t by c e n t r i f u g a t i o n and washed w i t h 2 per cent p e r c h l o r i c a c i d . The combined e x t r a c t and washings were n e u t r a l i z e d w i t h ammonium hydroxide, t r e a t e d w i t h an excess of magnesia mixture (96) and allowed to stand i n the r e f r i g e r a t o r o v e r n i g h t . The p r e c i p i t a t e was removed by 5 2 f i l t r a t i o n , washed w i t h d i l u t e ammonium hydroxide, and the combined f i l t r a t e and washings were t r e a t e d w i t h an excess o f the ammoniacal s i l v e r n i t r a t e reagent o f Graves and Kober (97)» A f t e r s t a n d i n g o v e r n i g h t i n t h e r e f r i g e r a t o r , the g e l a t i n o u s p r e c i p i t a t e o f the s i l v e r p u rines was rec o v e r e d by c e n t r i f u -g a t i o n and washed twice w i t h c o l d , d i l u t e ammonium h y d r o x i d e . The s i l v e r p u r i n e p r e c i p i t a t e was then decomposed by h e a t i n g w i t h 0 . 5 M HC1 and, a f t e r c o o l i n g , the p r e c i p i t a t e o f s i l v e r c h l o r i d e was removed by c e n t r i f u g a t i o n . The puri n e s o l u t i o n was d i l u t e d w i t h l i p a r t s of water and chromatographed by the i o n exchange procedure d e s c r i b e d above. The only purines found i n t h i s e x t r a c t were u r i c a c i d , guanine and adenine. They were i d e n t i f i e d by t h e i r p o s i t i o n i n the e l u t i o n sequence, t h e i r u l t r a v i o l e t a b s o r p t i o n s p e c t r a and by paper chromato-graphy i n t e r t i a r y - b u t a n o l - H C l ( 7 8 ) . I n a l l subsequent experiments, these t h r e e were the only p u r i n e s i d e n t i f i e d . I I . USE OP THE MUCOSAL SUSPENSION A. Media Krebs-Ringer carbonate or phosphate s o l u t i o n s (9i+), c o n t a i n i n g glucose {0.2%) and aureomycin (10 p a r t s per m i l l i o n ) , were used as the suspending media. The i n c l u s i o n o f aureomycin i n t he medium was intended t o minimize b a c t e r i a l growth and i s d i s c u s s e d i n subsequent s e c t i o n s . I n the phosphate-buffered s a l i n e , prepared In the d e s i g n a t e d manner (9lj.), c a l c i u m pre-c i p i t a t e d t o v a r y i n g degrees and f o r t h i s r e a s o n i t s concen-t r a t i o n was reduced t o one h a l f of the recommended v a l u e . E l l i o t mentions s i m i l a r d i f f i c u l t i e s w i t h phosphate-buffered 53 media and omits c a l c i u m e n t i r e l y ( 9 8 ) . The b i c a r b o n a t e b u f f e r appeared to be the most s a t i s f a c t o r y w i t h t h i s p a r t i c u l a r t i s s u e , g i v i n g b e t t e r pH c o n t r o l d u r i n g i n c u b a t i o n than the phosphate b u f f e r . The f a l l I n pH d u r i n g i n c u b a t i o n (post i n c u b a t i o n v a l u e s of pH 7 and s l i g h t l y below were observed) c o u l d be minimized i n f u t u r e work w i t h t h i s system by u s i n g media w i t h i n i t i a l pH values of 7 . 6 - 7 . 8 (98) and by u s i n g more d i l u t e suspensions of mucosa. T h i s l a t t e r course would have t o be f o l l o w e d by the combination of s e v e r a l r e p l i c a t e f l a s k s (or by use of l a r g e r f l a s k s ) to p r o v i d e s u f f i c i e n t l y l a r g e amounts of a c i d - s o l u b l e purines f o r i s o l a t i o n and a n a l y s i s . B. Procedure f o r a t y p i c a l experiment ( i ) I n c u b a t i o n I n a t y p i c a l experiment, 3«0 ml. p o r t i o n s of the mucosal sus p e n s i o n were added t o Warburg f l a s k s which contained t h e s u b s t r a t e s under t e s t . The p r e p a r a t i o n was then incubated i n the Warburg r e s p i r o m e t e r at 3 7 ° C. f o r 3 hours, except i n the time study experiments. When p o s s i b l e , the r e s p i r a t i o n of the t i s s u e was f o l l o w e d d u r i n g the i n c u b a t i o n . ( i i ) I s o l a t i o n and a n a l y s i s of p u r i n e s F o r the i s o l a t i o n of the p u r i n e s the suspending medium was r e c o v e r e d by c e n t r i f u g i n g the f l a s k contents and s t o r e d i n the f r o z e n s t a t e f o r subsequent examination. To i s o l a t e the a c i d - s o l u b l e n u c l e o t i d e f r a c t i o n by the method of LePage ( 3 ) , the t i s s u e was f i r s t d i s p e r e e d i n 3«0 ml. of c o l d 2 per cent p e r c h l o r i c a c i d and kept i n i c e f o r 15 - 20 minutes. The t i s s u e r e s i d u e Sk was recovered by c e n t r i f u g a t i o n and the c o l d e x t r a c t i o n was repeated twice w i t h 2 . 0 ml. p o r t i o n s of 2 per cent p e r c h l o r i c a c i d . The combined c o l d p e r c h l o r i c e x t r a c t s were c a l l e d the a c i d - s o l u b l e (AS) f r a c t i o n . The t i s s u e r e s i d u e was e x t r a c t e d w i t h 3«0 ml. of ij. per cent p e r c h l o r i c a c i d f o r 30 minutes a t 90° C. A f t e r c o o l i n g , the r e s i d u e was spun down and washed twice w i t h 2 ml. p o r t i o n s o f 2 per cent p e r c h l o r i c a c i d . The combined e x t r a c t and washings contained the n u c l e i c a c i d (NA) p u r i n e s , l i b e r -ated by h y d r o l y s i s from the p o l y n u c l e o t i d e s . The AS f r a c t i o n e x t r a c t s were a l s o heated a t 90° f o r 30 minutes t o l i b e r a t e the purines from the n u c l e o t i d e s . The purines were i s o l a t e d from the p e r c h l o r i c a c i d e x t r a c t s on the s m a l l Dowex-50 columns d e s c r i b e d p r e v i o u s l y . Adenine and guanine were contained i n the 6N HC1 e l u a t e s which were evaporated t o dryness a t reduced p r e s s u r e over s u l f u r i c a c i d and sodium hy d r o x i d e . The p u r i n e - c o n t a i n i n g r e s i d u e s were taken up i n approximately 0 . 5 ml. of water and p l a c e d on chromatograms w i t h the a p p l i c a t o r d e s c r i b e d e a r l i e r . Chromatograms were r u n f i r s t i n Wyatt's i s o - p r o p a n o l - H C l s o l v e n t ( 7 9 ) . I t was e s s e n t i a l t o dry thoroughly such chromatograms p r i o r t o r a d i o c a r b o n assay as t r a c e s o f t h i s s o l v e n t remaining on the f i l t e r paper d i s c s caused the appearance of s p u r i o u s counts. Adequate d r y i n g was achieved by hanging the papers i n a c u r r e n t o f a i r (such as produced i n a powerful fume c l o s e t ) f o r a t l e a s t 15 hours. 5 5 Purine areas on the chromatograms were l o c a t e d by the combined v i s u a l scanning and contact p r i n t i n g methods w i t h u l t r a v i o l e t l i g h t , and t h e n punched out as d i s c s , 26 mm. i n diameter. The d i s c s were assayed f o r carbon^-, and t h e i r p u r i n e contents determined s p e c t r o p h o t o m e t r i c a l l y . Prom these d a t a s p e c i f i c a c t i v i t i e s were c a l c u l a t e d . I n p r e l i m i n a r y experiments, the 0.1N HC1 e x t r a c t s from the s p e c t r o p h o t o m e t r i c a n a l y s i s were evaporated to dryness, rechromatographed i n the same or other s o l v e n t s and the counting, and p u r i n e a n a l y s i s repeated. I n subsequent experiments, s p e c i f i c a c t i v i t i e s were measured only on the rechromatographed p u r i n e s . The purines contained i n the medium o f the mucosa suspension were i s o l a t e d by making the medium 2 per cent i n p e r c h l o r i c a c i d and proceeding as i n the case of the AS f r a c -t i o n . U r i c a c i d was i s o l a t e d from the combined i n i t i a l e f f l u e n t s from the Dowex-50 columns of the AS and the medium f r a c t i o n s by u s i n g the ammoniacal s i l v e r n i t r a t e method d e s c r i b e d above. When f r e e of s i l v e r c h l o r i d e , the u r i c a c i d s o l u t i o n s were d i l u t e d t o 10 ml. and the u l t r a v i o l e t a b s o r p t i o n spectrum determined. Very c l o s e agreement w i t h the spectrum of pure u r i c a c i d was shown. A l i q u o t s of the u r i c a c i d s o l u -t i o n were evaporated f o r t r a n s f e r t o chromatograms and f o r mounting as i n f i n i t e l y - t h i n samples f o r C^" assay. Chromato-graphy w i t h b u t a n o l - a c e t i c a c i d s o l v e n t (82) d i d not a l t e r the s p e c i f i c a c t i v i t y of the u r i c a c i d . 56 ( i i i ) A n a l y s i s of centre w e l l c o n t e n t s 1 1 O x i d a t i o n of the i s o t o p i c s u b s t r a t e s was measured by the d e t e r m i n a t i o n of c a r b o n a t e - C 1 ^ trapped i n the centre w e l l of the Warburg cups. T h i s was done only when phosphate b u f f e r s were used as the suspending medium. The a l k a l i from the centre w e l l s was t r a n s f e r r e d t o v o l u m e t r i c f l a s k s w i t h 10 - 12 washings. The f l a s k s were made up t o volume and s u i t a b l e p o r t i o n s removed f o r r a d i o c a r b o n a n a l y s i s by the thick-sample method. C a r r i e r carbonate was added t o each sample b e f o r e p r e c i p i t a t i o n as barium carbonate. 5 . IS0T0PICALLY LABELLED SUBSTRATES A. Sodium Formate-C 1^ R a d i o a c t i v e sodium formate was obtained from Atomic Energy o f Canada L i m i t e d . Chromatography by the method of Kennedy and Barker (99) and radioautography showed the prep-a r a t i o n had a l l of i t s r a d i o a c t i v i t y present as formate. The r a d i o a c t i v i t y of formate was measured w i t h the sample mounted on f i l t e r paper t o permit comparison w i t h p u r i n e a c t i v i t i e s which were measured i n a s i m i l a r f a s h i o n . The s p e c i f i c a c t i v i t y of formate was 1 .59 x 1 0 ° c.p.m. p e r j j M and was used r o u t i n e l y i n 2 . 2 5 JJM p o r t i o n s per Warburg f l a s k . To permit comparison w i t h the r a d i o a c t i v i t y o f carbon d i o x i d e d e r i v e d from the b i o l o g i c a l o x i d a t i o n of formate-C 1^, The s p e c i f i c a c t i v i t y o f the s t o c k formate was determined a f t e r o x i d a t i o n t o carbon d i o x i d e by the m e r c u r i c s u l f a t e method ( 9 1 ) • 11 These assays were very k i n d l y performed by Dr. S. H. Zbarsky. 57 The s p e c i f i c a c t i v i t y , measured on " i n f i n i t e l y - t h i c k " samples was found t o be 0.32 x 10° c.p.m. perjaM. B. G l y c l n e - l - C 1 ^ C a r b o x y l - l a b e l l e d g l y c i n e was prepared by the r e a c t i o n of i s o t o p i c cyanide w i t h N-chlormethylphthalimide (100). The i s o t o p i c cyanide f o r t h i s r e a c t i o n was synthes-i z e d from barium carbonate- C ^ by the method of McCarter (101) as m o d i f i e d by Jeanes (102). The l a b e l l e d g l y c i n e produced i n t h i s way shown t o be chromat©graphically pure. Radioauto-graphs prepared from the chromatograms showed only one r a d i o -a c t i v e spot which c o i n c i d e d e x a c t l y w i t h the n i n h y d r i n s p o t . P r e p a r a t i o n A. Counted i n the form of " i n f i n i t e l y - t h i n " samples, t h i s p r e p a r a t i o n had a s p e c i f i c a c t i v i t y o f 2.81* x 10^ c.p.m. per ja M. P r e p a r a t i o n B. Samples were mounted on f i l t e r paper and i n d i c a t e d a s p e c i f i c a c t i v i t y of 2.61* x 10^ c.p.m. perjuM. C. Sodium B i c a r b o n a t e - C 1 ^ Sodium b i c a r b o n a t e - C 1 ^ was prepared by t r a p p i n g C 1^-carbon d i o x i d e , generated from barium carbonate-C i n the s t o i c h i o m e t r i c amount of sodium h y d r o x i d e . The r e a c t i o n was conducted i n the apparatus I l l u s t r a t e d i n F i g u r e 12. FIGURE 12. APPARATUS FOR THE PREPARATION OF SODIUM BICARBONATE-C 1^ 2 4 / 4 0 F J O I N T ALKALI T U B E B U L B FOR H 3 P 0 4 14 BAC 0 3 58 Procedure. I s o t o p i c barium carbonate was p l a c e d i n the tube h a v i n g the s i d e bulb,which contained an excess o f syrupy phosphoric a c i d . A volume of sodium hydroxide, e x a c t l y e q u i v a l e n t t o the number o f moles of barium carbonate present, was pl a c e d In the other tube and f r o z e n w i t h a Dry Icer-alcohol mixture. The apparatus was then evacuated t o approximately 1 mm. of mercury and the phosphoric a c i d t i p p e d onto the carbonate v e r y c a u t i o u s l y . A f t e r the r e a c t i o n was complete, the apparatus was allowed to stand a t room temperature f o r a t l e a s t 12 hours t o a l l o w complete a b s o r p t i o n of carbon d i o x i d e . I n p r e l i m i n a r y experiments almost q u a n t i t a t i v e y i e l d s of b i c a r b o n a t e were o b t a i n e d . B i c a r b o n a t e h a v i n g a s p e c i f i c a c t i v i t y of 1.12 x 10° c.p.m. peryj.M was prepared i n t h i s manner. 6. N0N-IS0T0PIC SUBSTRATES A. Glycinamide h y d r o c h l o r i d e The ammonolysis of g l y c i n e e t h y l e s t e r h y d r o c h l o r i d e was used t o prepare glycinamide h y d r o c h l o r i d e ( 1 0 3 ) . ^he product was r e c r y s t a l l i z e d from a methanol s o l u t i o n t o which ether was added u n t i l c r y s t a l l i z a t i o n was i n c i p i e n t . On paper chromatograms the product showed a s i n g l e y e l l o w spot w i t h n i n h y d r i n reagent. B. lL-amlno - 5-lmidazolecarboxamidine d i h y d r o c h l o r i d e T h i s compound was s y n t h e s i z e d by the method of Shaw (82) and was r e c r y s t a l l i z e d t w ice from methanol-ether, as above. The i d e n t i t y of the product was e s t a b l i s h e d by m e l t i n g p o i n t (2l|.2-2l|iL° C., (lOli) ) and by t h e f a c t t h a t i t was converted to adenine by f o r m y l a t i o n and c y c l i z a t i o n ( 1 0 5 ) . 59 RESULTS 1. CHARACTERISTICS OF THE INTESTINAL MUCOSA SUSPENSION A. Physical c h a r a c t e r i s t i c s The mucosal suspensions consist of small fragments of e p i t h e l i a l tissue ranging i n siz e from clumps of a few c e l l s to sheets of c e l l s i i - 6 mm. i n area. Low-power 12 photomicrographs of a t y p i c a l preparation are shown i n Figures 13 and l l i . D i f f u s i o n of gases to the deepest l y i n g c e l l s presents no problem i n these preparations since the largest fragments are thinner than the l i m i t i n g thickness of 0.5 mm. (69,98). 12 The photomicrographs were very kindly prepared by Dr. H. E..Taylor of the Department of Pathology. 6o PHOTOMICROGRAPHS OF A TYPICAL PREPARATION OF RAT INTESTINAL MUCOSA FIGURE 1 3 FIGURE II L B. R e s p i r a t o r y a c t i v i t y The mucosa suspensions r e s p i r e d a c t i v e l y f o r 3 - l | hours at gradually d e c l i n i n g r a t e s . R e s p i r a t o r y quotients ( Q 0 2 ^ » which averaged approximately 7 (values of 1 1 and 1 2 were a l s o recorded), were comparable w i t h the range of values Dickens and Weil-Malherbe (69) reported f o r mucosa from var-ious regions of the smal i n t e s t i n e . Aureomycin ( 1 0 p.p.m.) had no measurable e f f e c t on oxygen uptake i n these preparations. C. B a c t e r i a l contamination B a c t e r i a l contamination was small i n these prepar-a t i o n s . I n p r e l i m i n a r y experiments, i n which the t i s s u e 61 suspension was not washed, b a c t e r i a l countsl3 d i d not exceed 13 x 10 per ml. a t t h e end o f the i n c u b a t i o n p e r i o d . The suspension was r o u t i n e l y washed s e v e r a l times I n a l l subse-quent experiments. The b a c t e r i a l counts d i d not exceed 2 x 101 b a c t e r i a per ml. i n the washed p r e p a r a t i o n s . The b a c t e r i a l counts v a r i e d g r e a t l y below these l i m i t s and were g e n e r a l l y lowered by the a d d i t i o n of 10 p.p.m. of aureomycin t o the medium. Although probably unnecessary, aureomycin was r o u t i n e l y used i n the medium as a safeguard a g a i n s t b a c t e r i a l growth d u r i n g i n c u b a t i o n . The b a c t e r i a l contamination was co n s i d e r e d t o exert no s i g n i f i c a n t i n f l u e n c e on the metabolism of the p r e p a r a t i o n s i n view of the f a c t t h a t homogenates o f t h i s t i s s u e were u n i f o r m l y i n a c t i v e w i t h r e s p e c t t o the uptake of formate i n t o the p u r i n e s . The f a c t t h a t oxygen consumption decreased w i t h time supports t h i s c o n c l u s i o n . D. The purines of the mucosa sus p e n s i o n ( i ) Whole sus p e n s i o n A n a l y s i s of the t o t a l p u r i n e content of the incubated mucosal p r e p a r a t i o n by the i o n exchange procedure o u t l i n e d above showed t h a t only adenine, guanine, and u r i c a c i d were present i n demonstrable amounts. ( I i ) Medium The suspending medium, when r e c o v e r e d a f t e r i n c u b a t i o n , was found to be r i c h i n u r i c a c i d . Adenine and guanine were present a l s o and were presumed t o o r i g i n a t e from the l y s i s of some of the mucosal c e l l s d u r i n g i n c u b a t i o n . 13 Miss A i k o H o r i very k i n d l y performed the b a c t e r i a l counts. 62 The p y r i m i d i n e c y t o s i n e was found i n some experiments. ( i i i ) A c i d - s o l u b l e f r a c t i o n The a c i d - s o l u b l e f r a c t i o n o f t i s s u e s contains a complex mixture o f n u c l e o t i d e s of the pu r i n e and p y r i m i d i n e bases (1 ,2). Adenine and guanine were r e l e a s e d from t h e i r n u c l e o t i d e s by the h y d r o l y t i c t r e a t -ment mentioned p r e v i o u s l y . ( i v ) P o l y n u c l e o t i d e f r a c t i o n Adenine and guanine were l i b e r a t e d from DNA and RNA by the hot p e r c h l o r i c a c i d e x t r a c t i o n of the t i s s u e r e s i d u e . E. The o x i d a t i o n o f g l y c i n e - l - C 1 ^ and formate-C 1^-A n a l y s i s o f the contents of the centre w e l l s o f the r e s p i r o m e t e r f l a s k s showed t h a t the mucosal s u s p e n s i o n o x i d i z e d formate r e a d i l y . I n F i g u r e 15 the r a d i o a c t i v i t y r e c o v e r e d as carbonate from the centre w e l l s i s expressed as a percentage of the t o t a l r a d i o a c t i v i t y o r i g i n a l l y present as formate and Is p l o t t e d a g a i n s t time of i n c u b a t i o n . FIGURE 15. THE OXIDATION OF C"^-FORMATE TO C ^ p BY A SUSPENSION OF INTESTINAL MUCOSA hJ 63 Nakada and Weinhouse, who have s t u d i e d the o x i d a t i o n of formate i n i n d i v i d u a l t i s s u e s of the r a t ( 1 0 6 ) , r e p o r t t h a t homogenates of l i v e r and kidney were the most a c t i v e of the t i s s u e s t e s t e d . As whole c e l l p r e p a r a t i o n s were not employed i n t h e i r study and i n t e s t i n e was not examined, comparisons w i t h the present r e s u l t s can be only approximate. As may be seen i n F i g u r e If?, a f t e r 1 hour i n c u b a t i o n approximately 20 per cent of the formate s u b s t r a t e was o x i d i z e d , g i v i n g a c o n v e r s i o n c a p a c i t y ( " C C " ; microatoms of s u b s t r a t e converted t o product per hour per gram of dry t i s s u e (106) of approximately If?. Nakada and Weinhouse r e p o r t C C . v a l u e s f o r l i v e r and kidney homogenates of I4.8 and 23 r e s p e c t i v e l y . TABLE I I I THE OXIDATION OF FORMATE-C 1^ TO CARBON DIOXIDE-C 1^ Expt. Number of cen t r e w e l l s analysed %2 Average per-centage of. formate-C 1'+ a c t i v i t y i n centre w e l l 1 6 ca. 1 3 0 . 8 2 2 3 31.1* 3 5 6 1 8 . 7 S u b s t r a t e : 2.2f? jam sodium f o r m a t e - C 1 ^ ( 0 . 7 2 x 10 c.p.m.) per f l a s k . 61* I t would appear from the data of Table I I I t h a t the a b i l i t y of the mucosal su s p e n s i o n t o o x i d i z e formate does not depend on i t s r e s p i r a t o r y a c t i v i t y . The r e s p i r a t i o n o f the t i s s u e p r e p a r a t i o n was suboptimal i n the f i r s t two experiments c i t e d i n Table I I I , y e t the o x i d a t i o n o f formate was twice t h a t observed when r e s p i r a t i o n o c c u r r e d a t a h i g h e r r a t e o f Experiment 3« Nakada and Weinhouse (106) propose t h a t formate o x i d a t i o n occurs through a p e r o x i d a t i c a c t i v i t y o f c a t a l a s e . They f u r t h e r suggest t h a t the hydrogen peroxide necessary f o r t h i s r e a c t i o n may o r i g i n a t e i n the o x i d a t i o n of pu r i n e s by xanthine dehydrogenase. The present r e s u l t s may be e x p l a i n e d on t h i s b a s i s . As an adequate energy supply has been shown to be necessary f o r the maintenance of n u c l e i c a c i d s t r u c t u r e ( 1 0 7 ) , the low r e s p i r a t i o n r a t e s probably I n d i c a t e some break-down of the t i s s u e system. A g r e a t e r a v a i l a b i l i t y of p u r i n e s might t h e r e f o r e be expected i n the p o o r l y r e s p i r i n g p r e p a r a -t i o n s , which then could g i v e r i s e t o i n c r e a s e d amounts of hydrogen peroxide through the a c t i o n o f xanthine dehydrogenase. I n t e s t i n e has a h i g h xanthine dehydrogenase a c t i v i t y ( 7 3 ) • G l y c i n e - l - C 1 ^ " was o x i d i z e d t o only a very s l i g h t extent (approximately 0,2%) under these c o n d i t i o n s . 2 . PURINE SYNTHESIS IN MUCOSA SUSPENSIONS Purine s y n t h e s i s was s t u d i e d by i n c u b a t i n g the suspensions w i t h the p u r i n e p r e c u r s o r s , formate, g l y c i n e , and bi c a r b o n a t e , each l a b e l l e d w i t h r a d i o c a r b o n . An observed i n c o r p o r a t i o n o f i s o t o p e from these compounds by the pu r i n e s 65 of the mucosa c e l l s was considered t o i n d i c a t e t h a t p u r i n e s y n t h e s i s was t a k i n g p l a c e by the s o - c a l l e d "de novo pathway". The purines of the p r e p a r a t i o n were found t o be r a d i o a c t i v e a f t e r i n c u b a t i o n w i t h formate-C 1^", but b e f o r e s i g n i f i c a n c e could be a t t a c h e d t o t h i s o b s e r v a t i o n , I t was e s s e n t i a l t o e s t a b l i s h t h e i r r a d i o c h e m i c a l p u r i t y . T h i s was done by comparing the s p e c i f i c a c t i v i t i e s of the i s o l a t e d purines b e f o r e and a f t e r rechromatography i n d i f f e r e n t s o l -v e n t s . The s p e c i f i c a c t i v i t i e s of the NA purines were un-a l t e r e d by rechromatography, w h i l e those of AS adenine were Increased.. I n t h i s way the C1^" content of the i s o l a t e d p u r i n e s was shown t o be r e a l and not due t o accompanying contaminants. T h i s evidence i n d i c a t e d t h a t de novo p u r i n e s y n t h e s i s was o c c u r r i n g , but d i d not exclude the p o s s i b i l i t y of the i n c o r p o r -a t i o n o f C ^ - f o r m a t e by known exchange r e a c t i o n s . The l a t t e r process was shown to be of minor importance by a comparison of purine s y n t h e s i s from glycine-1-C 1^" and formate-C 1^". A. Radiochemical p u r i t y A comparison was made of the s p e c i f i c a c t i v i t i e s of adenine and guanine of b o t h AS and NA f r a c t i o n s , b e f o r e and a f t e r a second chromatographic p u r i f i c a t i o n as shown i n Table IV. AS Purines I t was noted t h a t i s o - p r o p a n o l - H C l chromatograms of the AS f r a c t i o n had l a r g e n i r i h y d r i n p o s i t i v e areas i n the v i c i n i t y of the p u r i n e s p o t s . I n order t o a v o i d the p o s s i -b i l i t y of i n t e r f e r e n c e from t h i s m a t e r i a l (presumably amino a c i d s ) , the p u r i n e areas were r o u t i n e l y rechromatographed i n 66 the n-butanol-ethanol system. T h i s s o l v e n t was found t o separate the n i n h y d r I n - p o s i t i v e m a t e r i a l from the p u r i n e s , l e a v i n g i t almost e n t i r e l y a t the o r i g i n . These amino a c i d areas were absent from chromatograms of the NA f r a c t i o n . The data of Table IV i n d i c a t e d t h a t rechromatography of the AS purines was d e s i r a b l e . Rechromatography i n the b u t a n o l s o l v e n t made the s p e c i f i c a c t i v i t y d ata of the AS f r a c t i o n more c o n s i s t e n t , c a using d u p l i c a t e d e t e r m i n a t i o n s t o agree more c l o s e l y (not shown) and i n some cases i n c r e a s i n g s p e c i f i c a c t i v i t i e s . T h i s would suggest t h a t u l t r a v i o l e t a b s o r b i n g i m p u r i t i e s were present on the i s o - p r o p a n o l chromatograms. NA Purines As can be seen from Table IV, rechromatography of the NA purines o b t a i n e d from iso-propanol-HC1 chromatograms was found t o be unnecessary s i n c e the redetermined s p e c i f i c a c t i v i t i e s were not s i g n i f i c a n t l y d i f f e r e n t from the i n i t i a l v a l u e s . On the b a s i s of these r e s u l t s , i t was concluded t h a t the 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 the p u r i n e areas on the chromatograms was p a r t of the p u r i n e s t r u c t u r e and not due t o accompanying contaminants. T n i s suggested t h a t p u r i n e s y n t h e s i s took p l a c e i n the suspension. The AS p u r i n e s i s o l a t e d from iso-propanol-H01 chroma-tograms r e q u i r e d an a d d i t i o n a l p u r i f i c a t i o n , but the s p e c i f i c a c t i v i t y of the n u c l e i c a c i d p u rines was u n a l t e r e d by rechrom-atography. Unless otherwise noted, the AS purines were rechromatographed r o u t i n e l y i n n - b u t a n o l - e t h a n o l . 67 TABLE IV RADIOCHEMICAL PURITY OP AS AND NA PURINES DERIVED PROM FORMATE-C1^. A COMPARISON OP SPECIFIC ACTIVITIES BEFORE AND AFTER RECRROMATOGRAPHY S p e c i f i c A c t i v i t y (;c A. I n i t i a l D e t e r m i n a t i o n 2 ~k 1 ..p.m. per mg.) x 10 B. Determined a f t e r Rechromatography^ adenine guanine adenine guanine AS Purines^" Experiment 1 1 2 . 6 1 5 . 1 2 3 . 5 H o. o 1 3 . 2 1 3 . 0 24.0 1 .9 1 .7 7 . 1 Experiment^ 2 3 . 3 5 . 2 5 . 5 8 . 9 0 . 6 0 . 7 0 . 7 0 . 8 6 . 1 I'.l 0 . 7 0 . 8 0 . 7 0 . 9 NA Purines Experiment 3 0 . 9 0 0 . 5 7 0 . 2 0 0 . 1 5 0 . 9 1 0.5b-0 . 1 8 0 . 1 6 Experiment k 1 . 0 3 1 .11 1 . 3 8 1 .13 1.17 1.05 i 4 t 1.19 1 Spectrophotometric e r r o r s were probably not i n excess of $% f o r adenine d e t e r m i n a t i o n and 10$ f o r guanine determin-a t i o n . 2 S p e c i f i c a c t i v i t y from i n i t i a l i s o - p r o p a n o l - H C l chromatograms (see, " I s o l a t i o n and analysTs of p u r i n e s " , p.53* 3 S p e c i f i c a c t i v i t i e s o f purines from (A) were r e d e t e r -mined a f t e r b e i n g rechromatographed i n n-butanol-ethanol except I n Experiment 1 where i s o - p r o p a n o l - H C l was used. k Counted by thin-sample method. 5 E r r o r o f net a c t i v i t y was l e s s than tl0% f o r guanine. 68 B. I n c o r p o r a t i o n o f f o r m a t e , g l y c i n e and c a r b o n a t e The p r o b a b i l i t y t h a t t h e de novo s y n t h e s i s o f p u r i n e s o c c u r r e d i n t h e mucosal p r e p a r a t i o n s r e c e i v e d s u p p o r t f r o m e x p e r i m e n t s i n w h i c h p u r i n e p r e c u r s o r s o t h e r t h a n f o r m a t e were i n c o r p o r a t e d . G l y c i n e - 1 - and b i c a r b o n a t e - C ^ l a b e l l e d t h e p u r i n e s o f m ucosal s u s p e n s i o n s w h i c h were a l s o i n c o r p o r a t -i n g f o r m a t e , as i l l u s t r a t e d i n t h e e x periments o f T a b l e s V and V I . When r a d i o g l y c i n e was used,as t h e l a b e l l e d s u b s t r a t e , rechromatography o f t h e A S p u r i n e s was e s s e n t i a l s i n c e a s m a l l amount o f g l y c i n e accompanied t h e p u r i n e s t h r o u g h t h e i r i s o l a -t i o n . On chromatograms run. i n t h e i s o - p r o p a n o l - H C l s o l v e n t , g l y c i n e had an R f v a l u e s u f f i c i e n t l y c l o s e t o t h a t o f a d e n i n e t h a t t h e i r s p o t s o v e r l a p p e d . A s o l v e n t system w h i c h i s o l a t e d t h i s h i g h l y a c t i v e g l y c i n e contaminant was o b v i o u s l y e s s e n t i a l . C a r t e r ' s jLso-amyl-d i s odium phosphate (81) o r n - b u t a n o l - e t h a n o l ( 8 2 ) , i n w h i c h g l y c i n e and R f v a l u e s o f 0.7 and 0.1 r e s p e c t i v e -l y , were found t o be e f f e c t i v e s o l v e n t systems i n t h i s r e s p e c t . I n t h e experiment d e s c r i b e d i n T a b l e V, t h e C1^"-f o r m a t e s u b s t r a t e had a s p e c i f i c a c t i v i t y a p p r o x i m a t e l y 1 t i m e s t h a t o f t h e C " ^ - g l y c i n e s u b s t r a t e . Assuming no d i l u t i o n f r o m endogenous s o u r c e s , t h e f o r m a t e s h o u l d g i v e r i s e t o p u r i n e s a p p r o x i m a t e l y X I L t i m e s as a c t i v e as t h o s e d e r i v e d f r o m g l y c i n e , s i n c e 2 m o l e c u l e s o f f o r m a t e a r e i n c o r p o r a t e d i n t h e p u r i n e r i n g ( p o s i t i o n s 2 and 8) f o r e v e r y m o l e c u l e o f g l y c i n e ( p o s i t i o n s 1*, 5> and 7 ) . Prom a c omparison o f t h e s p e c i f i c a c t i v i t i e s o f t h e a denine d e r i v e d f r o m t h e s e s u b s t r a t e s , i t 69 TABLE V A COMPARISON OP FORMATE-C 1^ AND GLYCINE - 1-C 1^ INCORPORATION BY THE AS PURINES f l a s k S u b s t rates S p e c i f i c A c t i v i t y (c.p.m. per mg.) x 10^" AS adenine AS guanine 1 C ^ - f o r m a t e 2 0 . 8 1*.07 2 C ^ - f ormate g l y c i n e 2 5 . 6 3 . 9 3 3 C ^ - g l y c i n e 0 . 7 5 0 . 1 8 * I* C ^ - g l y c i n e formate 0 . 8 5 0 . 1 8 medium: Krebs-Ringer carbonate s u b s t r a t e s , amount per f l a s k : g l y c i n e - l - C 1 ^ , 8 , 5 p . m ( 2 . 5 x 1 0 ° c.p..m.) sodium formate-C 1 1!-, 9 . 5 j j m ( 1 9 . 7 x 1 0 ° c.p.m.) g l y c i n e , 10jj.m . sodium formate, 10jam C 1 ^ assay: # e r r o r of net a c t i v i t y approximately - 20% 70 TABLE VI A COMPARISON OP FORMATE-C1^, GLYCINE, AND BI CARBONATE-C^M- INCORPORATION BY ADENINE Subs t r a t e s S p e c i f i c A c t i v i t y ( AS adenine c.p.m. per mg. )x 10"*^ NA adenine 1. C 1^-formate, g l y c i n e and b i c a r b o n a t e 1 .10 0 . 1 9 2 . C ^ - g l y c i n e , formate and b i c a r b o n a t e 0 . 1 0 0.08 * 3 . C ^ - b i c a r b o n -a t e , formate and g l y c i n e O.liL 0 . 1 5 -""»-Medium: Krebs-Ringer phosphate ( c a l c i u m omitted) s u b s t r a t e s (amount per f l a s k ) : sodium formate-C 1^-, 2 . 2 5 J^ m ( 3 . 5 8,x 1 0 ° c.p.m.) g l y c i n e - l - C 1 ^ , 2;2lj.jam ( 5 . 92 x 1 0 b c.p.m.) sodium b i c a r b o n a t e - C 1 ^ , 0.99jj.m ( 1 . 1 2 x 1 0 ° c.p.m.) n o n - i s o t o p i c s u b s t r a t e s were present i n t h e same •jj^ amounts. C ^ assay: f o r m a t e - d e r i v e d p u r i n e s , e r r o r of net a c t i v i t y l e s s than I l O $ ; g l y c i n e - a n d c a r b o n a t e - d e r i v e d p u r i n e s , e r r o r of net a c t i v i t y approximately ±30$. # e r r o r of net a c t i v i t y l e s s t h a n ± 1 9 $ . ## e r r o r of net a c t i v i t y l e s s t han 113$ 71 appeared t h a t g l y c i n e was used approximately one h a l f of the amount expected from the observed i n c o r p o r a t i o n of formate. The r e s u l t s w i t h guanine were s i m i l a r and i n d i c a t e d t h a t approximately 3 molecules of formate were used f o r each g l y c i n e molecule i n s t e a d of the t h e o r e t i c a l 2 & r a t i o . The experiment o u t l i n e d i n Table VI i n d i c a t e s t h a t approximately 2 molecules of formate were used f o r each molecule of g l y c i n e i n the f o r m a t i o n of AS adenine. I t shows a l s o a s i g n i f i c a n t uptake of i s o t o p e by adenine d u r i n g 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 - The i n c o r p o r a t i o n of formate i n t o AS adenine i n t h i s experiment was l e s s t h a n one t e n t h o f the u s u a l v a l u e (compare with'succeeding experiments) and was l i k e l y r e l a t e d t o the low oxygen uptake ( QQ£ l e s s t h a n 1 ) . I t appeared t h a t the p r e p a r a t i o n was f u n c t i o n i n g a t y p i c a l l y and f o r t h i s reason, comparisons between the t h r e e s u b s t r a t e s may be u n r e l i a b l e . The c o n t r i b u t i o n o f b i c a r b o n a t e cannot be compared w i t h those of the other s u b s t r a t e s because of the d i f f e r e n c e s i n counting techniques and f u r t h e r , because the b i c a r b o n a t e content d i m i n i s h e d p r o g r e s s i v e l y d u r i n g i n c u b a t i o n because of a c i d p r o d u c t i o n by the t i s s u e . With phosphate media the pH u s u a l l y f e l l d u r i n g i n c u b a t i o n , i n some cases t o as low as pH 6 . 9 . E l l i o t (98) has commented on the f a l l o f pH w i t h s i m i l a r media d u r i n g the i n c u b a t i o n of s l i c e s . I n s p i t e of the poor c o n d i t i o n s of t h i s experiment, s i g n i f i c a n t uptake of b i c a r b o n a t e - C ^ - was observed. The departure from the t h e o r e t i c a l r a t i o o f 2 : 1 f o r the i n c o r p o r a t i o n of formate and g l y c i n e may be a t t r i b u t a b l e 72 t o d i l u t i o n of the r a d i o a c t i v i t y of the s u b s t r a t e s by endo-genous pools o f these m e t a b o l i t e s , or p o s s i b l y t o some i n o s ^ i n i c a c i d t r a n s f o r m y l a s e a c t i v i t y (31). T h i s enzyme system has been shown to c a t a l y s e an exchange r e a c t i o n between the 2 - p o s i t i o n o f i n o s i n i c a c i d and formate. By the i n t e r r e l a t i o n -s h i p s between the p u r i n e n u c l e o t i d e s o u t l i n e d p r e v i o u s l y , such an exchange could p l a c e more formate i s o t o p e I n p u r i n e p o s i t i o n 2 of adenosine and guanosine. phosphates t h a n was d e r i v e d by s y n t h e s i s a l o n e . 3. SOME CHARACTERISTICS OF PURINE SYNTHESIS IN INTESTINAL MUCOSA SUSPENSIONS A. U n i f o r m i t y of the s u s p e n s i o n The e r r o r s inherent i n C 1 ^ assay and purine a n a l y s i s have been d i s c u s s e d i n previous s e c t i o n s , but an a d d i t i o n a l f a c t o r a l s o I n f l u e n c e s the s p e c i f i c a c t i v i t i e s of the p u r i n e s of r e p l i c a t e samples; namely, the homogeniety of the suspension. The e x i s t e n c e of d i f f e r e n c e s i n m e t a b o l i c a c t i v i t y between the mucosa from v a r i o u s regions of the s m a l l gut, measured i n terms of Q 0 £ by Dickens and Weil-Malherbe (69) make i t essen-t i a l t h a t t h e suspension be made completely homogeneous when i d e n t i c a l samples are t o be taken. A measure of the u n i f o r m i t y of the p r e p a r a t i o n and of the manner i n which i t was sampled i s found i n the d a t a of Table V I I . The v a r i a t i o n i n the r e p l i c a t e s , which do not vary more t h a n If? per cent from the mean, suggests t h a t the homo-gen i e t y of the p r e p a r a t i o n c o u l d be Improved. The agreement between d u p l i c a t e samples i n subsequent experiments was 73 v a r i a b l e but f r e q u e n t l y much c l o s e r than t h a t i n T a b l e V I I . I t would seem d e s i r a b l e t o fragment the t i s s u e more f i n e l y , the o b j e c t , of course, b e i n g t o o b t a i n a f r e e - c e l l s uspension. The use of c h e l a t i n g agents (108) may f a c i l i t a t e the fragmen-t a t i o n of t h e mucosal s t r i p s and a v o i d the mechanical damage which would i n c r e a s e w i t h a d d i t i o n a l m a n i p u l a t i o n of the p r e p a r a t i o n . TABLE V I I A COMPARISON OP REPLICATE SAMPLES S p e c i f i c A c t i v i t y (c.p .m, per mg.) x l O ' ^ Warburg v e s s e l AS adenine AS guanine NA adenine NA guanine4'" % of t o t a l f orm-a t e -C ^ i n centre w e l l 1 1 3 . 5 1 .15 O . l l * 0 . 0 5 1 1 8 . 6 2 l l * . 3 1 . 0 0 O . l l * o.ol*5 1 8 . 3 3 1 3 . 3 1 . 0 7 0 . 1 2 0.01*2 1 8 . 7 I* 1 1 . 2 0 . 9 5 0 . 1 1 0 . 0 3 9 1 8 . 7 5 124 0.81* 0 . 1 3 0.01*5 1 9 . 0 Average 1 2 . 5 1 . 0 0 0 . 1 3 0.01*1* 1 8 . 7 medium: Krebs-Ringer phosphate / s u b s t r a t e : sodium formate-C 4 , 2 . 2 5 p i (3*58 x 10 c.p.m. per f l a s k s o l v e n t system f o r chromatography: i s o - p r o p a n o l - H C l assay: -35- e r r o r o f net a c t i v i t y l e s s than -Values f o r NA pu r i n e s are averages of d u p l i c a t e a n a l y s e s . 71* B. A comparison of the s p e c i f i c a c t i v i t i e s of the s e v e r a l  p u r i n e f r a c t i o n s ( i ) A c i d - s o l u b l e and n u c l e i c a c i d p u r i n e s T y p i c a l v a l u e s f o r the AS and NA pu r i n e s are arranged f o r comparison i n F i g u r e 1 2 . I s o t o p i c formate was used as the s u b s t r a t e i n a l l cases and values were s i m i l a r . S e v e r a l d e v i a t i o n s o c c u r r e d from these g e n e r a l values as i s noted subsequently. The a c i d - s o l u b l e p u r i n e s were h i g h l y l a b e l l e d , the adenine and guanine of t h i s f r a c t i o n b e i n g approximately 1J_0 and l l i times more a c t i v e , r e s p e c t i v e l y ^ t h a n the corres p o n d i n g n u c l e i c a c i d p u r i n e s . AS adenine, which c o n s i s t e n t l y d i s p l a y e d the h i g h e s t renewal r a t e s , was 6 or more times as a c t i v e as AS quanine. NA adenine was u s u a l l y 2 - 3 times as a c t i v e as NA guanine. ( i i ) Purines i n the medium U r i c a c i d o c c u r r e d i n r e l a t i v e l y l a r g e amounts (60 - 70 i n the suspending medium f o l l o w i n g i n c u b a t i o n , but i t was only s l i g h t l y r a d i o -a c t i v e . Adenine and guanine were a l s o present i n the medium, i n amounts £ t o 10 times l a r g e r t h a n i n the AS f r a c t i o n . T h i s adenine f r a c t i o n was only s l i g h t l y r a d i o a c t i v e and the guanine was n o n - r a d i o a c t i v e s u g g e s t i n g that t h e s e compounds arose from the breakdown of t i s s u e d u r i n g i n c u b a t i o n and were, t h e r e f o r e , not p a r t i c i p a n t s i n the s y n t h e t i c p r o c e s s e s . C y t o s i n e ( n o n - r a d i o a c t i v e ) was a l s o found on chromatograms. of the supernatant f r a c t i o n . T able V I I I and IX permit a comparison of the s p e c i -f i c a c t i v i t i e s of the purines i n the medium w i t h other p u r i n e 75 FIGURE 16 TYPICAL SPECIFIC ACTIVITIES OF THE PURINE FRACTIONS 1 0 16 | -X 14 h o 10 >• H > 6 U "* 4 u b. 3 2 bl a. M 0 • AS ADENINE H AS GUANINE NA ADENINE NA GUANINE f r a c t i o n s . I t i s t o be noted i n Table IX, which summarizes the r e s u l t s of a time study experiment, t h a t the v a l u e s of the NA purines are a t y p i c a l , b e i n g approximately 10 times h i g h e r than u s u a l . T h i s a c t i v i t y remained unchanged on rechromatography. T n e purines of the medium were a p p a r e n t l y catabo-l i t e s , as suggested by t h e i r s p e c i f i c a c t i v i t i e s . The s p e c i f i c a c t i v i t i e s of u r i c a c i d and adenine of the medium were approximately 3 and 1 per cent, r e s p e c t i v e l y , of the AS adenine. I n Table V I I I the o b s e r v a t i o n t h a t u r i c a c i d has a h i g h e r a c t i v i t y than NA. adenine suggests t h a t i t was d e r i v e d p a r t l y from the a c i d - s o l u b l e f r a c t i o n of the s u r v i v i n g c e l l s . The data do not enable any c o n c l u s i o n s t o be made as t o whether 76 the C ^ - u r i c a c i d was produced endogenously and passed to the medium or whether i t was produced exogenously from p u r i n e m e t a b o l i t e s of formate which had "leaked-out" o f the mucosa c e l l s , as i n the case of E h r l i c h carcinoma c e l l s (109). TABLE V I I I A COMPARISON OP THE SPECIFIC ACTIVITIES OP URIC ACID OP THE MEDIUM, AS AND NA PURINES S p e c i f i c a c t i v i t y (c.p.m. per mg.) x 1 0 " ^ u r i c a c i d AS adenine AS guanine NA adenine NA guanine 0 . 3 3 1 3 . 3 1 .9 0 . 2 9 0 . 1 6 medium: Krebs-Ringer carbonate s u b s t r a t e : sodium formate-C 1^, 2 . 2 5 jam ( 3 - 5 8 x 1 0 ° c.p.m.) u r i c a c i d : was counted as the f r e e a c i d on p l a n c h e t s and d i s c s cut from chromatograms r u n i n b u t a n o l - a c e t i c a c i d ( 8 3 ) , w i t h agreement between b o t h r e s u l t s . C. Formate i n c o r p o r a t i o n w i t h r e s p e c t t o time The i n d i v i d u a l r e s u l t s o f f o u r t ime study experiments are shown i n F i g u r e s 17 to 2 0 . The averaged values o f t h e AS purines f o r the fou r experiments a r e shown i n F i g u r e 2 1 . There was c o n s i d e r a b l e v a r i a t i o n between the shape of i n d i -v i d u a l curves, but the averaged values i n d i c a t e d a t r e n d towards a l i n e a r uptake w i t h r e s p e c t t o time. F i g u r e 18 i n d i c a t e d a d e c l i n i n g r a t e of i n c o r p o r a t i o n a f t e r f o u r hours of i n c u b a t i o n . The p r e p a r a t i o n was d e t e r i o r a t i n g at t h i s time ( f a l l i n g pH and r a t e of r e s p i r a t i o n ) . \ TABLE IX A COMPARISON OF THE SPECIFIC ACTIVITIES OF ACID-SOLUBLE AND NUCLEIC ACID PURINES AND THE PURINES .OF THE MEDIUM p e r i o d of incub-a t i o n S p e c i f i c a c t i v i t y (c.p.m. per mg.) x 10 ^ % of f o r -mate-C 1 1* i n centre w e l l AS adenine ' AS guanine NA adenine NA" guanine i Med':."" adenine Med. guanine v. Med. u r i c 0 . 5 n r . 5 . 5 0 . 7 3 * 2 . 5 0 1 . 0 3 O . O I L i n a c t i v e — 1 0 . 2 1 .0 h r . 6 . 1 0 0.82 3.61+ 1.11 0 . 0 5 it 0 . 2 7 2 3 . 3 2 . 0 h r . 7 . 7 1 0 . 6 5 5 . 5 1 1 . 3 8 0 . 0 7 tt 0 . 2 6 2 7 . 0 3 . 0 h r . 9 . 6 2 0 . 8 8 5 . 6 i 1 .26 0 . 0 9 it 0 . 2 9 31-IJ-medium: Krebs-Ringer phosphate s u b s t r a t e : sodium formate-C 1^, 2 . 2 5 (3*58 x 10^ c.p.m.) per f l a s k s o l v e n t system f o r u r i c a c i d chromatography was n-butanol-ammonia ( 8 3 ) . e r r o r of net a c t i v i t y l e s s than * 10$ " " t 15$ '» . » t 20% C 1^ - assay: tt it tt 11 it it tt it \1 78 FIGURE 17. A TIME STUDY OF THE INCORPORATION OF FORMATE-C1^" INTO ADENINE AND GUANINE medium; Krebs-Ringer phosphate s u b s t r a t e : 2.25jlm sodium f o r m a t e - C 1 ^ (3.58 x 10 ,1 c.p.m.) per f l a s k C x ^ assay: - t h e 30 and 180 min. NA p u r i n e s p e c i f i c a c t i v i t i e s were determined from s i n g l e i n c u b a t i o n s w h i l e the other v a l u e s were averaged from d u p l i c a t e i n c u b a t i o n s - e r r o r s o f the net a c t i v i t y were l e s s t h an .-10$ f o r the AS guanine and l e s s t h an ±15$ f o r the NA pu r i n e s 79 FIGURE 18. A TIME STUDY OF THE INCORPORATION OF FORMATE-C 1^ INTO ADENINE AND GUANINE medium: Krebs-Ringer phosphate s u b s t r a t e : 2.25jJ-m sodium formate-C ^ (3.58 x 10° c.p.m.) •JJL per f l a s k C ^ assay: e r r o r o f the net a c t i v i t y was l e s s t han tlO% f o r 65 min. AS guanine The v a l u e s p l o t t e d a r e averages Obtained from d u p l i c a t e i n c u b a t i o n s . FIGURE 1 9 . A TIME STUDY OF THE INCORPORATION OF FORMATE-C 1^ INTO ADENINE AND GUANINE medium: Krebs-Ringer carbonate s u b s t r a t e : 2.25>jum sodium formate-C ^ ( 3 . 5 8 x 1 0 ° c.p.m.) I per f l a s k C1^- assay: e r r o r o f the net a c t i v i t y was l e s s t han ±10$ f o r t h e NA f r a c t i o n 81 FIGURE 2 0 . A TIME STUDY OF THE INCORPORATION OF FORMATE-C 1^ INTO ADENINE AND GUANINE mediumt Krebs-Ringer carbonate . s u b s t r a t e : 2.2$ jsm of sodium f o r m a t e - C 1 ^ ( 3 . 5 8 x 1 0 ° c.p.m.) •jj, per f l a s k C ^ assay: e r r o r o f net a c t i v i t y i s l e s s than I l 0 $ f o r adenine f r a c t i o n s and l e s s than tl$% f o r guanine f r a c t i o n s A l l v alues are averages of two i n c u b a t i o n s , one w i t h aureomycin and one without. Since t h e r e were no s i g n i -f i c a n t d i f f e r e n c e s between t h e s e p a i r s , they were t r e a t e d as d u p l i c a t e s and averaged. FIGURE 21. A TIME STUDY OF THE UPTAKE OF FORMATE-C 1^ INTO ADENINE AND GUANINE The d a t a shown In t h i s f i g u r e were obtained by av e r a g i n g the s p e c i f i c a c t i v i t i e s f o r each p a r t i c u l a r time i n t e r v a l from the f o u r p r e c e d i n g time s t u d i e s . 83 LePage has demonstrated w i t h g l y c o l y s i n g homogenates t h a t the p r o d u c t i o n of h i g h energy phosphate i s necessary f o r the maintenance of n u c l e i c a c i d s t r u c t u r e s (107). F o r these reasons and a l s o because the p o s s i b i l i t y of b a c t e r i a l i n t e r -f e r e n c e i n c r e a s e s w i t h time, the i n c u b a t i o n p e r i o d s were not extended beyond three hours. E x c l u d i n g the r e s u l t s of F i g u r e 18, which are a t y p i c a l , t h e r e seems t o be a time l a g i n the appearance of r a d i o a c t i v i t y i n the n u c l e i c a c i d p u r i n e s . T h i s may be another m a n i f e s t a t i o n of the o b s e r v a t i o n made i n experiments w i t h i n t a c t animals t h a t n u c l e i c a c i d p r e c u r s o r s pass through the a c i d - s o l u b l e n u c l e o t i d e p o o l b e f o r e e n t e r i n g the n u c l e i c a c i d f r a c t i o n . k. THE EFFECT OF ADDITIVES ON PURINE SYNTHESIS An attempt was madeto i n f l u e n c e the p u r i n e s y n t h e s i s of the mucosal suspension by the a d d i t i o n o f c e r t a i n compounds which were i n v o l v e d i n p u r i n e b i o s y n t h e s i s . G l y c i n e and glutamine, known t o c o n t r i b u t e t o the puri n e r i n g , were t e s t e d i n t h i s way, as were glycinamide and lj.-amino - 5-imidazolecarbox-amidine. The l a t t e r compound was c o n s i d e r e d l i k e l y t o i n f l u e n c e p u r i n e s y n t h e s i s because of i t s s t r u c t u r a l resemblance to adenine which may be seen i n the f o l l o w i n g formulas. NH2 H-N' l).-amino -5-imidazole-carb oxamid Ine NH 2 adenine H 8k The r i b o t i d e of glycinamide and i t s f o r m y l d e r i v a t i v e have r e c e n t l y been i m p l i c a t e d as i n t e r m e d i a t e s i h i n o s i n i c a c i d b i o s y n t h e s i s (21,33). I f they were t o have any e f f e c t , g l y c i n e , glutamine and glycinamide were expected t o exert a s t i m u l a t i n g i n f l u e n c e on formate-C^" uptake by the p u r i n e s . A l o w e r i n g i n formate-C"^ uptake was expected w i t h lj.-amino - 5-imidazole-carboxamidine i f t h i s compound was i n c o r p o r a t e d i n t o the pur-i n e s , s i n c e i t r e q u i r e s the a d d i t i o n of only one formate molecule t o complete the p u r i n e r i n g . The d a t a of Tables X - X I I i n d i c a t e t h a t the a d d i t i o n of any of the above compounds to the medium d i d not s i g n i f i c a n t l y i n f l u e n c e p u r i n e s y n t h e s i s i n the mucosal system. The v a r i a t i o n s observed i n these data c o u l d be e x p l a i n e d by the r e l a t i v e inhomogeniety of the suspensions of mucosa. TABLE X THE EFFECTS OF GLYCINE, GLUTAMINE, AND GLYCINAMIDE ON PURINE SYNTHESIS i ; (  A d d i t i v e s (10 M per f l a s k ) S p e c i f i c a c t i v i t y (c. p.m. per mg. ) x 10"*^ " f l a s k V 1 No. a c i d - s o l u b l e f r a c t i o n n u c l e i c a c i d f r a c t i o n g l y c i n e g l y c i n - glutamine adenine guanine adenine guanine amide 1 -- -- — 1 5 . 1 2 . 3 7 046 0.20 2 -- — i + 12 . 8 l o s t 0.2lj. 0.1l|. 3 -- — — 12 . 5 2 . 5 8 0.28 0 . 3 4 k -- 10 . 7 1-91* 0.22 0.12 5 - - ii+4 2 . 5 6 0.38 0 . 1 1 6 -- i + + 1 7 . 9 2 . 9 6 0.33 O.llj. 7 -- — — 1 7 . 0 2 . 0 9 049 0 . 2 2 8"- - — + 1 8 . 5 2 . 0 3 . O.lj.0 0.2li * f l a s k s 7 and 8 contained a d i f f e r e n t p r e p a r a t i o n of mucosa. medium: Krebs-Ringer b i c a r b o n a t e , s u b s t r a t e : sodium formate-G 1^, 2.2% p.M ( 3 . 5 8 x , 1 0 ° c.p.m.) per f l a s k s o l v e n t system f o r chromatography: Iso-propanol-HCl TABLE XI THE EFFECT OF GLYCINAMIDE ON PURINE SYNTHESIS Sub s t r a t e S p e c i f i c A c t i v i t y (c.p.m. per mg.) x 10~^: AS adenine AS guanine formate-C 1^- 1 3 . 2 1 .9 f o r m a t e - C 1 ^ glycinamide 1 3 . 0 3 . 8 medium; Krebs-Ringer carbonate s u b s t r a t e (amount per f l a s k ) ; sodium formate-C 9.7£jjLm ( 1 . 9 7 x 1 0 ° c.p.m.) • glycinamide, lOjJim C ^ assay.; i n f i n i t e l y ; * t h i n samples 87 TABLE X I I THE EFFECT. OF l*-AMINO-5-IMIDAZOLECARBOXAMIDINE ON PURINE SYNTHESIS S p e c i f i c a c t i v i t y (c.p.m. per mg.) x 10"^ S u b s t r a t e AS f r a c t i o n NA f r a c t i o n adenine guanine adenine guanine f o r m a t e - C 1 ^ 0.7 0.1 0.2 0.2 f o r m a t e - C 1 ^ p l u s iraidaz o1e carb ox-amidine 1.0 0.2 0.2 0.1 medium; Krebs-Ringer phosphate ( C a + + omitted) . s u b s t r a t e ; (amount per f l a s k ) ; sodium formate-C 2.2$ y.m (3.58 x 10° c.p.m.) . i m i d a z o l e carboxamidine, 2jum C ^ assay; v a l u e s are averages obtained from d u p l i c a t e , i n c u b a t i o n s . 88 5 . PURINE SYNTHESIS IN HOMOGENATES To a v o i d the p o s s i b i l i t y of c e l l u l a r i m p e r m e a b i l i t y to the t e s t substances, homogenates of mucosa were employed. The homogenates were prepared i n a l l - g l a s s homogenizers of the P o t t e r - E l v e h j e m type (110) i n an i s o t o n i c potassium c h l o r i d e b u f f e r of the f o l l o w i n g composition ( 1 1 1 ) : Potassium c h l o r i d e , 0 . 1 3 M Potassium phosphate b u f f e r (pH 7.1*), 0 . 0 3 M Magnesium c h l o r i d e , 0 . 0 0 3 M Potassium b i c a r b o n a t e , O.Ol* M Glucose, 0 . 0 1 M The homogenates r e s p i r e d a t a very low r a t e ( Q o 2 l e s s t h a n 1) and u n i f o r m l y f a i l e d t o i n c o r p o r a t e formate i n t o adenine and guanine of e i t h e r the AS or NA f r a c t i o n s . U r i c a c i d , a l s o not r a d i o a c t i v e , was found i n l a r g e amounts (approximately Ij.OO'S'per f l a s k , a s i x f o l d i n c r e a s e over the amount o r d i n a r i l y found i n the s u s p e n s i o n s ) . Wanner et a l . have noted t h a t kidney, h e a r t and l i v e r homogenates have an ab s o l u t e requirement f o r d i p h o s p h o p y r i d i n e n u c l e o t i d e ( 1 1 2 ) . F o l l o w i n g t h e i r s uggestions, the mucosal homogenates were f o r -t i f i e d w i t h : Diphosphopyridine n u c l e o t i d e , 0 . 0 0 0 2 M Cytochrome C, O.OOOOI4. M Adenosine t r i p h o s p h a t e , 0 . 0 0 2 M I n a d d i t i o n , the f o l l o w i n g compounds were i n c l u d e d i n the i n c u b a t i o n mixture, t o i n s u r e an adequate supply o f components f o r de novo pur i n e s y n t h e s i s : Ribose - 5-phosphate, 0 . 0 0 5 M Glutamine, 0 . 0 1 M G l y c i n e , 0 . 0 1 M 89 These a d d i t i v e s had no s t i m u l a t i n g e f f e c t on puri n e s y n t h e s i s and caused only a s l i g h t i n c r e a s e i n oxygen consumption. The i n a c t i v i t y o f the homogenates w i t h r e s p e c t t o p u r i n e s y n t h e s i s probably was connected w i t h the i n a b i l i t y of the system t o m a i n t a i n r e s p i r a t i o n . I n c r e a s i n g the f i n a l d i l u t i o n o f the homogenate (by u s i n g l e s s t i s s u e i n l a r g e r v e s s e l s ) from approximately 1 : 1 0 , as used above, t o 1 :100 would probably improve the r e s p i r a t o r y a c t i v i t y of the p r e p a r a t i o n ( 1 1 3 ) . P a t e r s o n and LePage have observed t h a t the de novo s y n t h e s i s of AS and NA purines from g l y c i n e - 2 - C 1 ^ i n tumour homogenates occ u r r e d o n l y t o a s m a l l extent under c o n d i t i o n s i n which C ^ - a d e n i n e was w e l l i n c o r p o r a t e d i n t o b o t h f r a c t i o n s ( 5 8 ) . 6 . INCORPORATION OF FORMATE-C 1^ BY THE PURINES OF INTESTINAL MUCOSA IN THE INTACT RAT The d i s t r i b u t i o n o f for m a t e - d e r i v e d r a d i o a c t i v i t y i n t he AS and NA pur i n e s of i n t e s t i n a l mucosa from the i n t a c t animal i s i n c l u d e d f o r comparison w i t h the r e s u l t s of the i n v i t r o experiments. T h i s comparison must be of a s u p e r f i c i a l nature only f o r s e v e r a l reasons, such as the d i f f e r e n c e s i n the experimental c o n d i t i o n s f o r the t i s s u e , and the d i f f e r e n c e s i n the time i n t e r v a l d u r i n g which formate was a v a i l a b l e t o the mucosa. The i n t e s t i n a l t i s s u e used i n t h i s experiment was obtained from an experiment conducted by D r. S.H. Zbarsky i n which male W i s t a r r a t s b e a r i n g the N o v i k o f f hepatoma were i n j e c t e d w i t h C ^ - f o r m a t e . These animals r e c e i v e d a s i n g l e dose of sodium formate-C 1^" a d m i n i s t e r e d sub cutaneous l y on the back. 90 The d a t a of Table X I I I do not show the l a r g e d i f f e r -ences between the s p e c i f i c a c t i v i t i e s of the AS and NA f r a c t i o n s observed i n the i n v i t r o experiments d e s c r i b e d i n t h i s t h e s i s and i n other i n v e s t i g a t i o n s (3 , 5 )• T i m e s t u d i e s w i t h i n t a c t animals have shown t h a t very r a p i d l y a f t e r the a d m i n i s t r a t i o n of a l a b e l l e d n u c l e i c a c i d p r e c u r s o r , the i s o t o p e c o n c e n t r a t i o n i n the a c i d - s o l u b l e n u c l e o t i d e s passes through a sharp maximum f o l l o w e d by a more gr a d u a l d e c l i n e (7, 8, llli.) • The appearance of the i s o t o p e i n the n u c l e i c a c i d f r a c t i o n i s more gr a d u a l , the maximum s p e c i f i c a c t i v i t y appearing w e l l a f t e r t h a t of the n u c l e o t i d e f r a c t i o n . Bennett has r e p o r t e d t h a t i n t h e n u c l e o t i d e f r a c t i o n of mouse stomach and i n t e s t i n e , AMP reached a maximum s p e c i f i c a c t i v i t y w i t h i n 2 hours a f t e r the a d m i n i s t r a t i o n of adenine-C 1^" (111).). The s p e c i f i c a c t i v i t y of n u c l e i c a c i d adenine showed a r a p i d i n i t i a l r i s e , but t h e r e a f t e r r o s e s l o w l y to the maximum v a l u e w i t h i n 2l\. hours and then d e c l i n e d s l o w l y a t a f i r s t o r d e r r a t e . I n the present experiment, because of t h e i n t e r v a l of 2^ hours between th e a d m i n i s t r a t i o n of the i s o t o p e and the s a c r i f i c e of the animal, the s p e c i f i c a c t i v i t i e s of the a c i d -s o l u b l e purines had undoubtedly f a l l e n below t h e i r maximum values and were approaching those of the n u c l e i c a c i d p u r i n e s . The r a t i o of the s p e c i f i c a c t i v i t i e s of AS adenine to AS guanine was approximately 1.1)., whereas i n the i n v i t r o experiments, t h i s v a l u e was approximately 6. T h i s d i s c r e p a n c y may i n d i c a t e some Impairment of guanine s y n t h e s i s i n the i n  v i t r o system. The r a t i o o f the s p e c i f i c a c t i v i t i e s of NA 91 adenine t o NA guanine were s i m i l a r t o those r e p o r t e d by Drochmans et a l . ( 6 7 ) . TABLE X I I I THE INCORPORATION OP FORMATE-C"^ BY THE PURINES OF INTESTINAL MUCOSA IN THE INTACT RAT R a t S p e c i f i c a c t i v i t i e s i n c.p.m. per mg. x 10"^ A c i d - s o l u b l e f r a c t i o n N u c l e i c a c i d f r a c t i o n adenine guanine adenine guanine 1 .(tumour) I*. 17 . 2 . 9 7 2 . 2 3 2 . 0 3 2 . ( c o n t r o l ) 3 . 6 2 . 2 . 5 2 2 . 1 1 2 ; 11* 3 .(tumour) i*.i*6 2.81* 2 . 8 6 2 . 7 6 I*, ( c o n t r o l ) l * . l 8 3 . 7 7 3 . 3 7 3 . 3 0 Each h o r i z o n t a l row of values was obtained from 1 r a t which r e c e i v e d f t s u b c u t a n e o u s l y 225.jam sodium formate-C ^ ( 3 . 5 8 x 1 0 ° c.p.m.). Animals were s a c r i f i c e d 2[j. hours l a t e r by b l e e d i n g under e t h e r a n a e s t h e s i a . I n t e s t i n a l mucosa was p l a c e d i n 2% p e r c h l o r i c a c i d and the purines prepared i n the u s u a l f a s h i o n . AS f r a c t i o n s , except No. 1* were r e chroma to graphed i n n - b u t a n o l - e t h a n o l . 92 DISCUSSION The g e n e r a l f i e l d o f n u c l e o t i d e and n u c l e i c a c i d metabolism i s very a c t i v e at p r e s e n t . The p r i n c i p a l areas of i n t e r e s t and expansion at t h i s time i n v o l v e the pathways of p u r i n e and p y r i m i d i n e b i o s y n t h e s i s , the i n t e r r e l a t i o n s h i p s between the components of the a c i d - s o l u b l e f r a c t i o n , and the f u n c t i o n a l r o l e s o f these compounds as coenzymes and b u i l d i n g b l o c k s i n p o l y n u c l e o t i d e s y n t h e s i s . R i b o - and d e s o x y r i b o -n u c l e i c a c i d b i o s y n t h e s i s and the b i o l o g i c a l f u n c t i o n s of the n u c l e i c a c i d s are areas i n which expansion i s imminent. Many of these s t u d i e s have progressed to the extent t h a t use of the i n t a c t animal must be bypassed i n favour of systems which gi v e the e x p e r i m e n t a l i s t g r e a t e r c o n t r o l over the r e a c t i o n system under study. F o r such reasons, the use of t i s s u e p r e p a r a t i o n s i n v i t r o has become e s s e n t i a l t o many aspects of s t u d i e s of n u c l e i c a c i d metabolism. I n t e s t i n a l mucosa has not h i t h e r t o been u t i l i z e d f o r i n v i t r o s t u d i e s of p u r i n e metabolism. The demonstration t h a t 93 b o t h a c i d - s o l u b l e and p o l y n u c l e o t i d e purines are r a p i d l y s y n t h e s i z e d i n the mucosal sus p e n s i o n suggests t h a t t h i s p r e p a r a t i o n may be of value i n s t u d i e s of p u r i n e n u c l e o t i d e b i o s y n t h e s i s and metabolism. In. t h i s f i e l d , comparisons between a v i a n and mammalian systems are d i s t i n c t l y needed and i n t e s t i n a l mucosa suspensions may have a u s e f u l f u n c t i o n i n t h i s r e s p e c t . I n v i t r o systems have been used i n m e t a b o l i c s t u d i e s of a n t I - m e t a b o l i t e s ( 6 2 , 115) w i t h the tumour chemo-therapy as a p o s s i b l e o b j e c t i v e ; i t i s suggested t h a t the mucosal suspension c o u l d be employed s i m i l a r l y . The apparent absence of hypoxanthine i n these prep-a r a t i o n s and the l a r g e amounts o f u r i c a c i d found present, e s p e c i a l l y I n homogenates, are i n d i c a t i v e of the h i g h l e v e l of xanthine oxidase i n the mucosa of the s m a l l i n t e s t i n e ( 7 3 ) • The occurrence of i n o s i n i c a c i d i n the a c i d - s o l u b l e f r a c t i o n of l i v e r and tumour has been shown by H u r l b e r t et a l . ( 1 ) , and by Edmonds and Lepage ( 5 ) , but was not d e t e c t e d i n these experiments where i t would appear as hypoxanthine i n the h y d r o l y s e d AS f r a c t i o n . The o x i d a t i o n of formate by the mucosal p r e p a r a t i o n i s t o be expected i n view of the apparent ease of t h i s r e a c t i o n i n most t i s s u e s ( 1 1 6 ) . As much as 30 per cent of the formate s u b s t r a t e was o x i d i z e d t o carbon d i o x i d e i n the i n c u b a t i o n per-i o d of 3 hours. T h i s c o n v e r s i o n i s not as g r e a t as t h a t observed by Nakada and Weinhouse (106) f o r formate o x i d a t i o n I n l i v e r and kidney homogenates, but compares f a v o u r a b l y w i t h values r e p o r t e d f o r other t i s s u e s by these authors ( i n t e s t i n e was not i n v e s t i g a t e d ) . Nakada and Weinhou.se c o n s i d e r t h a t formate i s o x i d i z e d through a p e r o x i d a t i c a c t i v i t y of c a t a -l a s e , the peroxide a r i s i n g by the a c t i o n of xanthine oxidase, presumably on p u r i n e s . The low l e v e l of g l y c i n e o x i d a t i o n observed i n t h i s work i s i n acco r d w i t h Greeriberg's r e p o r t t h a t t i s s u e p r e p a r a t i o n s decompose g l y c i n e t o carbon d i o x i d e t o a s m a l l extent ( l l 6 ) . Nakada and Weinhouse have r e p o r t e d h i g h r a t e s of g l y c i n e o x i d a t i o n f o r l i v e r and kidney prepar-a t i o n s , but low r a t e s f o r other t i s s u e s ( i n t e s t i n e was not examined). The study of n u c l e i c a c i d metabolism i n i n t e s t i n e has been c o n f i n e d t o ob s e r v a t i o n s of the i n c o r p o r a t i o n of i s o t o p e from l a b e l l e d p r e c u r s o r s i n the i n t a c t animal (I4., 9> 6I4., 6 5 , 6 7 , 117 - 1 2 3 ) . Of the v i s c e r a , i n t e s t i n e d i s p l a y s the h i g h e s t r a t e of s y n t h e s i s of DNA and RNA pu r i n e s from s m a l l molecule p r e c u r s o r s such as formate ( 6 7 , 117, 1 2 0 , 1 2 1 ) , g l y c i n e (118, 119, 1 2 3 ) , and ammonia ( 1 1 8 ) . R e l a t i v e t o l i v e r , i n t e s t i n e has a s m a l l e r a b i l i t y t o i n c o r p o r a t e pre-formed purines i n t o the n u c l e i c a c i d s ( 6 7 , 1 2 3 ) . In compar-i s o n w i t h other v i s c e r a , i n t e s t i n e i s s u p e r i o r i n i t s a b i l i t y t o i n c o r p o r a t e i s o t o p i c adenine i n t o the a c i d - s o l u b l e p u r i n e n u c l e o t i d e f r a c t i o n (1*,9)» T h e prominence of n u c l e i c a c i d s y n t h e s i s i n t h i s t i s s u e i s probably r e l a t e d t o the very h i g h m i t o t i c a c t i v i t y (61*, 65) The vigorous p u r i n e s y n t h e s i s demonstrated i n the i n t e s t i n e of the i n t a c t animal manifested i t s e l f i n the mucosal sus p e n s i o n i n v i t r o by the s y n t h e s i s of b o t h a c i d -95 s o l u b l e and n u c l e i c a c i d p u r i n e s . That the 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 the p urines of the p r e p a r a t i o n was t r u l y i n c o r p o r a t e d i n the p u r i n e molecule and was not due t o r a d i o -a c t i v e contaminants was e s t a b l i s h e d by demonstrating the r a d i o c h e m i c a l p u r i t y of the p u r i n e s . The c o n v e r s i o n o f the s u b s t r a t e r a d i o a c t i v i t y t o adenine and guanine c o u l d hot be a t t r i b u t e d t o b a c t e r i a l a c t i v i t y , as shown by the low b a c t e r i a l counts, d e c l i n i n g r e s p i r a t o r y a c t i v i t y and, most c o n c l u s i v e l y , by the i n a b i l i t y of homogenates t o perform t h i s c o n v e r s i o n . I t i s u n l i k e l y t h a t the i n c o r p o r a t i o n of f o r m a t e - C ^ i n t o adenine and guanine was due t o an exchange type of r e a c -t i o n such as t h a t observed between formate and p o s i t i o n 2 of i n o s i n i c a c i d by Buchanan and h i s coworkers (13, 31 (see r e f . 106, 107, .109) )• The exchange took p l a c e i n p i g e o n l i v e r p r e p a r a t i o n s i n which i n o s i n i c a c i d s y n t h e s i s was l i m i t e d by suboptimal amounts of b i c a r b o n a t e , and was d e t e c t e d by an extreme e l e v a t i o n of the r a t i o of formate t o g l y c i n e i n c o r -p o r a t i o n . I n the mucosal p r e p a r a t i o n s , g l y c i n e was I n c o r p o r -ated l e s s e f f i c i e n t l y t h an formate, but the r e l a t i v e amounts i n c o r p o r a t e d o f these p r e c u r s o r s were s u f f i c i e n t l y c l o s e t o the t h e o r e t i c a l p r o p o r t i o n s t o exclude a major i n c o r p o r a t i o n of formate by an exchange r e a c t i o n . A comparison of the r e l a t i v e i n c o r p o r a t i o n of l a b e l l e d formate and g l y c i n e i s complicated by the endogenous occurrence of these compounds which, of course, has the e f f e c t of l o w e r i n g the s p e c i f i c a c t i v i t y of the i s o t o p i c s u b s t r a t e s . That the i n c o r p o r a t i o n of g l y c i n e r e l a t i v e t o formate was observed t o be l e s s t h a n 96 t h e o r e t i c a l i n . t h e present experiment may be due to d i l u t i o n of the l a b e l l e d s u b s t r a t e by endogenous g l y c i n e . A comparison between b i c a r b o n a t e and the other p r e c u r s o r s cannot be made because of the d i f f e r e n t elk - c o u n t i n g techniques employed, and because the endogenous p r o d u c t i o n of carbon d i o x i d e g r e a t l y a f f e c t e d the s p e c i f i c a c t i v i t y of the bicarbonate-C H* s u b s t r a t e . The data presented f o r the uptake of C " ^ - b i c a r b o n a t e by the purines had a l a r g e s t a t i s t i c a l e r r o r , but i n d i c a t e d a s i g n i -f i c a n t i n c o r p o r a t i o n of t h i s purine p r e c u r s o r by n u c l e i c a c i d adenine. The i n c o r p o r a t i o n o f these t h r e e p r e c u r s o r s by adenine and guanine i s regarded as evidence o f a de novo s y n t h e s i s of pu r i n e s by the mucosal suspension. I n the growing number of r e p o r t s on n u c l e o t i d e metabolism, glycine-2-C''"^' has been used t o demonstrate the r a p i d t u r n o v e r of the AS pu r i n e s i n v i v o (5) and i n i s o l a t e d t i s s u e p r e p a r a t i o n s (3)» A d e n i n e - C ^ has a l s o been used s i m i l a r l y i n whole animal experiments ( 9 , I*) and i n i n v i t r o s t u d i e s (55)• T h e mucosal sus p e n s i o n d i s p l a y e d t h i s charac-t e r i s t i c a l s o , a c i d - s o l u b l e adenine and guanine b e i n g lj.3 and 3.I4. times more a c t i v e , r e s p e c t i v e l y , than the corresponding n u c l e i c a c i d p u r i n e s . I n the i n v i t r o experiments r e p o r t e d by LePage ( 3 , see Table V ), the AS : NA r a t i o s f o r adenine and guanine of mouse l i v e r c e l l s are 170 and 2 2 0 , r e s p e c t i v e l y . F o r adenine and guanine of the E h r l i c h mouse a s c i t e s c e l l carcinoma, these r a t i o s are 30 and 6 0 , r e s p e c t i v e l y . I n the mucosal suspension, the s y n t h e s i s of adenine from formate-C 1^" i s more r a p i d than t h a t of guanine, t h i s 97 b e i n g p a r t i c u l a r l y n o t i c e a b l e I n the a c i d - s o l u b l e f r a c t i o n . LePage found the r e v e r s e t o be t r u e i n suspensions of mouse tumour and l i v e r c e l l s w i t h g l y c i n e - 2 - as the l a b e l l e d s u b s t r a t e ( 3 ) . T o t t e r and Best (62) have i n v e s t i g a t e d formate I n c o r p o r a t i o n i n t o the n u c l e i c a c i d s of r a b b i t bone marrow c e l l s i n v i t r o and r e p o r t s p e c i f i c a c t i v i t i e s f o r RNA adenine 3 - 5 times those of of RNA guanine. The r e a s on f o r the divergence between the r e l a t i v e r a t e s of adenine and guanine s y n t h e s i s i n these r e p o r t s i s not apparent, but may be due, i n p a r t , to d i f f e r e n c e s i n the s i z e s of the pools of endogenous n u c l e o t i d e s . In the i n v i v o experiments d e s c r i b e d i n Table X I I I , the purines of i n t e s t i n a l mucosa a l l had very s i m i l a r s p e c i f i c a c t i v i t i e s , i n b o t h the AS and NA f r a c t i o n s . As e x p l a i n e d p r e v i o u s l y , t h i s i s probably due to the time i n t e r v a l between the a d m i n i s t r a t i o n of the formate-C"^ and the i s o l a t i o n of the p u r i n e s . The s p e c i f i c a c t i v i t y of the AS adenine was 1.1 t o 1 . 5 times h i g h e r than t h a t of AS guanine (compare w i t h the 6 - l 6 f o l d d i f f e r e n c e between these compounds i n the i n v i t r o experiments summarized i n F i g u r e l 6 . Assuming t h a t the s p e c i f i c a c t i v i t y of the NA purines i s r e l a t e d i n more or l e s s d i r e c t f a s h i o n to t h a t of the AS p u r i n e s , t h i s may be i n t e r p r e t e d as meaning t h a t guanine s y n t h e s i s i s suppressed i n the i n v i t r o -preparations. The low l e v e l of a c t i v i t y found i n the u r i c a c i d f r a c t i o n suggests t h a t i t i s a d e g r a d a t i o n p r o d u c t . S i m i l a r l y , the weak a c t i v i t y of the adenine and the absence of i s o t o p e 98 I n the guanine of the medium i n d i c a t e t h a t these p u r i n e s probably a r i s e by c a t a b o l i c p r o c e s s e s . The combined r e s u l t s of the time s t u d i e s shown i n F i g u r e 21 demonstrate that the AS purines are s y n t h e s i z e d at an approximately l i n e a r r a t e and u n d e r l i n e the d i f f e r e n c e between the r a t e s of s y n t h e s i s of adenine and guanine. The data of F i g u r e 20 i n d i c a t e a d e c l i n e i n the r a t e of s y n t h e s i s at 3 - 1* hours of i n c u b a t i o n , by which time the system was undoubtedly b r e a k i n g down. I n c u b a t i o n p e r i o d s cannot be extended without r i s k i n g b a c t e r i a l growth. The a t y p i c a l l y h i g h s p e c i f i c a c t i v i t i e s of the NA purines i n F i g u r e 18 i s unexplained. The a d d i t i o n of the s e v e r a l compounds to the i n c u b a t i o n mixture was undertaken i n the hope t h a t formate i n c o r p o r a t i o n would be i n f l u e n c e d i n such a way as t o i n d i c a t e an involvement of the a d d i t i v e i n p u r i n e s y n t h e s i s . However, formate i n c o r p o r a t i o n was not a f f e c t e d s i g n i f i c a n t l y by a d d i t i o n s of g l y c i n e , glutamine, glycinamide and Ij.-amino-5-imidazolecarboxamidine. Subsequent t o the i n i t i a t i o n of these s t u d i e s , s e v e r a l p u b l i c a t i o n s have shown t h a t i t i s u n l i k e l y t h a t f r e e glycinamide i s i n v o l v e d i n p u r i n e s y n t h e s i s ( 2 1 , 3 1 , 3 3 , 3 7 ) . Present i n d i c a t i o n s are t h a t g l y c i n e , by i n t e r a c t i o n w i t h 5-phosphoribosylamine, enters the b i o s y n t h e t i c pathway as glycinamide r i b o t i d e . The n e g a t i v e f i n d i n g s w i t h glycinamide i n the mucosal system are compatible w i t h these concepts. Abrams has shown t h a t jj.-amino -5~imidazolecarboxamide has no e f f e c t on formate uptake by bone marrow s l i c e s ( 6 l ) . 99 The amidine of t h i s compound was t e s t e d i n the mucosal susp e n s i o n because of i t s s t r u c t u r a l r e l a t i o n s h i p to p o s s i b l e adenine p r e c u r s o r s and the i n d i c a t i o n that i t may have a m e t a b o l i c f u n c t i o n i n c e r t a i n protozoa ( 3 8 ) • The l a c k of s i g n i f i c a n t e f f e c t s on p u r i n e s y n t h e s i s by the above a d d i t i o n s may be due t o f a c t o r s such as the f o l l o w i n g : (1) non-involvement of t h e compound In p u r i n e s y n t h e s i s (2) p e r m e a b i l i t y e f f e c t s (3) an a l r e a d y adequate endogenous p o o l of the a d d i t i v e or i t s m e t a b o l i t e s (Ij.) the r e a c t i o n sequences o f p u r i n e s y n t h e s i s may have r a t e -l i m i t i n g steps i n t h i s system subsequent to t h e p o i n t a t which the a d d i t i v e e n t e r s . I n view of the above demonstration t h a t a r a p i d de  novo s y n t h e s i s of p u r i n e s takes p l a c e i n the suspensions of i n t e s t i n a l mucosa, s e v e r a l a p p l i c a t i o n s o f t h i s system suggest themselves. As noted p r e v i o u s l y , t h e r e i s a need f o r compar-i s o n of the pathways of p u r i n e b i o s y n t h e s i s i n mammalian t i s s u e w i t h the known pathways i n a v i a n l i v e r ; the mucosal p r e p a r a t i o n may have a u s e f u l f u n c t i o n i n t h i s r e s p e c t . T h i s system may have an a p p l i c a t i o n i n the t e s t i n g of a n t i -m e t a b o l i t e s d i r e c t e d at n u c l e i c a c i d metabolism, w i t h the o b j e c t i v e of s e l e c t i n g compounds which are p o t e n t i a l l y u s e f u l i n cancer chemotherapy. I n c e r t a i n r e s p e c t s , i n t e s t i n a l mucosa bears a resemblance t o cancer t i s s u e , n o t a b l y , i n carbohydrate metabolism, i n the h i g h r a t e of c e l l d i v i s i o n , 100 and i n the prominence of n u c l e i c a c i d renewal; the l a t t e r two f e a t u r e s a r e r e l a t e d t o the a p p a r e n t l y continuous growth of i n t e s t i n a l mucosa i n the r a t (12I4.). F o r these reasons, t h i s system has a p o t e n t i a l a p p l i c a t i o n t o s t u d i e s of the m e t a b o l i c f e a t u r e s which a r e c h a r a c t e r i s t i c of r a p i d l y growing t i s s u e s and neoplasms. 101 SUMMARY The p r e p a r a t i o n of suspensions of mucosa from the s m a l l i n t e s t i n e of t h e r a t i s d e s c r i b e d and methods are r e p o r t e d f o r the use of t h i s system i n i n v i t r o s t u d i e s of n u c l e i c a c i d metabolism. Some f e a t u r e s of p u r i n e metabolism i n these p r e p a r a t i o n s are d e s c r i b e d and a demonstration of de novo p u r i n e s y n t h e s i s i s presented. The mucosal sus p e n s i o n i s a whole c e l l p r e p a r a t i o n c o n s i s t i n g of s m a l l fragments of mucosa r a n g i n g i n s i z e from clumps of a few c e l l s t o sheets l\. - 6 mm. . The suspensions of mucosa r e s p i r e d a c t i v e l y f o r 3 - h hours w i t h i n i t i a l Q Q 2 v a l u e s of approximately 8. B a c t e r i a l contamination was not great i n these p r e p a r a t i o n s and was shown t o have no s i g n i f i -cant i n f l u e n c e on the measurements of p u r i n e s y n t h e s i s . The mucosal suspensions o x i d i z e d g l y c i n e - l - C 4 t o a s m a l l extent only, but v i g o r o u s l y o x i d i z e d f o r m a t e - C ^ to carbon d i o x i d e -c4. A procedure i s presented f o r the s e p a r a t i o n , by i o n exchange chromatography of the i n d i v i d u a l p u r i n e s from s o l u t i o n s 102 c o n t a i n i n g u r i c a c i d , xanthine, nypoxanthine, guanine and adenine. When t h i s method was a p p l i e d to incubated suspensions of i n t e s t i n a l mucosa, the only purines demonstrable were adenine, guanine and u r i c a c i d . Techniques were developed f o r r o u t i n e use which permitted the i s o l a t i o n , d e t e r m i n a t i o n , and r a d i o c a r b o n assay of the p u r i n e s of the a c i d - s o l u b l e , n u c l e i c a c i d and medium f r a c t i o n s of mucosal suspensions c o n t a i n i n g 150 - 200 mg. of f r e s h t i s s u e . The de novo s y n t h e s i s of purines by t h i s system was demonstrated by measuring the i n c o r p o r a t i o n of s e v e r a l C^"-l a b e l l e d p u r i n e p r e c u r s o r s i n t o the t i s s u e p u r i n e s . I t was necessary t o e s t a b l i s h the f o l l o w i n g t h r e e p o i n t s which are fundamental t o the c o n c l u s i o n t h a t the observed i n c o r p o r a t i o n was due t o de novo p u r i n e s y n t h e s i s by the mucosal t i s s u e : (a) r a d i o c h e m i c a l p u r i t y of the i s o l a t e d p u r i n e s (b) n o n - p a r t i c i p a t i o n of exchange r e a c t i o n s (c) e x c l u s i o n of b a c t e r i a l i n f l u e n c e The c r i t e r i o n of r a d i o c h e m i c a l p u r i t y employed was the un-changed s p e c i f i c a c t i v i t y of the p u r i n e s a f t e r b e i n g s u c c e s s -i v e l y chromatographed on f i l t e r paper i n . d i f f e r e n t s o l v e n t systems. The c o n c l u s i o n t h a t the i n c o r p o r a t i o n of formate-ill-C took p l a c e by de novo pur i n e s y n t h e s i s r a t h e r than by a process of exchange was based on the o b s e r v a t i o n t h a t g l y c i n e -and bicarbonate-C 1^" were a l s o i n c o r p o r a t e d i n t o the p u r i n e s . I n one experiment, 3 - 1 * molecules o f formate were i n c o r p o r a t e d f o r each molecule of g l y c i n e used i n p u r i n e s y n t h e s i s and v a l u e s approximating the t h e o r e t i c a l 2:1 r a t i o 103 were observed i n another experiment. These valu e s are s u f f i c i e n t l y c l o s e t o the t h e o r e t i c a l r a t i o o f formate t o g l y c i n e i n c o r p o r a t i o n t o exclude a major i n c o r p o r a t i o n o f formate by exchange r e a c t i o n s of the i n o s i n i c a c i d t r a n s -formylase type. The i n a b i l i t y of homogenates of mucosa t o i n c o r p o r a t e i s o t o p i c formate r u l e s out the p a r t i c i p a t i o n of b a c t e r i a i n t h i s p r o c e s s . Adenine, guanine and u r i c a c i d were found i n the suspending medium, but were only weakly r a d i o a c t i v e , suggest-i n g t h a t they o r i g i n a t e d l a r g e l y i n the breakdown of t i s s u e d u r i n g i n c u b a t i o n and were not p a r t i c i p a n t s i n a n a b o l i c p r o c e s s e s . The a c i d - s o l u b l e p u r i n e s were h i g h l y l a b e l l e d , the adenine and guanine of t h i s f r a c t i o n h a v i n g s p e c i f i c a c t i v i t i e s approximately I4.0 and lL|. times those of n u c l e i c a c i d adenine and guanine, r e s p e c t i v e l y . I n the a c i d - s o l u b l e f r a c t i o n , adenine c o n s i s t e n t l y had the h i g h e s t renewal r a t e , b e i n g 6 or more times as a c t i v e as guanine. The r a p i d t u r n -over of the a c i d - s o l u b l e purines r e l a t i v e t o the n u c l e i c a c i d p u r i n e s suggested t h a t the former bears a p r e c u r s o r r e l a t i o n -s h i p towards the l a t t e r . The i n c o r p o r a t i o n of f o r m a t e - C ^ by purines w i t h r e s p e c t t o time showed an approximately l i n e a r t r e n d , w i t h a d e c l i n e i n r a t e appearing a f t e r 3 - k hours of i n c u b a t i o n . I n s e v e r a l experiments an i n i t i a l l a g i n the r a t e of s y n t h e s i s of n u c l e i c ' a c i d purines was apparent. Purine s y n t h e s i s , as measured by the uptake of f o r m a t e - C 1 ^ i n t o purines was u n a f f e c t e d by the a d d i t i o n of the lOlj. f o l l o w i n g substances t o the i n c u b a t i o n mixture: g l y c i n e , glycinamide, glutamine, and l|-amino-£-imidazolecarboxamidine. Homogenates of i n t e s t i n a l mucosa, prepared i n b u f f e r e d I s o t o n i c potassium c h l o r i d e and f o r t i f i e d w i t h DPN, cytochrome C, and ATP, f a i l e d t o i n c o r p o r a t e formate-C 1^" i n t o p u r i n e s . The purines were prepared from the i n t e s t i n a l mucosa of i n t a c t animals which had r e c e i v e d subcutaneously a d m i n i s t e r ed formate-C 1^" 2lj. hours p r i o r t o s a c r i f i c e . Adenine and guanine of b o t h the a c i d - s o l u b l e and n u c l e i c a c i d f r a c t i o n s were shown t o have very s i m i l a r s p e c i f i c a c t i v i t i e s . The r e l a t i v e l o w e r i n g of the a c t i v i t i e s of the a c i d - s o l u b l e purines i n t h i s experiment was a t t r i b u t e d t o the 2I4. hour i n t e r v a l . Comparison o f purine s y n t h e s i s from f o r m a t e - C 1 ^ i n the i n t a c t animal w i t h t h a t of the i n v i t r o system suggested t h a t guanine s y n t h e s i s was suppressed i n the l a t t e r . The p o s s i b l e a p p l i c a t i o n o f t h i s t i s s u e system t o s t u d i e s of pur i n e b i o s y n t h e s i s i n the mammal i s d i s c u s s e d , and i t s use i n s t u d i e s of n u c l e i c a c i d a n t i m e t a b o l i t e s and i n the s c r e e n i n g of anti-tumour compounds i s suggested. 1 105 BIBLIOGRAPHY 1. H u r l b e r t , R. B., Schmitz, H., Brumm, A. P.. and P o t t e r , V. R., J . B i o l . Chem., 2 0 $ , 23 (1951*). 2 . Schmitz, H., H u r l b e r t , R . B., and P o t t e r , V. R., J . B i o l . Chem., 2 0 9 , 1*1 (1951*). 3 . LePage, G. A., Cancer Res., 13_, 178 ( 1 9 5 3 ) . li_. M arrian, D. H., Biochim. et Biophys. A c t a , 13_,282 (1951*). 5 . Edmonds, M. P., and LePage, G. A., Cancer Res., 15 , 93 ( 1 9 5 5 ) . 6. 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Chem., I83., 251 ( 1 9 5 0J. 12l|.. Leblond, C. P., and Stevens, C. E., Anat. R e c , 1 0 0 , 357 ( 1 9 4 8 ) . Reprinted from Biochim. et Bioph. Acta VOL. 18 (1955) SHORT COMMUNICATIONS, PRELIMINARY NOTES 44I In vitro s y n t h e s i s o f p u r i n e s b y r a t i n t e s t i n a l m u c o s a * In vitro studies of purine and nucleic acid synthesis from precursors such as glycine, formate and carbon dioxide have been carried out principally with pigeon liver preparations. In the small number of such investigations involving mammalian tissues, regenerating liver1, bone marrow 2, 3 and tumor cells4 have been utilized. Certain characteristics of rat intestinal mucosa suggested that this tissue would be suitable for studies of purine biosynthesis, e.g., the very high rate of cell division5, the high uptake of purine precursors in the intact animal 6,', 8 and the high metabolic activity in vitro9. Further, the mucosa can be prepared with little damage as a suspension in which the tissue particles are sufficiently small so that diffusion is not limiting. This report describes the preparation of such suspensions and some observations of purine biosynthesis in the acid-soluble nucleotides and the mixed nucleic acid fractions of these preparations. A male Wistar rat was killed by a blow on the head. The small intestine was removed immediately, chilled and cut into 10 cm segments. These were flushed free of contents with the ice-cold medium described below and, after being split open, were applied to a cold glass slab, mucosa upwards. The mucosa was then removed with a microscope slide, as described by DICKENS9, suspended in 4-5 volumes of ice-cold medium and the strips of mucosa freed by gentle mixing. The tissue was washed several times by centrifuging at low speed followed by resuspension in the cold medium. The volume was made up to approximately 25 ml and the mucosa strips con-verted to a fairly homogeneous suspension by gently and repeatedly drawing the preparation into and expelling it from a syringe without a needle. The material was then strained through gauze. Throughout all operations the preparation was kept cold. The'small intestine from a 200 g rat provided a suspension containing 25-45 mS dry weight of tissue per 3.0 ml portion. Krebs-Ringer phosphate or bicarbonate solutions containing aureomycin (10 ppm) and glucose (2 mg/ml) were used as the suspending medium. The mucosa suspensions respired actively at a gradually declining rate for 3-4 hours with initial Qo2's of 6-8. Bacterial counts**, which did not exceed 4-5 • io 6 bacteria per ml at the end of 3 hours incubation, indicated that bacterial contamination in these preparations was not great and would not affect the results significantly. The suspension of mucosa was added in 3.0 ml portions to Warburg cups which contained 2.25 fiM of sodium formate-14C (8.1 • io 6 cpm) and during incubation at 370 C, oxygen consumption was routinely followed. After incubation, the tissue was recovered by centrifugation of the cup contents and the purines of the acid-soluble and nucleic acid fractions obtained by the perchloric acid extraction method described by LEPAGE4. The purines of each fraction were separated by paper chromatography using isopropanol-HC1 1 0 . The specific activities of the purines of the nucleic acid fraction were unaltered by re-chromatography in butanol-ethanol-water11, but it was found necessary to rechromatograph the acid-soluble purines in this solvent. The purine areas on the chromatograms were located with U.V. light, and for analysis a disc, 26 mm in diameter, was punched out of each area. After determining the radioactivity of the discs, they were extracted with 0.1 M H Q and the purine content of the extracts measured in the usual way by U.V. absorption. The radioactivity determinations were corrected for absorption by the paper discs with empirically determined factors. The uptake of formate in the purines was considered to be a measure of de novo synthesis in this system. That radioactive formate was readily incorporated into the purines of both the acid-soluble nucleotides and nucleic acids is shown in the data of Table I which is typical of a T A B L E I PURINE SYNTHESIS BY REPLICATE SAMPLES OF AN INTESTINAL MUCOSA SUSPENSION* Specific activity in cpmjmg • io~* pj^fey Acid-soluble fraction** Nucleic acid fraction*** adenine guanine adenine guanine I 50-7 3-7 0.41 0.16 2 53-7 3-3 0.40 0.15 3 49-9 3-5 °-35 0.14 4 42.0 3-i o-34 0.13 5 46.5 2.4 0.40 0.15 * Incubated at 37° C for 3 h with 2.25 fiM sodium formate-1 4C (8.1 • i o 6 cpm). ** Chromatographed in isopropanol-HCl only. * * * Average of duplicate determinations. 442 SHORT COMMUNICATIONS, PRELIMINARY N O T E S V O L . 18 (1955) number of such experiments. The variation in the replicates indicates that the present method of preparation does not produce a completely homogeneous suspension. Aureomycin was shown to have no effect on the rate of incorporation of formate by the purines. Glycine-1- 1 4C is also incorporated into the purines of this system but at about half the rate of formate. The addition of glycine, glycinamide and glutamine, singly or in combination, appeared to have no significant effect on the incorporation of formate. The data of Table I and the rate study shown in Table II (in which a different mucosa preparation was used) indicate that the purines of the acid-soluble fraction are synthesized rapidly. This observation is in accord with reports of the early labelling of the nucleotide pool in i i i i ;o 1 2 ' 1 3 > 1 4 and in vitro by purine precursors4 and free purines 1 5. Adenine was more active than guanine in both fractions, a finding in agreement with the observations of TOTTER3 and ABRAMS8 for nucleic acid purines derived from formate- 1 4C in their in vitro experiments with bone marrow. In contrast, LEPAGE 4 has reported that in mouse liver and mouse tumors glycine-2- 1 4C is incorporated to a greater extent in guanine. T A B L E II RATE OF PURINE SYNTHESIS IN AN INTESTINAL MUCOSA SUSPENSION* Time of incubation h Specific activity in cpmlmg • icr -4 Acid-soluble fraction Nucleic acid fraction adenine guanine adenine guanine °-5 1.2 O trace O I.O 1.8 trace trace trace 2.0 6-5 0.7 0.16 0.07 3-0 I3.6 0.29 0.12 4.0 I7.6 !-9 033 0.22 * The values presented are averages of duplicate incubations made for each period. Each vessel contained 2.25 /j,M of sodium formate- 1 4C (8.1 • i o 6 cpm). It has been shown that rat intestinal mucosa, prepared in the form of a suspension, will synthesize acid-soluble and nucleic acid purines and appears to be a useful mammalian system for in vitro studies of the synthesis of nucleic acids and their components. A. R. P. PATERSON*** Department of Biochemistry, Faculty of Medicine, S. H. ZBARSKY University of British Columbia, Vancouver [Canada) 1 P. REICHARD AND S. BERGSTROM, Acta Chem. Scand., 190 (1951) 5. 2 R. ABRAMS AND J. M. GOLDINGER, Arch. Biochem., 30 (1951) 261. 3 J . ' R . TOTTER AND A. N. BEST, Arch. Biochem. Biophys., 54 (1955) 31^-4 G. A . LEPAGE, Cancer Research, 13 (1953) 178. 5 C. E . STEVENS, R. DAOUST AND C. P. LEBLOND, /. Biol. Chem., 202 (1953) 177. 6 P. DROCHMANS, D. H. MARRIAN AND G. B. BROWN, Arch. Biochem. Biophys., 39 (1952) 310. 7 P. REICHARD, /. Biol. Chem., 179 (1949) 733. 8 R. ABRAMS, Arch. Biochem. Biophys., 33 (1951) 436. 9 F. DICKENS AND H. WEIL-MALHERBE, Biochem. J., 35 (1941) 7. 1 0 G. R. WYATT, Biochem. J., 48 (1951) 584. 1 1 H. K. BERRY, H. E . SUTTON, L. CAIN AND J. S. BERRY, University of Texas Publication, No. 5109 (1951) 22. 1 2 R. B. HURLBERT AND V. R. POTTER, /. Biol. Chem., 195 (1952) 257. 1 3 D. H. MARRIAN, Biochim. Biophys. Acta, 13 (1954) 2 ^ 2 -1 4 E . L. BENNETT, Biochim. Biophys. Acta, 11 (1953) 487. 1 5 E . GOLDWASSER, Nature, 171 (1953) I 2 ^-Received August 19th, 1955 * This work was supported by the National Research Council of Canada. ** The bacterial counts were kindly performed by Miss Aiko Hori . *** National Research Council of Canada Fellow, 1954-55. [Reprinted from the Journal of the American Chemical Society, 75, 5753 (1953).] Copyright J953 by the American Chemical Society and reprinted by permission of the copyright owner. The Preparation of 2-C14-Adenine BY A. R. P. PATBRSON AND S. H. ZBARSKY RECEIVED JUNE 25, 1953 As a preliminary to a study of the metabolism of the purines, with especial reference to the 2-position of the ring, the synthesis of adenine labeled in the 2-position with C u was undertaken. The method described by Shaw, 1 in which 4-amino-5-imidazole-carboxamidine is formylated and the product cyclized to give adenine, appeared to be suitable since by using C I 4-formic acid for the formylation 2-labeled adenine would be obtained. A n advan-tage of this method is that the isotope would be introduced at a late step in the synthesis, thereby minimizing losses of radioactive material. The undesirable feature of the method, however, as far as economy of radioactive material is concerned, is that the formylation is carried out with a large excess of 98% formic acid in the presence of acetic anhydride. This would necessitate the use of an inordinately large amount of C 1 4-formate in order to obtain adenine with appreciable radioactivity. In order to avoid the use of such a large excess of formic acid, experiments were carried out to study the feasibility of formylating the carboxamidine with an aqueous solution of formic acid, since such conditions have been used to formylate other amines. 2 3 The formylation reaction was found to proceed in 6 ¥ formic acid, and by using this modification it was possible to obtain 2-C 1 4 -adenine in yields of 60-65%, based on the car-boxamidine used. The unreaeted C l 4-formate can be recovered almost quantitatively and used for further preparations of labeled adenine. Method.—A solution of 0.200 g. of 4-amino-5-imidazole-carboxamidine dihydrochloride1 in 2.0 ml. of 2 0 % formic acid was placed in a reaction tube made from the outer member of a 24/40 standard taper joint. To this solution was added 0.170 g. of potassium formate, making the solu-tion 6.3 M with respect to formate. The solution was then boiled gently under reflux for 4 hours. The formamido de-(1) E . Shaw, J. Biol. Chem., 188, 439 (1950). (2) V. M Clark and H. M Kalckar, J Chem. Soc. 1029 (1950) (3) R. Abrams and L. Clark, T H I S JOURNAL, 73, 4609 (1951). rivative was not isolated but was cyclized to adenine by di-luting the solution to 8 ml. with water, adding sufficient potassium bicarbonate to neutralize the formic acid and to make the solution 0.5 M in bicarbonate, and then boiling under reflux for 1 hour. An amount of hydrochloric acid slightly less than that required to neutralize the solution was added, and the solution was concentrated under reduced pressure to a volume of 2-3 ml. On placing the solution in the refrigerator for several hours crude adenine precipitated. This material was collected by centrifugation, washed 3 times with ice-cold water and dried in vacuo. The super-natant and wash liquids were saved for the recovery of un-reaeted formate. The crude material was sublimed at 220° and a pressure of 1 mm. to give 0.083 g. of pure adenine, a yield of 61% based on the carboxamidine. Yields of 40-42% were obtained when the formylation was carried out with 4.0 M formic acid. Anal. Calcd. for C 6 H 6N 6: C , 44.44. Found: C , 44.27. The compound formed a picrate which melted with de-composition at 2 8 6 - 2 8 7 ° . 1 Admixture with picrate pre-pared from authentic adenine did not depress the m.p. The ultraviolet absorption spectrum and R< values obtained by paper chromatography4 were identical with those of authen-tic adenine. 2-C u-Adenine was prepared by using C14-potassium for-mate in the above procedure. In a typical experiment, adenine having a specific activity of 1.055 X 106 c.p.m. per mM was synthesized and the formate recovered from the re-action mixture had a specific activity of 1.025 X 106 c.p.m. per mM. The unreaeted C1 4-formate in the supernatant fluid and washings after separation of the crude adenine was recov-ered almost quantitatively by steam distillation.' For fur-ther use in preparing radioactive adenine, the steam distil-late was titrated with standard potassium hydroxide solu-tion and concentrated to small volume under reduced pres-sure. The concentrate was then transferred to the reaction tube and evaporated to dryness. The appropriate amount of 4-amino-5-imidazolecarboxamidine dihydrochloride was added, followed by hydrochloric acid equivalent to the for-mate present less the amount of hydrochloric acid present as the dihydrochloride salt. The procedure outlined above was then followed for the remainder of the synthesis. Acknowledgment.—This work was supported by grants from the National Research Council of Canada. (4) J . D. Smith and R. Markham, Biochem. J., 46, 509 (1950). (5) S. Weinhouse and B. Friedmann, J. Biol. Chem., 197. 733 (1952) DEPARTMENT OP BIOCHEMISTRY FACULTY OF MEDICINE THE UNIVERSITY OF BRITISH COLUMBIA VANCOUVER 8, BRITISH COLUMBIA, CANADA 

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