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In vitro studies of purine nucleotide biosynthesis in rat intestinal mucosa Somerville, Ronald Lamont 1957

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IK VITRO STUDIES  OF PURINE NUCLEOTIDE BIOSYNTHESIS  IN RAT INTESTINAL MUCOSA by Ronald Lamont Somerville A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n the Department of Bloohemistry We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA September, 19.57 ABSTRACT The de novo pathway of purine nucleotide biosynthesis in rat intestinal mucosa has been studied using respiring whole c e l l tissue suspensions and cell-free extracts prepared in isotonic sucrose or buffered saline. The antibiotic azaserine (O-diazoacetyl L-serine) was found to exert an inhibitory effect on the in vitro incorporation of formate-C 1 4 into the acid-soluble and nucleic acid purines of intact cells when injected intraperitoneally into rats one hour before the animals were sacrificed. Such an inhibition could not be demonstrated i f azaserine and a labelled precursor were added to incubation vessels simultaneously with tissue suspensions prepared from normal animals. This indicated that azaserine had to be in contact with a tissue constituent for a period of time before the enzymes concerned were affected. Studies with cell-free extracts of mucosal tissue showed that glycine-l-C 1 4 could be metabolized in small amounts to a form which displayed some of the properties of glyeinamide ribotide, a known intermediate in the de_ novo synthesis of Inosinic acid by pigeon li v e r enzymes. Because azaserine does affect de novo synthesis of purines and because isotopic glycine i s transformed to a form having properties in common with those reported for glyeinamide ribotide, i t appears possible that the enzymatic steps of purine synthesis in mucosal tissue are similar to those already known for pigeon l i v e r . The level of free glycine in mucosal tissue has been measured by a method not previously employed. The results obtained are intermediate i n value to those of the two groups who have studied the problem independently. An average value of 0.42.5 mg. free glycine per gram (fresh weight) of mucosal tissue was obtained. In presenting t h i s thesis i n p a r t i a l fuf#rfment of the requirements f o r an advanced degree at the University of B r i t i s h Columbia, I agree that the Library s h a l l make i t f r e e l y available for reference and study. I further agree that permission for extensive copying of t h i s t h e s i s for scholarly purposes may be granted by the Head of my Department or by his representative. It i s understood that copying or publication of t h i s thesis for f i n a n c i a l gain s h a l l not be allowed without my written permission. Department of The University of B r i t i s h Columbia, Vancouver 8, Canada., Date A CEH OWLED GMEHT S The author wishes to express his appreciation of the advice and encouragement given by Dr. S, H. Zbarsky during the course of this research. Thanks are also due to Dr. A. R. P. Paterson fo r his assistance with c e r t a i n phases of the work and to Dr. J. J . R. Campbell for the generous use of cold room f a c i l i t i e s i n the Department of Dairying. This research was supported by a grant from the n a t i o n a l Research Council. TABLE OF CONTENTS Page INTRODUCTION . . . . 1 EXPERIMENTAL A. MATERIALS AND METHODS I. Preparations from small intestine (a} Whole c e l l suspensions • 1$ (b) Homogenates and extracts 15 I I . Substrates and reference compound (a.) Substrates 16 (b) Reference compound 19 III. A n a l y t i c a l methods , 21 IV. Metabolism experiments (a) Respiring whole c e l l s of rat i n t e s t i n a l mucosa . . . . . . . . . . 21 (b) C e l l - f r e e extracts of rat i n t e s t i n a l mucosa 22 B. RESULTS I. E f f e c t of azaserine on de novo purine synthesis (a.) Azaserine added to tissue suspension 25 (b; Azaserine e f f e c t as shown by i n vivo preincubation 25 II . Attempted biosynthesis of a l i p h a t i c r i b o t i d e s (&) Extracts prepared i n is o t o n i c sucrose 28 (b) Extracts prepared i n buffered saline 50 II I . Glycine content of mucosal tissue 52 DISCUSSION . 54 SUMMARY 40 BIBLIOGRAPHY i 42 TABLES I. Metabolic origins of the positions of the purine r i n g structure 5 I I . P a r t i a l summary of published data regarding a l i p h a t i c r i b o t i d e s . 9 II I . Enzymes i d e n t i f i e d i n c e l l - f r e e mucosal preparat-ions . , 11 Page IV. Effect of azaserine on the incorporation of formate-C 1 4 into the acid-soluble and nucleic a c i d purines of r e s p i r i n g whole c e l l s of rat i n t e s t i n a l mucosa 24 V. E f f e c t of preincubation i n vivo with azaserine on the incorporation of l a b e l l e d precursors into the acid-soluble and nucleic acid purines of r e s p i r i n g whole c e l l s of rat i n t e s t i n a l mucosa 27 VI. F i x a t i o n of g l y c i n e - l - C 1 4 into a form not retained by Dowex .501 (NH 4) at pH 3.3.5 by isotonic sucrose extracts of mucosal tissue . . 29 VII. Paper chromatographic properties of glycine and GAR 30 VIII. F i x a t i o n of g l y c i n e - l - C ^ into a form not retained by Dowex 0^W (1H"4) at pH 3.35 by buffered saline extracts of mucosal tissue . . 31 IX. Glycine content of r a t i n t e s t i n a l mucosa . . . 33 FIGURE I. The de novo pathway of purine nucleotide biosynthesis . 5 ADDENDUM Dr. G. A. LePage, i n a personal communication to Dr. A. R. P. Paterson, has indicated that the compound previously identified by him as glyeinamide ribotide (GAR) i s actually formyl-glycinamide ribotide. These results are i n press (Cancer Research). INTRODUCTION N u c l e o t i d e s occupy p o s i t i o n s of paramount Importance i n c e l l u l a r p h y s i o l o g y because they f u n c t i o n as i n t e r m e d i a t e s i n the metabolism of c e r t a i n w i d e l y d i s t r i b u t e d coenzymes and of the p o l y n u c l e o t i d e s i d e o x y r i b o n u c l e i c a c i d (DNA) and r i b o -n u c l e i c a c i d (RNA). Many of the better-known n u c l e o t i d e coenzymes are of e s t a b l i s h e d importance i n e l e c t r o n t r a n s p o r t and carbo-hydrate metabolism, but i n r e c e n t years a group of newly char-a c t e r i z e d compounds have been d i s c o v e r e d and found to p l a y key r o l e s i n p h o s p h o l i p i d , amino a c i d and f a t s y n t h e s i s and break-down (1). The d e o x y r i b o n u c l e i c a c i d s are s i g n i f i c a n t p a r t i c i p -ants i n the mechanisms whereby the v a r i o u s forms of l i v i n g matter pass on to t h e i r progeny t h e i r v a r i o u s s p e c i f i c a l l y o r ganized p a t t e r n s of growth and metabolism and are prominent c o n s t i t u e n t s of the n u c l e i of a l l c e l l s (2,3). The r i b o n u c l e i c a c i d s , a l s o p r e s e n t i n a l l c e l l s , d e r i v e b i o l o g i c a l importance from t h e i r known involvement i n p l a n t v i r u s m u l t i p l i c a t i o n and morphogenesis, both of whieh are aspects of a t h i r d and l a r g e r problem, that of p r o t e i n s y n t h e s i s (4). The more prominent s t r u c t u r a l and metabolic f e a t u r e s of RNA and DNA have been e x p e r t l y reviewed i n a r e c e n t p u b l i c a t i o n by Cohn and V o l k i n (5). N u t r i t i o n a l s t u d i e s extending over a p e r i o d of h a l f a century have shown that w i t h the e x c e p t i o n of some f a s t i d i o u s m i c r o b i o l o g i c a l s p e c i e s , organisms are able to accomplish n u c l e i c a c i d b i o s y n t h e s i s from simple, r e a d i l y a v a i l a b l e met-a b o l i t e s . Workers u s i n g p u r i f i e d and f u l l y c h a r a c t e r i z e d d i e t s e s t a b l i s h e d t hat n u c l e i c a c i d f o r m a t i o n occurred w e l l i n the - 2 -presence of ammonia, phosphate, and simple carbon sources supplied i n the d i e t , although no indications concerning the mechanisms by which small molecules were assembled to form the complex polynucleotides were obtained from this type of study. Modern concepts of the steps involved i n the i n t e r -mediary metabolism of nucleotides can be said to date from the application of isotopic tracers as biochemical tools and the demonstration by Barnes and Schoenheimer (6) that admin-iste r e d -ammonium c i t r a t e l a b e l l e d nucleic acid purines and pyrimidlnes i n the rat and pigeon. With the exception of some very recent work (7) studies of nucleic acid metabolism have been mainly concerned with the purine-containing constituents of these maeromoleeules. The carbon and nitrogen sources of the various positions of the purine r i n g are known from i n vivo studies of u r i c acid pre-cursors and from i n v i t r o studies using pigeon l i v e r enzymes. This information i s summarized i n Table I . P a r t i a l e l ucidation of the mechanisms by which the i n d i v i d u a l carbons and nitrogens are assembled to form the purines, purine nucleotides and polynucleotides has been made possible through the use of i s o t o p i c tracers i n soluble enzyme systems coupled with the techniques of ion-exchange and paper chromatography and u l t r a v i o l e t spectrophotometry. More than one pathway of purine nucleotide biosynthesis i s known, but that which i s considered (45) to be quantitatively most im-portant i s the de novo pathway, which has been described i n TABLE I Metabolic origins of the positions of the purine r i n g structure. Purine P o s i t i o n Metabolic Source Reference 2 and 8 "formate" 8 4 -C00H (glycine) 9 -CH - ( " ) 9 7 glycine 10 3 and 9 glutamine (amide) 11 1 aspartic acid 12 i co 2 9 a series of publications from the laboratories of Greenberg and Buchanan (see figure I ) . Ribose-jj-phosphate (A), which may be formed oxi-datively from glucose-6-phosphate v i a 6-phosphogluconate and ribulose -5-phosphate (13)» anaerobically from fructoses-phos-phate (14), from ribose and ATP by kinase action (15)> or from ribose-l-phosphate by the action of phosphoribomutase (16), reacts with ATP to y i e l d 3-phosphoribosylpyrophosphate (B) by donation of a pyrophosphate group of ATP to carbon 1 of rlbose-5-phosphate. This reaction, discovered by Kornberg and co-workers (17) as well as independently by Remy et a l . (18) i s of considerable importance i n the u t i l i z a t i o n of free pyrim-idines and purines as well as being an i n i t i a l step i n the de novo synthesis of purine nucleotides (19,20,21). The next step involves the enzymatic pyrophosphorolysis of PRPP to l i b -erate glutamic acid, inorganic pyrophosphate and a product not - 4 -yet isolated from incubation mixtures which i s presumed to be 5-phosphor!bosylamine (C ) ( 22 ) . As demonstrated by Greenberg and his collaborators (23,24), the elements of glycine, then formate are added to produce glyoinamide r i b o t i d e (D) and formylglycinamide r i b o t i d e (E). These findings were rapidl y confirmed by Hartman et a l (25), who also implicated the a n t i -b i o t i c azaserine as exerting an i n h i b i t o r y action on the de  novo synthesis of purine nucleotides at a metabolic s i t e sub-sequent to the formation of FGAR. This compound i s metabolized further by enzymes present i n pigeon l i v e r to produce (od-K-formyl) -glycinamidine r i b o t i d e (F) through transfer of the amide nitrogen of glutamine to FGAR; ATP i s required f o r the conversion ( 2 6 ) . Elimination of the elements of water to ef f e c t r i n g closure of FGAM y i e l d s 5-aminoimidazole r i b o t i d e (G ) (27 ) , which then reacts with bicarbonate, forming 5- a mino-4-imidazole carboxylic acid r i b o t i d e (H) (28) . .The l a t t e r compound then reacts revers-i b l y with L-asparate i n the presence of ATP to produce 5-amino-4-imidazole-jI-suecinocarboxamide r i b o t i d e (I)(29), which i s cleaved i n a reaction thought to be i d e n t i c a l to that cata-lyzed by adenylosuccinase (50) to y i e l d 5-amino-4-imidazole carboxamide r i b o t i d e (J) and fumaric acid ( 51 ) . Inosinic acid (L), the f i r s t compound synthesized having a complete purine r i n g , i s formed by transfer of a formyl group to the amino function of AICAR followed by r i n g closure. IMP can be con-verted to 5.1-adenylic acid (N) v i a adenylosuccinic acid (M)(52) or to 5 T-guanylic acid (P) after oxidation to xanthosine d i -phosphate ( 0 ) ( 3 5 ) . FIG-TIRE I. The de novo pathway of purine nucleotide biosynthesis. o 9 HO-P-O-CH^  Q HOP-0-CHZ Q 0 HO-P-O-CH OH OH R-5»-P (A) C H O cw2 R-5-P FGAM (F) H 2 N N H R-5:P AIR (Gr) (o) \^ (L) MP » AMP-5 W A M P " " P o _ OP-O-p-OH 2 , 0 N H 2 OH OH OH OH PRPP (B) 0 CH ? CHO NH FGAR (E) V t O O C / N R-5:P C-AIR (H) 0 0 . R-5:P C H FAICAR (K) OH OH PRA (C) / N H 2 CH; R-5-P GAR (D) H O O C - C H C H ^ C O O H NH R-S-P SAICAR (I) R-5-P AICAR (J) - 6 -Other known pathways of purine nucleotide biosyn-thesis involve d i r e c t reaction of free purines with PRPP (19,20) as well as the conversion of purine nucleosides produced by nucleoside phosphorylase or phosphatase action to 3'-nucleotides by the action of ATP and s p e c i f i c kinases (34,33). Results published recently from the laboratory of Ochoa (36,37,38) describe the i s o l a t i o n and p u r i f i c a t i o n of an enzyme from Azotobacter v i n e l a n d i l which catalyzes the synthesis of highly polymerized polynucleotides from nucleoside 3'-diphos-phates with release of orthophosphate. The enzyme, named polynucleotide phosphorylase, has subsequently been described by other workers (39,40) and represents the f i r s t example of the net synthesis of RHA by c e l l - f r e e preparations. Experiments demonstrating the renewal of RHA nucleotides by c e l l - f r e e preparations. Experiments demonstrating the renewal of RHA nucleotides by c e l l - f r e e systems from animal tissue have also been reported (41,42,43,44), and Kornberg (43) has described c e l l - f r e e preparations from Escherichia c o l i which incorporate deoxynucleotides into DHA, apparently by increasing the chain s i z e . Many of the steps described i n the above discussion of the de novo pathway have been elucidated with enzymes ex-tracted from the l i v e r of pigeons, a species i n which purine metabolism i s very prominent, as shown by the high production of u r i c acid, which acoounts f o r over 90% of the nitrogen excreted. The phenomenon of uricotelism i s a general one for birds (46) and i s r e f l e c t e d i n t h e i r dietary requirement: - 7 -f o r g l y c i n e , one m o l e c u l e o f w h i c h i s r e q u i r e d f o r the ex-c r e t i o n o f t h r e e moles o f ammonia as u r i c a c i d . Thus g l y c i n e o c c u p i e s a p o s i t i o n i n a v i a n m e t abolism s i m i l a r i n c e r t a i n r e s p e c t s to t h a t o f a r g i n i n e i n mammalian m e t a b o l i s m . S t u d i e s o f the de novo pathway of p u r i n e n u c l e o t i d e f o r m a t i o n w i t h enzymes f r o m mammalian t i s s u e are c o n s p i c u o u s l y l a c k i n g , a l t h o u g h i s o t o p i c f o r m a t e , g l y c i n e , and 4<-amino-5-imidazole-carboxamide (47) are a l l known to be p u r i n e p r e c u r s o r s i n the i n t a c t r a t . I n d i c a t i o n s o f s i m i l a r i t i e s i n the b i o s y n t h e t i e mechanisms l e a d i n g to p u r i n e n u c l e o t i d e s have been o b t a i n e d w i t h enzymes from y e a s t (48) and E. c o l i (49), but a need s t i l l e x i s t s f o r c o m p a r a t i v e work w i t h o e l l ^ - f r e e systems from o t h e r s p e c i e s . C e r t a i n f e a t u r e s of the p h y s i o l o g i c a l and b i o c h e m i c a l a c t i v i t i e s o f the mucous membrane of the s m a l l i n t e s t i n e p o i n t e d to the use o f t h i s t i s s u e as a source o f enzymes f o r the s t u d y o f de_ novo p u r i n e n u c l e o t i d e f o r m a t i o n i n the mammalian organism. The d a i l y r a t e o f c e l l f o r m a t i o n i n the e p i t h e l i u m and l a m i n a p r o p r i a o f r a t s m a l l i n t e s t i n e was a s s e s s e d h i s t o l o g i c a l l y by Le B l o n d and Stevens (.50), who showed t h a t the e n t i r e i n t e s t i n a l e p i t h e l i u m was renewed i n 1.35 to 1.57 d a y s . The h i g h r a t e o f c e l l p r o l i f e r a t i o n was c o r r e l a t e d w i t h the s y n t h e s i s o f deoxy-r i b o n u c l e i c a c i d u s i n g r a d i o a u t o g r a p h t e c h n i q u e s i n t i s s u e s e c t i o n s t r e a t e d w i t h r i b o n u c l e a s e (51) a f t e r a n i m a l s had been i n j e c t e d w i t h p 2 2 - l a b e l l e d i n o r g a n i c phosphate. E x a m i n a t i o n of u n t r e a t e d r a d i o a u t o g r a p h s showed t h a t l a r g e amounts of RNA were - 8 -also synthesized by i n t e s t i n a l mucosa. A n a l y t i c a l determin-ations of the concentration of DNA i n rat small intestine (52) demonstrated that, of 13 tissues studied, small intestine was exceeded only by thymus and bone marrow i n the amount present; an average of 129.2jj-g* of DNA-phosphorus per lOOmg of fresh tissue was detected. In common with l i v e r , pancreas, and r e t i c u l o e n d o t h e l i a l c e l l s , small in t e s t i n e has a high ( 7 3 . 2 ^ ^ RNA-phosphorus per 10G mg fresh tissue) concentration of RNA, which i s probably related to the glandular function of the organ. Thus we have an example of a rapidly d i v i d i n g mass of tissue which synthesizes large amounts of nucleic acid at a very rapid rate; that this synthesis may occur de_ novo from small precursors i s known from i n vivo studies showing that glycine ( 53 ) . formate (54) and phosphate (55) are i n -corporated into the polynucleotides. It has also been shown 14 i n this laboratory (56) that formate-C i s r e a d i l y incorpor-ated i n v i t r o into acid-soluble and nucleic acid purines. It was therefore of inte r e s t to see whether the intermediates i n de novo purine nucleotide synthesis by i n t e s t -i n a l enzymes are the same as those described for pigeon l i v e r , so a series of experiments were undertaken designed to demon-strate the occurrence of GAR and FGAR i n mucosal ti s s u e . Aside from their importance as intermediates i n purine nucleo-tide formation, these a l i p h a t i c r i b o t i d e s deserve attention because they are known to accumulate i n pigeon l i v e r enzyne preparations carrying out de_ novo synthesis of i n o s i n i c acid (25 ) , i n E h r l i c h ascites c e l l s ( 57 ) , and i n bacteria (58) TABLE II P a r t i a l Summary of Published Data Regarding A l i p h a t i c Ribotides Buchanan (59) Greenberg (60) LePage (57) Skipper (58) Source of enzyme Ethanol fractions of pigeon l i v e r extract Dowex-treated, dialyzed extraot of pigeon l i v e r acetone powders E h r l i c h ascites carcinoma Wild type Escherichia c o l i Assay used Ion exchange Chemical Ion exchange Paper chromat-ographic Compounds isolat e d GAR and FGAR (Uo isomers) GAR and FGAR (Two isomers of each) GAR only Formylglyoinamide riboside and FGAR Effeot of bicarbon-ate Synthesis of GAR and FGAR enhanced i n presence of bicarbonate Omission of bicarbonate i n -creases a l i p h a t i c r i b o t i d e f o r -mation Orcinol reaction Hydrolysis of a l l samples i n 1.3K HCl for 40 min. necessary to give correct values Hydrolysis for 40 min. i n 1.5E HCl gives low y i e l d s . Periodate values correct Orcinol reaction works d i r e c t l y (Ho hydrolysis) — Hydrolysis products GAR: glycine, ammonia, free ribose Formylglyc inamide riboside: glycine, glycinamide, X Behaviour on ion-exchange columns --- Formylglycinamide riboside held + by Dowex 50 (2JH4) Formylglycinamide riboside i s non-ionizable - 10 -when these systems are poisoned by the a n t i b i o t i c azaserine ( O-diazoacetyl L-serine). In addition, c e r t a i n anomalies and contradictions exist i n the l i t e r a t u r e regarding the chemical and biochemical behaviour of GAR, FGAR, and t h e i r derivatives, so i t was deemed advisable to reinvestigate t h e i r properties as much as possible with a new system. Some of the outstanding differences are shown i n Table I I , i n addition to some other pertinent information. Ho integrated studies of the general enzymology of i n t e s t i n a l mucosa have been carried out, although a number of workers have i d e n t i f i e d i n d i v i d u a l enzymes i n preparations from this t issue. Most of the enzymes studied were ones which might be assumed to p a r t i c i p a t e i n the digestive process; work aimed at d i s c l o s i n g the presence of those functioning anabolically has not been extensively undertaken. A thorough l i t e r a t u r e search revealed 17 instances where c e l l - f r e e systems from mucosa had been used (Table I I I ) . Comparison and c o r r e l -ation of the methods of enzyme preparation described i n the l i t e r a t u r e pertinent to Table III provided the basis f o r some of the methodology used i n the present study. Intestine i s not a popular source of enzymes for a number of reasons, one of which i s the occurrence of many di f f e r e n t c e l l types arranged i n such a way as to prevent f r a c t i o n a t i o n to y i e l d one homogeneous type. The i n t e s t i n a l "membrane", through which nutrients pass during the digestive process, consists of e p i t h e l i a l c e l l s , i n t e r s t i t i a l t issue, - 11 -TABLE III Enzymes Identified i n Cell-Free Mucosal Preparations Enzyme Reference leucine aminoexopeptidase 61 tripeptidse 61 glycyl-L-leuoine dipeptidase 62 prolidase 63 a l k a l i n e phosphatase 64.65 66 phosphodiesterase adenosine deaminase 67 cholinesterase 68,69,70 thymidine phosphorylase 71 r i b o f l a v i n phosphokinase 72 inosine diphosphate kinase 73 hexokinase 74 pentokinase 75 ,transoarbamylase 76 xanthine oxidase 77 pentose phosphate isomerase 78 and an endothelium of blood and lymph c a p i l l a r y composed of r e t i c u l o e n d o t h e l i a l c e l l types. The v i l l u s , one of the main morphologically distinguishable structures, consists of a frame-work of r e t i c u l a r tissues containing many leucocytes, separated from the lumen of the gut by a continuous layer of columnar c e l l s . The surface c e l l s are of four kinds: " p r i n c i p a l " or "columnar absorbing" c e l l s , goblet c e l l s (which secrete mucus)1 Paneth c e l l s , and enterochrome or Basal granular c e l l s . The function of the l a s t two types i s at present obscure ( 7 9 ) . The p o s s i b i l i t y of an enzymatic as well as physio-l o g i c a l and morphological difference between the d i f f e r e n t c e l l types of i n t e s t i n a l mucosa seems probable i n the l i g h t of the - 12 -o l a s s i c a l work of van Genderen and Engel ( 80 ) . These workers measured various enzyme a c t i v i t i e s i n thin sections cut with a microtome from the surfaoe of the v i l l i to the muscularis. Depending p a r t l y on the dietary state of the animals used, gradations i n enzymatic a c t i v i t y were observed i n tissue sectioned from rat duodenum and ileum. Unfortunately no further work along these l i n e s has been done, but i t seems l i k e l y that a p p l i c a t i o n of modern enzymologieal techniques to such a study would y i e l d s i g n i f i c a n t information about the physiology of i n t e s t i n a l mucosa, i n p a r t i c u l a r , one would expect to f i n d a high concentration of enzymes concerned with nucleic acid and protein synthesis i n the c e l l s of the crypts of Lieberkuhn ( 51 ) . Another d i f f i c u l t y i n working with mucosal extracts i s that of the rapid endogenous breakdown of proteins and nucleic acids by enzymes of i n t e s t i n a l and pancreatic j u i c e . This necessitates rapid work at low temperatures during and after extraction when studying an enzyme a c t i v i t y whose re-sistance to autolysis i s not known. A th i r d aspect of metabolic studies with mucosal extracts i s the p o s s i b i l i t y of excessive d i l u t i o n of radio-active substrates by substances already present i n a crude extract or produced endogenously under the conditions of incubation. Since glycine - 1-C l 4was to be used as tracer molecule i n the present work, i t was deemed necessary to redetermine the l e v e l of free glycine i n mucosal t i s s u e . - 13 -Todd and Talman (81) found free glycine levels ranging from 76 to 103 mg. % (fresh weight basis), depending on the nature of the d i e t . However, the r e s u l t s of these authors are open to some question since the method they employed fo r glycine analysis has been shown (83) to be highly susceptible to serious a n a l y t i c a l errors unless stringent precautions are observed. Awapara and co-workers have determined the free glycine concentration of rat ileum by two dimensional paper chromatographic methods ( 84 ) . Calculations based on the data presented by these authors indicate a free glycine l e v e l of 12.1 mg. %. In the present studies, the microdiffusion method of Schwartz et a l . (85) has been used to obtain information regarding the free glycine l e v e l of rat i n t e s t i n a l mucosa. Experimental data collected during the course of the present investigations indicates that a l i p h a t i c r i b o t i d e s are probable products of glycine metabolism i n c e l l - f r e e ex-tracts of rat i n t e s t i n a l mucosa and that azaserine i s e f f e c t -ive i n preventing de novo synthesis of purine nucleotides from isotopic glycine and formate. Mucosal extracts prepared i n i s o t o n i c suerose or buffered saline and incubated at 37°C« In the presence of ribose-j>-phosphate, glutamine, a source 14 of high-energy phosphate and glycine-l-C produce small quantities of material which i s not retained by Dowex 30W (HH4) at pH 3.35 and which behaves when ohromatographed on anion-exehange columns and f i l t e r paper i n a manner si m i l a r to - 14 -authentic glycinamide ribotide prepared from azaserine-inhibited E h r l i c h ascites carcinoma. Azaserine, injected i n t r a p e r i t o n e a l l y at a l e v e l of 20 mg./kg., was able to i n h i b i t the i n v i t r o incorpor-ation of formate-C 1 4" into the acid-soluble and nucleic acid purines of r e s p i r i n g whole c e l l s of i n t e s t i n a l mucosa prepared from the s a c r i f i c e d r a t , although no such inhib-i t i o n could be demonstrated when azaserine was added to c e l l suspensions prepared from the small intestines of untreated animals. Because de_ novo purine nucleotide biosynthesis i n i n t e s t i n a l mucosa i s susceptible to the i n h i b i t o r y e f f e c t s of azaserine and since c e l l - f r e e systems from th i s tissue do produce small amounts of material from l a b e l l e d glycine which resembles glycinamide r i b o t i d e , one may ten t a t i v e l y conclude that the pathways by which small molecules are assembled to form purine nucleotides i n mucosal c e l l s are simi l a r to those already known from studies with pigeon l i v e r enzymes. The question of the l e v e l of free glycine i n In-t e s t i n a l tissue has been reinvestigated. The r e s u l t s ob-tained show glycine to be present at an average concentration of 4 times that previously reported. - 13 -EXPERIMENTAL A. MATERIALS AND METHODS I. Preparations from small in t e s t i n e (a) Whole C e l l Suspensions Male rats of the Wistar s t r a i n weighing between 180 and 200 gm. were used throughout the present study. The animals were k i l l e d by a blow on the head, decapitated and bled, and the small intestine from the pylorus to the caecum was removed, cut into 20cm. segments, and c h i l l e d on cracked i c e . The i n t e s t i n a l contents were flushed out using c h i l l e d isotonic sodium chloride. The mucosa was removed by s l i t t i n g the i n t e s t i n a l segments lengthwise, spreading them out mucosa side up on a cold porcelain surface, and scraping with a freshly broken microscope s l i d e , as recommended by Dickens and Weil-Malherbe ( 8 6 ) . For use i n manometric experiments, 9 volumes of Krebs-Ringer phosphate-saline buffer were added to the mucosa and a uniform suspension prepared by repeated passage through a syringe without a' needle. (b) Homogenates and Extracts A l l operations p r i o r to incubation were carried out at 0 - 5 ° . Rat i n t e s t i n a l mucosa, removed as above, were d i s -integrated either by mechanical s t i r r i n g at low speed for 15 minutes (65) or by treatment with a Potter-Elvejhem homo-genizer with a t e l f l o n pestle (10 passes). In early experi-ments isotonic sucrose was used as a suspending medium (3 parts of medium to 1 of tissue) but t h i s was discontinued - lb -when i t was found that a greater u t i l i z a t i o n of isotopic glycine occurred i f the buffered s a l t s medium of Schulman et a l . ( 87 ) was employed. Higher concentrations of tissue (2:1) were l a t e r adopted, and mechanical s t i r r i n g was d i s -continued when h i s t o l o g i c a l examination^ revealed the presence of i n t a c t c e l l s and clumps of c e l l s i n the homo-genate. The disrupted c e l l suspension was centrifuged at 600X g. i n a S e r v a l l SS-1 angle centrifuge to remove a con-siderable portion of the insoluble material: the opalescent supernatant was then transferred to l u s t e r o i d u l t r a c e n t r i -fuge tubes and spun at 1 0 0 , 0 0 0 X g. i n a Spineo Model £ ultracentrifuge (preparative head) for 30 minutes. At the end of this time a brown p e l l e t had sedimehted out to leave a cl e a r , straw-coloured supernatant, which was used d i r e c t l y as a source of enzymes. The protein concentration of t h i s solution, measured speetrophotometrieally ( 8 8 ) , was J^-^Omg./ml. I I . Substrates and Reference Compounds (a) Substrates The ATP used was a product of the N u t r i t i o n a l Biochemical Corporation; Descending chromatography on Whatman #1 f i l t e r paper using isobutyric a c i d : ammonia: water (89) as solvent resolved an applied spot (j? drops of a 1% solution) into two ultraviolet-absorbing spots of equal i n t e n s i t y corresponding i n mobility to that expected §• preparations were mounted and stained by Miss Ruth Hofer of the Department of pathology through the cooperation of Dr. H.E. Taylor, who interpreted the s l i d e s . - 17 -for ATP and ADP. Neither adenosine, adenine, noi? AMP were present as contaminants. Ribose-5-phosphate was a product of Schwartz Laboratories and was supplied as the c r y s t a l l i n e barium s a l t . For use, the compound was converted to the potassium s a l t according to the following procedure: 5.4 millimoles of the barium s a l t were dissolved i n 9 ml. of d i s t i l l e d water and 4.5 ml. of IN HCl. This solution was passed through a column of Dowex 50W (200-400 mesh, X8, hydrogen form) of dimensions 1.2 cm. by 5 cm. The column was then washed with 21 ml. of water and the washings and i n i t i a l eluate combined. An aliquot of this solution was removed for pentose analysis and paper chromatography and the remainder neutralized with a measured volume of 20% KOH. After analysis by the o r c i n o l method (90) of the aliquot removed using 1-arabinose as standard, d i s t i l l e d water was added to the main solution so that the f i n a l concentration of pentose was 0.05M. This solution was frozen u n t i l required as a substrate. A sample of the free acid was chromatographed on Whatman f l f i l t e r paper which had been previously washed with 1% oxalic acid and d i s t i l l e d water. The solvent used was n-butanol: propionic ac i d : water, which i s known (91) to d i f f e r e n t i a t e ribose-5-phosphate from ribulose -5-phosphate, the chief contaminant of such commercial preparations. Phosphate-containing compounds were located on the chromat-ogram using the Hanes-Isherwood molybdate spray according to the procedure of Bandurski and Axelrod (92). No phosphate-- 18 -containing impurities were detected. 3-phosphoglyceric acid was a product of the Nu t r i t i o n a l Biochemical Corporation supplied as a c r y s t a l -l i n e barium s a l t . Assuming the compound to be the d i -hydrate ( 93 ) , a 0.1M solution of the potassium s a l t was prepared for use as follows: 3.576 gm. were dissolved i n 16 ml. of d i s t i l l e d water and 16 ml. of In HCl. This solution was passed through a column of Dowex 50W (22-400 mesh, x8 , hydrogen form, 1 cm. by 13 em.). The column was washed with 50 ml. of d i s t i l l e d water and the i n i t i a l eluate and washings combined, neutralized with 20f. KOH, and diluted to 100 ml. This solution was frozen u n t i l required for use. L-glutamine was a product of the N u t r i t i o n a l Bio-chemical Corporation. Because of the reported (94) occurr-ence of glutamic acid and asparagine as contaminants In commercial samples of glutamine, a 1% aqueous so l u t i o n of the sample was subjected to descending paper chromatography using phenol-water as developing solvent (95). When the chromatogram was developed with ninhydrin* a small spot with Rf value i d e n t i c a l to that of authentic glutamic acid was observed. L-azaserine was a g i f t from Parke, Davis and Company^. A soluti o n of 1.3 mg. i n 200 ml. of 0.1M sodium § The azaserine was obtained through the courtesy of Dr. L. M. Long. phosphate buffer was examined spectrophotometrically using a Beckman DK-2 Ratio Recording Spectrophotometer. As ex-pected ( 9 6 )i a sharp absorption maximum was observed at 250 millimicrons, having an E ^ ° c m # of 1510. The l a t t e r value i s somewhat higher than that i n the l i t e r a t u r e , poss-i b l y because the reading was taken at pH 7 .4 instead of pH 7 . 0 . 14 Formate-C was obtained from Atomic Energy of o Canada, Limited. Samples showed a c t i v i t i e s of 3 - 5 X 10 counts/min./millimole when measured as i n f i n i t e l y thin samples i n a windowless gas-flow counter. G l y c i n e - l - C 1 4 was synthesized by Dr. A.r-R. P. Paterson* Paper chromatography i n phenol-water revealed one radioactive spot which coincided exactly with a well-defined ninhydrin-positive area. The s p e c i f i c a c t i v i t y , as determined by count-ing of an aliquot and photometric analysis (97) was 7.4 X 1 0 * counts/min./micromole. (b) Reference Compound It was deemed advisable to have an authentic sample of glyeinamide r i b o t i d e available as a chromatographic stand-ard, so a preparation of this compound was undertaken using an adaptation of the method of Greenlees and LePage (57)» even though some doubt now exists as to whether the compound isolated by these authors i s actually GAR#. Four Swiss mice § G. A. LePage, personal communication to Dr. A. R. P. Paterson. - 20 -(UBC s t r a i n ) tearing 7-day Eh r l i e h ascites carcinoma were injected i n t r a p e r i t o n e a l l y with 5 micrograms each of azaserine as a solution i n isotonic s a l i n e . An hour l a t e r each animal 14. , was injected with an aqueous solut i o n of glycine-l-C (72 micrograms, containing 6.51 J. 10^ eounts/min.). After a further hour had elapsed, the animals were k i l l e d by c e r v i c a l d i s l o c a t i o n , the ascites f l u i d removed, oentlifuged to separate the c e l l s , and the supernatant discarded. The pooled c e l l s were extracted twice at 0° C. with 2% HC104 fb£ c l 5 minute periods and the acid extract neutralized with 20% KOH, using bromcresol purple as an in t e r n a l i n d i c a t o r . The KC10 which preci p i t a t e d was discarded and the extract applied to a Dowex 1 column (200-400 mesh, formate form, 1 cm. by 10 cm.). E l u t i o n and c o l l e c t i o n of f r a c t i o n s was carried out as de-scribed by Greenlees and LePage (57) and each f r a c t i o n was analyzed for u l t r a v i o l e t absorption at 260 millimicrons and for r a d i o a c t i v i t y . Two peaks of r a d i o a c t i v i t y were detected: the minor one, containing approximately 4.6 ZI10 4 counts/min., appeared i n tubes 7-16 i n c l u s i v e ; the major one, containing 5.7 X 10* counts/min., was eluted into tubes 24-50. Both peaks were freed of ultraviolet-absorbing impurities by l y o p h i l i z a t l o n of the pooled contents of the tubes involved and rechromatography on Dowex 1 (200-400 mesh, formate form* 1 cm. by 20 cm.). The major peak, assumed to be GAR, gave positi v e tests for pentose and phosphate and migrated as a single radioactive spot when subjected to paper chromatography using isobutyric a c i d : ammonia: water as solvent. The - 2 1 solution containing the compound was l y o p h i l i z e d , taken up i n 2 ml. of d i s t i l l e d water, and stored at 2 ° C. II I . A n a l y t i c a l Methods Determination of free glycine. Descending paper chromatography using phenol-water as developing solvent demonstrated the presence of appreciable quantities of free glycine i n the supernatant f l u i d remaining af t e r p r e c i p i t a t i o n of the proteins of soluble extracts of rat i n t e s t i n a l mucosa with phosphotungstic a c i d . A precise measure of the quantity of glycine present was made possible by adapt-ing the microdiffusion method of Schwartz ejt a l . (85) for the determination of plasma glycine to mucosal extracts. Highly reproducible r e s u l t s were obtained over a range of concentrat-ions from 0 - . 1 0 micromoles of glycine/ml. Photometric measure-ments were made with a Beckman Model B spectrophotometer. IV. Metabolism Experiments (a) Respiring whole c e l l s of rat i n t e s t i n a l mucosa. Mucosal suspensions prepared i n Krebs-Ringer phosphate were added i n 3 ml. portions to Tifarburg cups containing sodium 1 4 rv formate-C and during incubation at 37 C., oxygen consumption was routinely followed. A f t e r incubation, the tissue was recovered by centrifugation of the cup contents and the purines of the acid-soluble and nucleic acid f r a c t i o n s obtained from the c e l l s by the method of Lepage (98) . The purines of each f r a c t i o n were separated by paper chromatography using isopropanol: HCl (99)» located on the dried - 22 -chromatogram with ultraviolet light, cut out, eluted from the paper with O.IH HCl, and rechromatographed. In order to bring the extracted compounds to constant specific activity, the acid-3oluble purines were rechromatographed in isobutyric acid: ammonia: water (89): the nucleic acid purines were rechromat-ographed in isopropanol: HCl: water. For analysis a disc 26mm. in diameter containing the respective purines was punched out, the radioactivity determined, and the purine concentration measured by ultraviolet spectrophotometry using a Beokman DZ«2 Ratio Recording Spectrophotometer after elution with 0.1H HCl. An empirically determined correction factor^ was employed to eliminate the self-absorption of the punchouts, thus bringing a l l counts to a common baseline. (b) Cell-free extracts of rat intestinal mucosa. To demonstrate aliphatic ribotide biosynthesis by crude extracts of rat intestinal mucosa, the assay of Hartman et a l . (5) was employed. These workers demonstrated that GAR and FGAR were not retained by columns of Dowex 50 (NH )^ at pH 3.34 and could thus be separated from unchanged glycine-l-C 1' which had been used as a substrate. In the present series of experiments, Dowex 50W (NH )^ columns, 0.8 X 6.0 cm., prepared and operated according to the procedures of Hirs, Moore and Stein (100) were employed. When these columns were eluted with 0.05M ammonium formate buffer at pH 3.35, glycine was eluted § As determined by Mr. K. G. Scrimgeour, the correction factor for Whatman #1 f i l ter paper was 2.7. in a symmetrical peak when 80-115 ml. of elutriant had. passed through. Incubation was carried out In 25 ml. Erlenmeyer flasks at 5 7 ° C. with shaking. Each vessel contained, in a volume of 5 ml., the following quantities of materials expressed in micromoles: g lycine- l -C 1 4 ( 21 ) , L-glutamine (24), L-azaserine ( 8 ) , potassium 5-phosphoglycerate ( 7 2 ) , potassium ribose-5-phosphate ( 3 2 ) , and disodium ATP (4-8). Where isotonic sucrose had been used as extracting medium, the following amounts of inorganic salts were also added: KHCO ( 9 0 ) , sodium phosphate buffer, 5 pH 7.4 ( 5 0 ) , KOI (200 ) , sodium formate (1?) and MgClg (16). The volume of mucosal extract used per vessel was 3 . 3 ml. After 90 min. the reaction mixture was deproteinized (unless otherwise specified) with 0.3 ml. of 30% trichloroacetic acid. The protein-free supernatants were applied to Dowex 50W (NH4) columns and the first 6 ml. of eluate collected. A suitable aliquot was then plated in duplicate, evaporated to dryness under an infra-red lamp, and assayed for radioactivity. B. RESULTS I. Effect of azaserine on de_ novo purine synthesis (a) Azaserine added to tissue suspension. Azaserine is reported to have a marked inhibitory effect on de_ novo synthesis of adenine and guanine in Ehrlich and 6C3HED ascites tumors (101) as well as in slices of Flexner-Jobling carcinoma and rat spleen (102) . Bennet e_t a l . showed 14 that azaserine inhibited utilization of formate-C and - 24 -,glycine-l-C" L* for purine synthesis i n the tumors, i n t e s t i n e s , l i v e r s and spleens of mice bearing Sarcoma 180 (103). Since azaserine also causes the accumulation of a l i p h a t i c r i b o t i d e s i n pigeon l i v e r enzyme systems synthesizing i n o s i n i c acid (59), i n E h r l i c h ascites c e l l s (57) and i n E. c o l i (58), i t was of interes t to test the p o s s i b i l i t y of an i n h i b i t i o n by azaserine of de novo purine biosynthesis In r e s p i r i n g whole c e l l s of rat i n t e s t i n a l mucosa. A metabolism experiment was therefore 14 carried out i n the Warburg apparatus using formate-C as a purine precursor. Levels of azaserine known to be s u f f i c i e n t to i n h i b i t de_ novo, purine synthesis i n the inta c t mouse (57) were used i n ce r t a i n incubation vessels. The results of t h i s experiment are shown i n Table IV. TABLE IV Eff e c t of added azaserine on the incorporation of formate-C into the acid-soluble and nucleic acid purines of r e s p i r i n g whole c e l l s of rat i n t e s t i n a l mucosa. 2.8 ml. of mucosal tissue suspension was Incubated at 37 C. for 90 minutes i n the presence of 10 micromoles of solium formate-C 1 4 (0.10 ml., containing 1.1 X 10' counts/min.). Azaserine, when added, was as an isotonic saline solution. Results are expressed as counts/min./ micromole. Treatment Aoid- Soluble Nucleic Acid Adenine Guanine Adenine Guanine Hone 5,630 728 878 357 Azaserine (1 microgram) 5,610 402 878 374 Azaserine (2 micrograms) 9,260 1,040 1,440 623 - 25 -The results of Table IY show that azaserine is not effective in preventing de novo purine synthesis from formate-Cl4 in whole c e l l suspensions of rat intestinal mucosa when substrate, inhibitor and tissue suspension are i n i t i a l l y present in the incubation vessel. G-reenlees and LePage (57) found i t necessary to preineubate ascites tumor cells with azaserine, in vitro or in vivo, before the cells were exposed to a labelled purine precursor, in order to demonstrate certain aspects of the effect of the antibiotic on de novo purine syn-thesis. Using glyeine -2-C l 4 as a purine precursor, these workers showed that azaserine reacted with a constituent of tumor cells during the f i r s t 8*10 minutes of contact to cause inhibition of de novo purine synthesis by what appeared to be an irreversible binding mechanism. This suggested that a similar situation might exist with respect to the enzymes of rat intestinal mucosa which carry out de_ novo purine nucleotide biosynthesis, so an in vivo preincubation with azaserine by means of intraperitoneal injection an hour before the death of the animal was adopted i n the next experiment. (b) Azaserine effect as shown by i n vivo preincubation. Bennett, Schabel and Skipper have published data showing that injected azaserine reduces the in vivo incorporation of glycine-l-C 1 4 and formate-C 1 4 into tumor and intestinal nucleio acids of cortisone-treated Wistar rats bearing sub-cutaneous implants of a human sarcoma (105). However, the same authors had previously shown that cortisone treatment alone produced a marked depression of formate-C incorporation into v i s c e r a l nucleic acids (104), so that the p o s s i b i l i t y of the azaserine-induced i n h i b i t i o n being an a r t i f a c t cannot be overlooked. The following experiment was therefore per-formed: Two adult male Wistar rats which had been l i t t e r mates were obtained. One was injected i n t r a p e r i t o n e a l l y with a saline solution of azaserine at a l e v e l of 20 mg./kg. while the other was untreated. After an hour had elapsed, both animals were s a c r i f i c e d , their small intestines removed and the mucosa used to prepare c e l l suspensions as previously described. The c e l l suspensions were incubated i n the presence of isotopic glycine and formate i n the Warburg apparatus f o r a period of 2& hours. At the end of this time the c e l l s were removed and treated to obtain the acid-soluble and nucleic acid purines, which were then p u r i f i e d by paper chromatography and assayed for s p e c i f i c a c t i v i t y . The r e s u l t s of this ex-periment are shown i n Table V. The data of this table c l e a r l y demonstrates that azaserine i s e f f e c t i v e i n preventing de_ novo purine nucleotide biosynthesis from formate-C 1 4. With g l y c i n e - l - C 1 4 as precursor, the i n h i b i t o r y effect of azaserine i s not evident except i n the case of the acid-soluble adenine; however, glycine incorporation was i n no case extensive, so that f a i l u r e to demonstrate an azaserine effect i n these cases i s probably related to the rather low uptake of l a b e l l e d precursor. Whether t h i s i s due to permeability effects or to excessive d i l u t i o n by endogenous TABLE V E f f e c t of p r e i n c u b a t i o n i n v i v o w i t h a z a s e r i n e on the i n c o r -p o r a t i o n of l a b e l l e d p r e c u r s o r s i n t o the 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 of r e s p i r i n g whole c e l l s of r a t i n t e s t i n a l mucosa. See t e x t f o r experimental d e t a i l s . R e s u l t s shown are counts/min./ micromole and are the averages o f d u p l i c a t e d e t e r m i n a t i o n s . P r e c u r s o r Treatment' A c i d -Soluble N u c l e i c . A c i d . Adenine Guanine Adenine Guanine Formate-C 1 4 , None 58,800 8,150 1,720 700 A z a s e r i n e 1 ,755 517 94 202 G l y e i n e - l - C 1 4 None 1,725 245 50 60 A z a s e r i n e I65 356 150 78 m a t e r i a l i s not known, although, as w i l l be shown l a t e r , c o n s i d e r -able amounts of f r e e g l y c i n e are presen t i n mucosal t i s s u e . In g e n e r a l , however, the r e s u l t s are i n accor d w i t h those p u b l i s h e d by other workers (57, 58) , namely that a z a s e r i n e does e f f e c t the enzyme system concerned w i t h the s y n t h e s i s of purine n u c l e o t i d e s from elementary p r e c u r s o r s . When these r e s u l t s a re considered i n the l i g h t of those of the p r e v i o u s experiment, i t appears that i t i s necessary f o r the a n t i b i o t i c to be i n c o n t a c t w i t h a c e l l u l a r c o n s t i t u e n t f o r a f i n i t e p e r i o d o f time before i n h i b i t i o n can be demonstrated. In t h i s r e s p e c t a l s o the mode of a c t i o n of a z a s e r i n e i s s i m i l a r to that r e p o r t e d f o r E h r i c h c e l l s . - 28 -I I . Attempted biosynthesis of a l i p h a t i o r i b o t i d e s A series of i n v i t r o experiments were car r i e d out using c e l l - f r e e extracts of rat i n t e s t i n a l mucosa to see i f d i r e c t evidence could be obtained for the p a r t i c i p a t i o n of GAR and FGAR i n the de novo pathway of purine nucleotide biosynthesis. Certain d i f f i c u l t i e s inherent i n the properties of the crude system employed prevented the obtaining of completely unequivocal evidence for a l i p h a t i o r i b o t i d e f o r -mation. However, isotopic glycine does appear to be metab-ol i z e d by mucosal extracts incubated i n the presence of azaserine to produce small quantities of material which display properties similar to those of GAR prepared from E h r l i c h a s c i t e s c e l l s . (a) Extracts prepared i n isotonic sucrose. Four separate experiments were performed using mucosal extracts prepared with isotonic sucrose as the sus-pending medium. The incubation procedures and conduct of the assays have been described under "Materials and Methods". The r e s u l t s of one such experiment are shown i n Table VI. The figures of this table show that a modest but f i n i t e f i x a t i o n of glycine has taken place. To ascertain whether GAR was present, the i n i t i a l eluate from a separate experiment where the isotope incorporation was more appreciable was evaporated to dryness, taken up i n a small volume of water, applied to the o r i g i n of a paper chromatogram and developed using isobutyric acid: ammonia: water as solvent. - 29 -TABLE ¥1 F i x a t i o n o f g l y c i n e - l - C i 4 i n t o a form not r e t a i n e d "by Dowex 50W (NR4) a t pH 3.35 by i s o t o n i c s u c r o s e e x t r a c t s o f mucosal t i s s u e . I n c u b a t i o n was c a r r i e d out f o r 90 minutes a t 3 7 0 C. 22.5 micromoles o f g l y c i n e , c o n t a i n i n g 7.2 X 1 0 4 c o u n t s / m i n . were used as s u b s t r a t e . F l a s k C ontents v Counts f i x e d F u l l system > Minus a z a s e r i n e Minus enzyme 511 384 GAR p r e p a r e d from E h r l i c h c e l l s and g l y c i n e were r u n on the same chromatogram as markers. R a d i o a c t i v e compounds were l o c a t e d a f t e r thorough d r y i n g w i t h the a i d o f a w i n d o w l e s s g a s - f l o w chromatogram s c a n n e r . GAR and the m a t e r i a l from the Dowex 50W (NfiJ) column had Rf v a l u e s o f 0.26 and 0.25, r e s p e c t i v e l y , whereas g l y c i n e had an Rf o f 0.46. When the r a d i o a c t i v e a r e a c o r r e s p o n d i n g to the "GAR" produced by mucosal e x t r a c t s was e l u t e d f r o m the chromatogram by d e s c e n d i n g chromatography, e v a p o r a t e d to d r y n e s s , r e s p o t t e d on a new chromatogram and d e v e l o p e d u s i n g b u t a n o l : 17.6N a c e t i c a c i d : w a t e r ( 1 0 5 ) , r a d i o a c t i v i t y c o u l d be d e t e c t e d o n l y a t Rf O .38 , w h i c h c o r r e s p o n d e d t o t h a t of g l y c i n e . T h i s would appear t o i n d i c a t e t h a t d e g r a d a t i o n o f any GAR p r e s e n t had o c c u r r e d a t some s t a g e subsequent to the f i r s t c h r o m a t o g r a p h i n g . The paper, c h r o m a t o g r a p h i c p r o p e r t i e s of g l y c i n e and GAR, as d e t e r m i n e d w i t h t h r e e d i f f e r e n t s o l v e n t systems, are summarized i n T a b l e V I I . - R O -TABLE VII Paper chromatographic properties of glycine and GAR. The composition of the solvents was as follows: Solvent 1: isobutyric a c i d : concentrated ammonium hydroxide: water (66: 1: 33); Solvent 2: n-butanbl: 17.6N acetic a c i d : water (2: 1: 1) ; Solvent 3: isopropanol: water: concentrated ammo nium hydroxide ( 7 0 : 30: 13 j . A l l 'ratios are in volume pro-portions. Solvent 1 Solvent 2 Solvent 3 Glycine GAR .26 .46 .16 .38 .72 .64 (b) Extracts prepared i n buffered s a l i n e . The use of isotonic sucrose as a suspending medium for mucosal extracts was discontinued when i t was discovered that the pH of the extract had a marked tendency to drop during the period from the time of extraction to the time of incubation. The buffered s a l t s medium described by Sehulman, Sonne, and Buchanan (87) was therefore adopted. In addition to improved control of pH during and after extraction, t h i s medium offered the added advantage of simplifying considerably the incubation procedure by obviating the necessity of adding c e r t a i n s a l t s to the incubation vessels. Table VIII shows re s u l t s obtained when mucosal extracts are prepared using the second type of medium. The data of this table again demonstrate a small but 1 4 d e f i n i t e f i x a t i o n of glycine-l-C" 1^ into a form which behaves on Dowex 50 ion-exchange columns i n a manner si m i l a r to that - C I -TABLE VIII Fixation of glycine-l-C into a form not retained by Dowex 50W (NH/) at pH 3 '35 by buffered saline extracts of mucosal t i s s u e . Incubation was carried out for 90 minutes at 37° C. 22 micro-moles of glycine, containing 6.5 X 105 counts/min. were used as substrate. The reaction was stgpped by placing the incubation vessels i n a deep freeze at -15 C. The frozen solutions were treated with 0.4 ml. of 12N HCl to p r e c i p i t a t e the proteins during thawing. The protein-free supernatants were placed d i r e c t l y on Dowex 50W (NH|) columns for assay. Counts i n i n i t i a l eluate F u l l System Boiled Control Difference 1,377# 560 817 reported f o r GAR. When the pooled radioactive material comprising the i n i t i a l eluate from two small Dowex 50W columns was adjusted to pH 9 with concentrated KOH, applied to a column of Dowex 1 (200-400 mesh, formate form, 1 cm. by 10 cm.) and eluted according to the procedure of Greenlees and Lepage ( 57 ) , one peak of radio-a c t i v i t y appeared i n pr e c i s e l y the same po s i t i o n on the ion-exchange chromatogram as authentic GAR prepared from azaserine-treated E h r l i c h c e l l s . Some ultraviolet-absorbing material was present as a contaminant of the peak. When the contents of the tubes comprising the peak were l y o p h i l i z e d , taking up i n a small volume of water, applied to the o r i g i n of a paper chromatogram and developed using isopropanol: water: concentrated § Average of two determinations. - 32 -ammonium hydroxide (106) as solvent, the r a d i o a c t i v i t y migrated as a single spot at the same rate as authentic GAR. In addition, six separate ultraviolet-absorbing spots appeared on the chromatogram, although none of these coincided with the peak of r a d i o a c t i v i t y . The r e s u l t s of the preceding sections obtained with c e l l - f r e e extracts of rat i n t e s t i n a l mucosa provide evidence of a preliminary nature that GAR i s a metabolite of glycine i n this system. A compound i s produced enzymatically i n small amounts which i s not retained by Dowex 50W (NH4) ion-exchange columns at pH 3»35i i s eluted from Dowex 1 (formate) columns at the same rate as authentic GAR prepared from E h r l i c h a s c i t e s c e l l s , and which migrates on paper i n two solvent systems at the same rate as authentic GAR. II I . Glycine content of mucosal tissue The o r i g i n a l purpose for undertaking the analysis of the free glycine content of i n t e s t i n a l mucosa was to correct for the d i l u t i o n of isotopic glycine by non-radioactive material present i n crude tissue extracts. After the f i r s t few analyses had been performed, i t became evident that the d i l u t i o n would be i n s i g n i f i c a n t insofar as the purposes of the assay were concerned. However, because the reported l i t e r a t u r e values (81, 84) did not agree with each other or with those i n i t i a l l y obtained i n the present work, a number of analyses were performed from time to time i n order to obtain a true picture of the amount of glycine present. The values obtained are shown i n Table IX. - 33 -TABLE IX Glycine content of rat i n t e s t i n a l mucosa. Analyses were performed by the method of Schwartz, Robertson and Holmes ( 8 5 ) . Results are expressed as mg. glycine/ gm. of fresh mucosal t i s s u e . Means of Preparation Glycine content Homogenizing 11 11 11 S t i r r i n g Average Reported (81) " (84) O.638 O.385 0.467 0.167 0.828 O.365 0.425 0 .76- 1.03 0.121# Some of the variations i n the r e s u l t s of Table IX are probably due to the absorption of water by mucin associated with mucosal tissue (107) during the preparation of the sample. It w i l l be noted, however, that most of the values obtained are intermediate between those of the two groups of authors who have studied the l e v e l of this amino acid previously. ft Calculated from the authors' data. - 34 -DISCUSSION The results of the present investigation show that de novo purine biosynthesis by whole c e l l s of rat i n t e s t i n a l muoosa i s susceptible to i n h i b i t i o n by azaserine only under certain conditions. In p a r t i c u l a r , a dramatic reduction i n the incorporation of formate-C 1 4 - into the acid-soluble and nucleic acid purines can be demonstrated i n mucosal tissue i f the animal i s injected on hour before death with azaserine at a l e v e l of 20 mg./kg. On the other hand, c e l l - f r e e extracts of i n t e s t i n a l mucosa incubated i n the presence of azaserine and suitable substrates do not produce correspondingly large amounts of a l i p h a t i c r i b o t i d e s when assayed by the procedure of Hartman, Levenberg and Buchanan ( 5 9 ) . The f a i l u r e of such a system to accumulate these metabolites may be ascribed to at l e a s t three reasons. The f i r s t of these i s the demonstrated a b i l i t y of c e r t a i n amino acids to a l l e v i a t e i n h i b i t i o n of growth of azaserine-treated E. c o l i . Tryptophan, phenylalanine and tyrosine (108) , as well as glutamic acid, glutamine and methionine (105) were a l l e f f e c t i v e at low concentrations i n reversing azaserine-induced i n h i b i t i o n of E. c o l i . With the exception of glutamine (109) . for which azaserine i s a compet-i t i v e i n h i b i t o r , no investigations have been made as to the mode of action of the remaining amino acids. Certain of those mentioned, namely glutamic acid, glutamine, phenylalanine and methionine, have been detected by paper chromatographic methods i n rat ileum. The study (84) was not an exhaustive one since 63.8% of the t o t a l amino nitrogen remained un-accounted f o r , so the p o s s i b i l i t y of free tryptophan and tyrosine also being present must be considered. One might therefore i n f e r that f a i l u r e to observe a large azaserine eff e c t with c e l l - f r e e mucosal preparations i s due to the presence of i n t e r f e r i n g amino acids. A second reason for non-accumulation of glycinamide ri b o t i d e may be that de_ novo synthesis of purine nucleotides i n the c e l l - f r e e system was reduced or i n h i b i t e d by the presence of free purines. That preformed purines may i n h i b i t the uptake of elementary precursors into the polynucleotides of r a t i n t e s t i n a l mucosa has been demonstrated by i n vivo experiments using i s o t o p i c a l l y l a b e l l e d glycine and formate. Abrams (54) showed that injected adenine brought about a 40/L i n h i b i t i o n of glyeine-l-C"'" 4' incorporation into i n t e s t i n a l polynucleotide adenine with a concurrent i n h i b i t i o n of glycine uptake into polynucleotide guanine of 5 l f . . Goldthwait and Bendich (53) investigated the ef f e c t of adenine on the incorporation of 14 C -formate into the v i s c e r a l nucleic acids of r a t s . By ad-ministering varying doses of unlabelled adenine to pairs of rats receiving simultaneously a standard dose of l a b e l l e d formate, these workers demonstrated a decrease of formate - r e -incorporation into nucleic acids. These two sets of r e s u l t s oan be explained by assuming either an i n h i b i t o r y e f f e c t of free adenine on nucleic aeid synthesis or a "sparing" action of adenine on the need f o r de_ novo synthesis from glycine or formate. The former p o s s i b i l i t y i s considered u n l i k e l y for two reasons: f i r s t , l a b e l l e d adenine i s known to be extensively incorporated into i n t e s t i n a l RNA and DNA (53 , 54 , 110); 14 secondly, simultaneous administration of adenine and formate-C produces a decreased formate incorporation into nucleic acid purines r e l a t i v e to thymine when compared to a s i m i l a r ex-periment without adenine. For example, i n intestine ( 5 3 ) , a f t e r administration of f ormate-C"1"4 and adenine, the r a t i o ^ o f adenine: thymine r a d i o a c t i v i t y was 0.75• When no adenine was employed, this r a t i o rose to 2.0. The "sparing" action of preformed purines on synthesis de novo from l a b e l l e d precursors has also been observed i n Lactobacillus casei (111) , yeast ( 112) , and E. c o l i ( 113) , thus suggesting a widespread occurrence of the phenomenon. Gots and Gollub (114) noted that i n E. c o l l , the synthesis of 4-amino-5-imidazole carboxamide r i b o t i d e , a known (115) intermediate i n the de_ novo synthesis of purine nucleotides j i s completely stopped by minute quantities of any purine that can be u t i l i z e d for normal nucleic acid synthesis. They suggest a mechanism of feed-back control of biosynthesis whereby the presence of u t i l i z a b l e purines i n h i b i t s de novo synthesis at a„level previous to AICAR formation. F a i l u r e - 37 -to detect the formation of large amounts of aliphatic ribo-tides in high-speed supernatants of rat intestinal mucosa incubated i n the presence of azaserine and suitable substrates may indicate that de novo purine synthesis in this system is inhibited at a stage prior to the formation of glyeinamide ribotide. In this connection, endogenous production of acid-soluble adenine and guanine during the period of incubation was considerable, no doubt because of the presence in mucosal tissue of enzymes which catalyze the breakdown of polynueleo-14 tides (116, 117). Incorporation of formate-C into acid-soluble or nucleic acid purines did not occur in crude c e l l -free extracts of rat intestinal mucosa. The present state of our knowledge of the enzymie mechanisms whereby such compounds as adenine and glycine are incorporated Into purine nucleotides would seem to indicate that pyrophosphorolysis of 5-phosphoribosylpyrophosphate to yield AMP (in the case of adenine) (20) or 5-phosphoribosylamine (in the ease of de_ novo synthesis) (118) is the site of compet-it i o n between the u t i l i z a t i o n of free purines and de novo synthesis. If i t is assumed that the de_ novo pathway is operative in cell-free extracts of mucosal tissue and that azaserine is able to affect the enzymes of this pathway in intestine, a third explanation for the lack of aliphatic ribotide accumulation then becomes possible. Azaserine is known to affect two enzyme-- 38 -catalyzed reactions of the de novo pathway, the condensation between PRPP and glutamine to y i e l d PRA, glutamic acid and inorganic pyrophosphate, and the transfer of the amide nitrogen of glutamine to FGAR to produce FGAM. Studies with pigeon l i v e r enzymes have shown that the l a t t e r reaction i s more susceptible than the former to the action of azaserine (109), although PRA formation was d e f i n i t e l y i n h i b i t e d by the presence of azaserine. Goldthwait (22), using PREP u t i l i z a t i o n as an index, showed that azaserine i n h i b i t e d PRA production dZ% i n the presence of an equimolar amount of glutamine, whereas Levenberg e_t a l . (109), measuring glutamate production spec-trophotometrically, observed only a 48f. i n h i b i t i o n for the same reaction. The degree of i n h i b i t i o n depended on the r e l a t i v e i n i t i a l concentrations of azaserine and glutamine. Since a detailed study of the enzymology of the de novo pathway i n i n t e s t i n a l mucosa has not been carried out, i t must be considered possible that azaserine could i n h i b i t the PRPP-glutamine reaction to such an extent that very l i t t l e PRA would become available to react with l a b e l l e d glycine to form l a b e l l e d GAR. In any event, the above three explanations w i l l have to await further p u r i f i c a t i o n of the enzyme system before they can be subjected to experimental t e s t . Such p u r i f i c a t i o n would appear to involve removal of nucleic acid contaminants which contribute to the large endogenous production of acid-soluble purines as well as extensive d i a l y s i s to eliminate any amino acids and coenzymes which might adversely a f f e c t the sub-strates added to the reaction mixture. - 39 -The values obtained for the free glycine l e v e l of rat i n t e s t i n a l mucosa are intermediate between those previously reported. It i s f e l t that the sup e r i o r i t y of the a n a l y t i c a l method employed over those of past workers has resulted i n a closer approximation to the actual concentration of glycine i n i n t e s t i n a l mucosa. - 40 -SUMMARY 1. When the a n t i b i o t i c azaserine i s added to r e s p i r i n g whole-c e l l suspensions of rat i n t e s t i n a l mucosa prepared i n Erebs-Ringer phosphate buffer, there i s no i n h i b i t i o n of de novo purine nucleotide biosynthesis from isotopic formate. 2. If azaserine i s injected i n t r a p e r i t o n e a l l y one hour before the death of the animal, i t i s possible to demonstrate an almost complete i n h i b i t i o n of formate uptake Into the tissue 14 purine nucleotides. Incorporation of glycine-l-C into the ; aeid-soluble adenine i s also greatly decreased. 3 . A study has been made of the a b i l i t y of crude extracts of rat i n t e s t i n a l mucosa prepared either with isotonic sucrose or 14 buffered saline to incorporate glyelne-l-C into a form not retained by Dowex 50W (NH4) at pH 3 . 3 5 . This assay method has been previously applied by other workers as a means of detecting glycinamide r i b o t i d e formation i n tissue extracts. Small amounts of l a b e l l e d glycine were incorporated into such a form i n a number of experiments. 4. The paper chromatographic behaviour of glycine and glycinamide ri b o t i d e prepared from azaserine-inhibited E h r l i c h ascites c e l l s has been studied i n three d i f f e r e n t solvent systems. 5. Paper chromatographic and ion-exchange methods have been used to study the material produced from l a b e l l e d glycine by mucosal extracts. Although the material was degraded to what appeared to be glycine when chromatographed on paper using isobutyrie acid: ammonia: water as solvent, i t was shown to behave on Dowex l(formate) ion-exchange columns - 41 -and i n two solvent systems on paper i n a manner si m i l a r to that of material prepared from E h r l i c h ascites c e l l s . It i s therefore tentatively suggested the glycinamide r i b o t i d e i s a metabolite of glycine i n i n t e s t i n a l mucosa. 6 . The l e v e l of free glycine i n mucosal tissue has been reinvestigated by a modified microdiffusion method. The r e s u l t s obtained show that t h i s amino acid i s present In an average concentration of 0.425 mg. per gram of fresh tissue. This figure i s intermediate i n value to those reported i n the l i t e r a t u r e . - 42 -BIBLIOGRAPHY 1. Kalckar> H. M, and Klenow, H . , Ann. Rev. Biochem. 2£, 527 ( W 4 ) . 2 . Hotchkiss, R. D . , in The Nucleic Acida, vol. II, ed. by Chargaff, E . , and Davidson, J . N. Academic Press, New York. 1955. p. 435. 3 . Butler, J . A. V . , and Davidson, P. F . , in Advances in Enzymology, vol. 18, ed. by Nord, F. F. Inter science, New York. 1957. p. 161. 4. Brachet, J . , in The Nucleic Acids, vol. II, ed. by Chargaff, E . , and Davidson, J . N. Academic Press, New York. 1955. P. 475. 3 . Cohn, W. E . . and Volkin, E . , Ann. Rev. Biochem. 26 , 491 (1957) . 6. Barnes, F. W., and Schoenheimer, R., J . Biol . Chem. 151, 123 (1943) . 7. Carter, C. E . , Ann. Rev. Biochem. 25 , 123 (1943) . 8. Sonne, J . C , Buchanan, J . M., and Delluva., A . M . , J . Biol . Chem. 173, 81 (1948) . 9* Buchanan, J . M., Sonne, J . C , and Delluva, A. M. , J . Biol . Chem. 173, 69 (1948). 10. Shemin, D., and Rittenberg, D., J . Biol . Chem. 16J7, 875 (1947) . 1 1 . Sonne, J . C , Lin, I . , and Buchanan, J . M. , J . Am. Chem. Soc. 75 , 151b (1953) . 12. Levenberg, B. , Hartman, S. C , and Buchanan, J . M. , Federation Proc. 14, 243 (1955) . 13 . Horecker, B. L . , Smyrniotis, p. Z . , and Seegmiller, J . E . , J . Biol . Chem. 193, 383 (1954) . 14. Racker, E . , de la Haba, G. , and Leder. I. G. , Arch. Biochem. Biophys. 48 , 238 (1954) . 15. Lampen, J . 0 . , in Phosphorus Metabolism, vol. II, ed. by McElroy, W. D . , and Glass, B. , Johns Hopkins Press, Baltimore. 1952. p. 3^3 • l b . Klenow, H. , Arch. Biochem. Biophys. 46 , 186 (1953) . - 43 -17..Kornberg, A . , Liebermann, I . , and Simms, E. S., J . Am. Chem. Soo. 76, 2027 18. Remy, C. H., Remy. W. T . , and Buchanan, J . M. , J . Biol . Chem. 217, 885 (1955) . 19. Korn, E. D., Remy, C. K . , Wasilejko, H. C . . and Buchanan, J . M. , J . Biol . Chem. 217, 875 (1955) . 20. Kornberg, A . , Liebermann, I . , and Simms, E. S,, J . Biol . . Chem. 213_, 417 (1955) . 21 . Liebermann, I . , Kornberg, A . , and Simms, E. S., J . Biol . Chem. 215, 403 (1955) . 22 . Goldthwait, D. A . , J . Biol . Chem. 222, 1051 (1956) . 23 . Goldthwait, D. A . , Peabody, R. A . , and Greenberg, G. R., in Amino Acid Metabolism, ed. by McElroy, W. D., and Glass, B. , Johns Hopkins Press, Baltimore. 1955. P. 765. 24. Goldthwait, D. A . , Peabody, R . A . , and Greenberg, G. R., J . Am. Chem. Soc. 76 , 5258 (1954) . 25. Hartman, S. C , Levenberg, B. , and Buchanan, J . M. , J . Am. Chem. Soc. 77, 501 (1955) . 2.6. Buchanan, J . M. , Levenberg, B. , and Lukens, L. If., Abstracts, American Chemical Society, 128th Meeting, Minneapolis. 12 C (1955) . 27 . Levenberg, B. , and Buchanan, J . M., J . Am. Chem. Soc. 78, 504 (1956) . 28 . Lukens, L. H . , and Buohanan, J . M., J . Am. Chem. Soc. 79, 1511 (1957) . 29. Lukens, L. If., and Buchanan, J . M. , Federation Proc. 15 , 305 (1956) . 30 . Carter, C. E . , and Cohen, L. H . , J . Am. Chem. Soc. 77, 499 (1955) . ~ " 3 1 . Mil ler, R. W., Lukens, L. U . , and Buchanan, J . M., J . Am. Chem. Soc. 79 , 1513 (1957) . 32 . Carter, C. E . , and Cohen, L. H. , J . Biol . Chem. 222, 17 (1956) . 3 3 . Moyed, H. S., and Magasanik, B. , Federation Proc. 15, 318 (1956) . - 44 34 . Greenberg, G. R., J . B i o l . Chem. 212, 4 2 3 (19^6). 35. Kornberg, A., and P r i c e r , W. £., J . B i o l * Chem. 193, 481 (1951). 36 . Grunberg-Manago, M., and Ochoa, S., J . Am. Chem. Soc. 77, 3165 (1955). 37 . Grunberg-Manago, M., O r t i z , P. J., and Ochoa, S., Science 122, 90? (1955) . 38 . Grunberg-Manago, M., Ortiz, p. J., and Ochoa, S., Biochim. et Biophys. Acta 20, 269 (1956) . 39 . L i t t a u e r , U. Z., Federation Proc. 15, 302 (1956). 40. Beers, R. F., nature 177, 790 (1956) . 41. Potter, V. R., Hecht, L. I., and Herbert, E., Biochim. et Biophys. Acta 20 , 439 ( I 956 ) . 42. Heidelberger, C , Harbers, E., Leibman, K. C , Takagi, Y., and Potter, V. R., Biochim. et Biophys. Acta 20, 445 (1956) . . — 43. Canellakis, E. S., Biochim. et Biophys. Acta 23 , 2 l8 (1957) . 44. paterson, A. R. P., and LePage, G. A., Cancer Res. 17, 409 (1957) . 45 . Kornberg, A., i n The Chemical Basis of Heredity, ed. by McElroy, W. D., and Glass, B., Johns Hopkins Press, Baltimore. 1957. p. 579. 46. Almquist, H. J•, Federation Proc. 1, 269 (1942). 47. M i l l e r , C. S., Gurin. S., and Wilson, D. W., Science 112, 654 (1950) . 48* Williams, W. J., and Buchanan, J . M., J. B i o l . Chem. 202, 253 (1953) . 49. Love, S. H., J . Bact. 72 , 628 (1956) . 50. LeBlond, c. P.. and Stevens, c. E., Anat. Rec. 100, 357 (1948) . 51. LeBlond, C. P., Stevens, C. E., and Bogorooh, R., Science 108, 531 (1948) . 5 2 . Thompson, R. Y., Heagy, F. C., Hutchinson, W. C . and Davidson, J . N., Biochem. J. 5 3 , 460 (1953) . - 45 -5 3 . Goldthwait, D. A., and Bendioh, A., J . B i o l . Chem. 196, 841 (1932). 34 . Ateams, R., Arch. Biochem. Biophys. £3> 436 (1931). 5 5 . Stevens, C. E., Daoust, R.. and LeBlond, C. P., J . B i o l . Chem. 202, 177 (1953) . 56 . Paterson, A. R. P., and Zbarsky, S. H., Biochim. et Biophys. Acta 18 , 441 (1955) . 5 7 . Greenlees, J . , and LePage, G. A., Cancer Res. 16, 808 (1956) . 38 . Tomisek, A. J ., Kel l y , H. J ., and Skipper, H. E., Arch. Biochem. Biophys. 64, 437 (1936 ; . 59 . Hartman, S. C , Levenberg, B., and Buchanan, J . M., J . B i o l . Chem. 221, 1057 (1956) . 60. Peabody, R. A., Goldthwait, D. A., and Greenberg, G. R,, J . B i o l . Chem. 221, 1071 (1956) . 6 1 . Smith, E. L., and Bergmann, M., J . B i o l . Chem. 138, 789 (1941). 62. Smith, E. L . , J . B i o l . Chem. 176, 9 (1948) . 63 . Smith, E. L., and Bergmann, M., J . B i o l . Chem. 15_3, 627 (1944). 64. Schmidt, G., and Thannhauser, S. J . , J . B i o l . Chem. 149, 369 (1943) . 65. Morton, R. K., Biochem. J . 5_7, 595 (1954) . 66. Hilmoe, R. J . , and Heppel, L. A., i n Methods i n Enzymology, v o l . II, ed. by Colowick, S. P., and Kaplan, H . , Academic Press, Hew York. 1955. P« 5^ 9 • 67i Kalckar, H. M., J . B i o l . Chem. 167, 461 (1947) . 68. Aldridge, W. E., Biochem. J. 37_, 692 (1954). 69. Davidson, A* N., Biochem. J . 5 4 , 583 (1953) . 70. Ord, M. G., and Thompson, R. H. 3 . , Biochem. J . 46 , 346 (1950) . ~~ 71-. Friedkin, M., and Roberts, D., J . B i o l . Chem. 207, 245 (1954). 72. Englard, S., Federation Proc. 11, 208 (1952) . 73 . Krebs, H. A., and Hems, R., Biochim. et Biophys. Acta 12, 172 (1953) . - 46 -74. Hele, M. P., Biochem. J . 55 , 857 (1953). 75. Hele, M. P., nature 166, 786 (1950). 76. Lowenatein, J. M., and Cohen, P. P., J. B i o l . Chem. 220, 57 (1956). 77. Richert, D. A., and Westerfeld. W. W., Proc. Soc. Exptl. B i o l . Med. 83> 726 (1953). 78. Dickens, F., and Williamson, D. H., Biochem. J . 64, 567 (1956). — 79. Davson, H. A Textbook of Physiology. J . A. C h u r c h i l l and Son. London. 1954. p. 297. 80 . van Genderen, H., and Engel, C , Enzymologia 5 , 71 (1938 -9 ) . 81 . Todd, W. R., and Talman, E., Arch. Biochem. 22 , 386 (19.49). 82 . Alexander, B., Landwehr, G., and Seligman, A . M., J . B i o l . Chem. 160, 51 (1945) . 83 . Christensen, H. N., Riggs, T. R., and Ray, N. E., Anal. Chem. 25 , 1521 (1951) . 84. Awapara, J . , Landua, A. J., and Fuerst, R. E., Biochim. et Biophys. Acta 5 , 457 (1950) . 85 . Schwartz, T. B., Robertson, M. C , and Holmes, L. B., J. Lab. and C l i n . Med. 46 , 657 (1955) . 86. Dickens, F., and Weil-Malherbe, H. , Biochem. J. 55_, 7 (1941) . 87. Schulman, M. P., Sonne, J . C , and Buchanan, J. M., J . B i o l . Chem. 196, 499 (1952) . 88 . Layne, E., i n Methods i n Enzymology, v o l . I l l , ed. by Colowiok, S. P., and Kaplan, N., Academic Press, New York. 1957. p. 447. 89 . Pabst Laboratories Circular OR-10. Pabst Brewing Company, Milwaukee. 1956. 90. Volkin, E., and Cohn, W. E., i n Methods of Biochemical Analysis, v o l . I, ed. by Glick, D. Inter science, New York. 1954. p. 287. 91 . Benson, A. A., In Methods in-Enzymology, v o l . I l l , ed. by Colowick, S. P., and Kaplan, N. Academic Press, New York. 1957. p. H O . - 47 -92. Bandurski, R. S., and Axelrod, B., J. B i o l . Chem. 193, 405 (1951) . 93. Forreat, I. S., and Neuberg, C , Biochim. et Biophys. Acta 11 , 388 (1953) . 94. l e i s t e r , A., Phys i o l . Rev. 36 , 103 (1956) . 95 . Block, R. J., Durrum, E. L., and Zweig, G., A Manual of Paper Chromatography and paper Electrophoresis. Academic Press, Hew York. 1956. p. 77 . 96. Fusari, 5. A., Frohardt, R. P., Ryder, A., Haskell, T. H., Johannessen, D. V/., Elder. C. C . and Bartz, Q. R., J. Am. Chem. Soc. 76 , 2878 (1954). 97. Moore, S., and Stein, W. H., J. B i o l . Chem. 176, 3&7 (1954) . 98 . Lepage, G. A., Cancer Res. 13 , 178 (1953) . 99 . Wyatt, G. R., Biochem. J . 48 , 584 (1951) . 100. Hirs, C. H. W., Moore, S.{ and Stein, W. H., J . B i o l . Chem. 19_5, 669 (1952) . 101. Lepage, G. A., and Greenlees, J., Cancer Research Suppl. Ho. 3 , P. 102 (1955) . 102. Heidelberger, C , and K e l l e r , R. A., Cancer Research Suppl. Ho. 3 , p. 106 (1955) . 103. Bennett, L. L., Sohabel, F. M., and Skipper, H. E., Arch. Biochem. Biophys. 64 , 423 (1956) . 104. Skipper, H. E., M i t c h e l l , J. H, Bennett, L. L., Newton, M. A., Simpson, L., and Eidson, M., Cancer Res. 11 , 145 (1951) . 105. Goldthwait, D. A., Peabody, R. A., and Greenberg, G. R., J. B i o l . Chem. 221, 535 (1956) . 106. Markham, R., and Smith, J. D., Biochem. J . 5 2 , 552 (1952) . 107. Moe, H., Internat. Rev. Cytol. IV, 299 (1955) . 108. Kaplan, L.. and Stock, C. C , Federation Proc. 1£, 239 ( I 9 5 4 ) . 109. Levenberg, B., Melnick, I.., and Buchanan, J. M., J. B i o l . Chem. 223, 163 (1957) . 110. Smellie, R. M. S., and Davidson, J . H., Experientia 12, 422 (1956) . - 48 -1 1 1 . B a l i s , M. E., Levin, D. H., Brown, G. B., E l i o n , G. B., vanderWerff, H., and Hitchings, G. H., J . B i o l . Chem. 19_6, 729 ( 1 9 5 2 ) . 1 1 2 . Kerr, S. E., Seraidarian, K., and Brown, G. B., J . B i o l . Chem. 1 8 8 , 2 0 7 ( 1 9 5 1 ) . 1 1 3 . Bolton, E. T., Abelson, P.. H., and Aldous, E., J . B i o l . Chem. 19_8, 1 7 9 ( 1 9 5 2 ) . 114. Gots, J . S., and Gollub, E., Proc. Amer. Assoc. Cancer Res. 2 , 2 0 7 ( 1 9 5 7 ) . 115. Schulman, M. P., and Buchanan, J . M . , J. B i o l . Chem. 1 9 6 , 5 1 3 ( 1 9 5 2 ) . 1 1 6 . A l l f r e y , V. G., Mirsky, A. E., and Stern, J., Adv. i n Enzymology 1 6 , 441 ( 1 9 5 5 ) . 1 1 7 . Cohn, W. E., and Carter, C. E., Nature 1 6 7 , 4 8 3 ( 1 9 3 1 ) . 118. Goldthwait, D. A., Greenberg, G. R., and Peabody, R. A., Biochim. et Biophys. Acta 18, 148 ( 1 9 5 5 ) . 

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