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Home , Inosinic acid, Nucleotide

Mechanisms of Synthesis of in Heart Muscle Extracts

David A. Goldthwait

J Clin Invest. 1957;36(11):1572-1578. https://doi.org/10.1172/JCI103555.

Research Article

Find the latest version: https://jci.me/103555/pdf MECHANISMS OF SYNTHESIS OF PURINE NUCLEOTIDES IN HEART MUSCLE EXTRACTS1 BY DAVID A. GOLDTHWAIT2 (From the Departments of and Medicine, Western Reserve University, Cleveland, Ohio) (Submitted for publication February 18, 1957; accepted July 18, 1957)

The key role of ATP, a purine , in 4. or + PRPP -> AMP the conversion of chemical energy into mechanical or (IMP) + P-P. work by myocardial tissue is well established (1, The third mechanism of synthesis is through the 2). The requirement for purine nucleotides has phosphorylation of a purine (8, 9): also been demonstrated in the multiple synthetic 5. + ATP -, AMP + ADP. reactions which maintain all animal cells in the Several enzymatic mechanisms are known which steady state. Since the question immediately arises result in the degradation of purine nucleotides and whether the purine nucleotides are themselves in . The of adenylic acid is a steady state, in which their rates of synthesis well known (10): equal their rates of degradation, it seems reason- 6. AMP -* IMP + NH8. able to investigate first what mechanisms of syn- Non-specific (11) as well as spe- thesis and degradation may be operative. cific 5'- (12) have been described At present, there are three known pathways for which result in dephosphorylation: the synthesis of purine nucleotides. The first is 7. AMP -> Adenosine + (Pi). the synthesis de novo of the purine ring from small Adenosine can be converted by the , nu- molecular weight precursors, such as , cleoside phosphorylase (13), to the free base and formate, CO2, and (3). a sugar derivative: The initial steps in this synthesis (4, 5) are as 8. Adenosine or + Pi -> Adenine or Hy- follows: poxanthine + -l-Phosphate. 1. Ribose-5-phosphate + ATP3 -> 5-Phospho- Finally, adenosine may be converted to inosine ribosylpyrophosphate (PRPP) + AMP. by an enzyme (14): 9. Adenosine -) Inosine + NH3. 2. PRPP + glutamine -- 5-phosphoribosyla- mine (PRA) + . The main purpose of the experiments reported 3. PRA + glycine + ATP -* glycinamide ribo- in this paper is to indicate which of the tide + ADP + Pi. that catalyze the synthesis of purine nucleotides Glycinamide ribotide is converted to formyl gly- are present in heart muscle extracts. Observations cinamide ribotide and then through a series of on some of the degradative steps are also presented. steps to adenylic and guanylic acids (6). The second pathway available for the synthesis EXPERIMENTAL of nucleotides is via the condensation of a free pu- rine base with PRPP (7): Enzyme preparation. Three fresh pig hearts were maintained at 00 C. immediately after removal for one- 1 This work was supported by grants from the Ameri- half hour. After the fat was removed, they were ground can Heart Association and the American Society. together in a refrigerated electric meat grinder. Three 2This work was done during the tenure of an Estab- hundred Gm. of ground meat was then suspended in lished Investigatorship of the American Heart Association. 600 ml. of 0.05 M potassium phosphate buffer pH 7.4 8 Abbreviations are as follows: ATP, adenosinetri- and homogenized for 30 seconds in a Waring blendor phosphate; ADP, adenosinediphosphate; AMP, adeno- with a Variac setting of 60 volts. The fluid portion of sinemonophosphate; IMP, inosinic acid; PRPP, 5-phos- the homogenate was squeezed through two layers of phoribosylpyrophosphate; PRA, 5-; cheese cloth and centrifuged in a Sorvall Centrifuge R-5-P, ribose-5-phosphate; Pi, inorganic phosphate; P-P, (Type SS-1) powered by 60 volts for 15 minutes. A ; NH,, ammonia; and PGA, 3-phospho- portion of the supernatant solution was kept frozen at glyceric acid. - 130 C. and the remainder was dialyzed against 20 liters 1572 NUCLEOTIDE IN HEART MUSCLE EXTRACTS 1573 of distilled water for 18 hours. To the dialyzed enzyme, radioactivity determinations by an end-window counter. potassium phosphate buffer pH 7.4 was added to make a Other aliquots were used for the determination of spectra final concentration of 0.05 M. All operations were car- and the estimation of the amount of material recovered. ried out at + 2° C. The enzyme solution was then ly- An extinction coefficient for AMP and IMP of 14.8 was ophilized and the dry powder was stored at - 13° C. employed. The results reported in the tables repre- Prior to each experiment, the dry powder was dissolved sent single experiments. Preliminary experiments with in water. Fifty mg. of powder per ml. resulted in a the different precursors yielded comparable results. concentration of 28.5 mg. per ml. Substrates. Adenine-8-CM and adenosine-8-CM were obtained from the Schwarz Laboratories. Adenine-8-C' RESULTS was deaminated to hypoxanthine--C" by a modification Synthesis of nucleotides by the de novo pathway of the method of Davoll (15). These purine derivatives were diluted with carrier so that the final specific ac- Incubation of glycine-1-Q' with the fresh ex- tivity was between 3,000 and 4,000 counts per minute per tract (Table I) and no substrate additions re- micromole. AMP' was synthesized by the method of sulted in 0.003 micromole of glycine fixed in one Eggleston (16). Glycine-l-CO' was synthesized by the hour. Addition of method of Sakami, Evans, and Gurin (17) and had a ribose-5-phosphate (R-5-P), specific activity of 21,500 counts per minute per micro- ATP and glutamine increased fixation to 0.006 mole. PRPP was prepared enzymatically by a slight micromole. Substitution of 5-phosphoribosylpyro- modification of the procedure of Kornberg, Lieberman, phosphate (PRPP) for R-5-P increased fixation and Simms (18). ATP was purchased from Pabst, Inc., by slightly more than three-fold while substitution and potassium phosphoglyceric acid (PGA) was pre- of pared from the barium salt as described previously (19). 5-phosphoribosylanine (PRA) for PRPP and Methods. All reaction mixtures were incubated at 370 glutamine again increased fixation by approxi- C. in air. For determination of the amount of glycine-l- mately three-fold to 0.066 micromole. It should C14 incorporated into the glycinamide ribotides and other be emphasized that glycine fixation represents in- compounds, the reaction filtrate was treated with nin- corporation into any compound in which the gly- hydrin to decarboxylate the glycine-1-C14, and the re- cine is not An maining radioactivity was counted (19). The column decarboxylated by ninhydrin. at- chromatographic isolation of the glycinamide ribotides tempt at identification of the radioactive products has been described (20). For the paper chromatographic by ion exchange chromatography and paper chro- separation of other compounds, the following solvent matography was unsuccessful. No radioactivity systems were employed: a) butanol saturated with water, 15 N NH4OH (100: 1); b) n-propanol, 15 N NH4OH, was found at the points of elution of glycinamide water (60:30: 10); c) isopropanol, concentrated HC1, ribotide or its formyl derivative. Thus the gly- HO (170:60:20). The amount of nucleotide synthe- cine incorporation, stimulated by purine pre- sized from radioactive adenine, hypoxanthine or adeno- cursors, could have been in other intermediates sine was determined by the application of a 0.2 ml. ali- quot of the reaction filtrate to a 5-cm. starting line on and the 0.006 micromole of glycine fixed when Whatman No. 1 paper. The filtrate was chromato- R-5-P and glutamine were added probably repre- graphed by the descending method using solvent A, which resulted in movement of nucleosides and free bases off the starting line, but no movement of nucleotides. It TABLE I was necessary to allow the solvent to run off the paper Fixation of glycine-l-CH in the presence of purine for an estimated front distance of 45 cm. in order to get nucleotide precursors * good separation of some of the nucleosides from the nu- cleotides. Solvent B was also employed in some ex- Glycine Additions fixed periments as a check on the isolation of nucleotides. It was possible with solvent B to separate the nucleotides micromoles into one band containing adenylic acid and another con- None 0.003 taining inosinic acid, ADP and ATP. After acid hy- R-5-P + glutamine 0.006 PRPP + glutamine 0.020 drolysis of the material from the second band, and re- PRA 0.066 chromatography of the bases in solvent system A, the radioactivity of the inosinic acid and a mixture of ADP * Each reaction vessel contained: ATP, 0.8 micromole; and ATP could be estimated. Paper electrophoretic PGA, 7 micromoles; glycine-1-C04, 5 micromoles; MgC2, 5 micromoles; Tris buffer pH 7.0, 20 micromoles; un- separations of nucleotides were done with 0.02 M sodium dialyzed pig heart extract, 4.1 mg. protein. Additions citrate buffer, pH 3.5. Radioactive compounds were were: R-5-P, 5 micromoles; glutamine, 5 micromoles; eluted from the paper by soaking overnight in 3 ml. of PRPP, 1.2 micromoles; PRA, approximately 1 micromole. The final volume was 1 ml. and the reaction mixture was 0.1 N HC1. Aliquots were dried on glass planchets for incubated for 1 hour at 370 C. 1574 DAVID A. GOLDTHWAIT

TABLE II dialyzed extract. Synthesis proceeded in a linear Synthesis of nuceotides from C1' adenine and PRPP * fashion (experiment 3) under these conditions for at least one hour. That the C" incorporation 0'4 UN.V adenine absorb- represents net synthesis rather than exchange is Experi- incor- ing meat Conditions Time porated material suggested by the net increase with time of nucleo- tide material as determined by optical mTao- miTao- density mix. mole moles (O.D.) at 260 mp. This increase probably repre- 1 Undialyzed Extract 60 0.11 sents synthesis of purine nucleotides, mainly from Undialyzed Extract + PRPP 60 0.17 2 Undialyzed Extract + PRPP 60 0.13 the C" adenine-PRPP reaction, and secondarily Dialyzed Extract + PRPP 60 0.20 from seme unlabeled precursor. 3 Dialyzed Extract + PRPP 15 0.035 0.182 DialyzedExtract + PRPP 30 0.074 0.240 When ribose-5-phosphate and ATP were sub- Dialyzed Extract + PRPP 45 0.112 0.288 stituted for PRPP, synthesis of purine nucleotides Dialyzed Extract. + PRPP 60 0.164 0.360 was effective (Table III, -experiment 1). There * Each reaction vessel contained: adenine-8-C4, 1 micro- appeared to be an optimal level of ATP as was mole; MgClt, 2.5 micromoles; Tris buffer pH 7.0, 20 micro. indicated by Kornberg, Lieberman, and Simms moles; dialyzed heart muscle extract, 5.7 mg.; PRPP addition, 0.75 micromole. The final volume was 0.50 ml. using a pigeon liver enzyme (18). Not only is and vessels were incubated at 370 C. adenine utilized in the synthetic reaction, but also hypoxanthine can be incorporated (Table III, sents a maximum value for un- experiment 2). der these conditions. The nucleotide products recovered from the enzymatic reaction between adenine and PRPP Synthesis of nucleotides by the reaction of urine were identified as adenylic acid and inosinic acid. bases with PRPP In Table IV, it is clear from the total amounts All of the experimental results presented in recovered that the main product was adenylic acid. Tables II and III indicate that an enzyme is pres- Furthermore the higher specific activity of ade- ent in heart muscle which catalyzes the synthesis nylic acid suggests that it is the precursor of ino- of purine nucleotides from PRPP and adenine or sinic acid. Adenylic acid was identified chromato- hypoxanthine. In experiment 1, Table II, in un- graphically by its movement in solvent system B, dialyzed extracts, addition of PRPP increased by its movement on paper electrophoresis, and by synthesis by 55 per cent. Dialyzed extract (ex- and identification of the free base as periment 2) appeared to be more active than un- adenine in solvent system A. Inosinic acid, the only nucleotide product of the reaction between hypoxanthine and PRPP, was identified by paper TABLE III chromatography in solvent system B, by spectra, Synthesis of nuceotides from C14 adenine, C14 hypoxanthine, PRPP and R-5-P * and by movement in paper electrophoresis at pH 3.5. C14 purine Exderi- incor- Synthesis of nucleotides from adenosine and ATP meat Conditions porated It is evident from the experimental results pre- miaco- sented in Table V, that an adenosine ex- 1 Adenine + R-5-P + 0.4 micromole ATP 0.047 in Adenine + R-5-P + 0.8 micromole ATP 0.023 ists heart muscle. Experiment 1 shows the Adenine + R-5-P + 2.0 micromoles ATP 0.020 synthesis of nucleotide from C14 adenosine as a Adenine + PRPP 0.160 2 Adenine + PRPP 0.160 function of time. Under these conditions, the Hypoxanthine + PRPP 0.130 synthesis is not linear. This is probably not due to insufficient ATP, as an ATP regenerating sys- * Each reaction vessel contained MgCI2, 2.5 micromoles; Tris buffer pH 7.0, 20 micromoles; and dialyzed heart tem (PGA and the required enzymes) is present, muscle extract, 5.7 mg. Additions were R-5-P, 2.5 micro- moles; ATP as indicated in table; PGA, 14 micromoles; but is more likely due to levels of adenosine in- PRPP, 0.75 micromole; adenine-8-C4, 0.98 micromole; sufficient to saturate the enzyme because of the hypoxanthine-8-C4, 0.70 micromole in a final volume of 0.50 ml. strong competing reaction catalyzed by adenosine NUCLEOTIDE METABOLISM IN HEART MUSCLE EXTRACTS 1575 deaminlase. If one assumes the enzyme istatu- TABLE TV rated for the first 15 minutes, then minimum NsUck.0ktC'rou ofth eadiox between PRPP rate of synthesis per horw is 0.37 micronole R-Re 4 C4 adeniUeP sults of experiment 2' indicate that ATP is re- Nucleoddes quired for synthesis of' mwle6des fnrm C0' ade-. -nosinrc Adenylic nosine. Isolation of C0' invine And hyponhne aged acdd (experiment 2) after i one-hour izlubafin is Ruin solvent B 0.15 0.22 strong evidence Or the presence f adenosne de- Micromoles CH incorporated 0.06 0.14 Specific activity in aminase and nucleoside phosphorylase. No C'4- dounts/Min./iinole 940 3,260 adenosine was recovered. The, total C1 recoveries Per cent of specific ct vt in this experiment were 87 and 90 per cent. In of originaladenine 24 83 experiment 2 the am~unt of nucleotide synthesis * Products were recovered from an experiment similar is decreased because one-half the amount of Cd to that in Table III, experiment 2. substrate was employed. Results of. experiment 3 indicate that different levels of ATP are not vent B, and of the free bases of the nucleotides in critical in the reaction. Again radioactive inosine solvent A, and also by paper electrophoresis of the and hypoxanthine were recovered as well as 0.26 nucleotides. After a one-hour incubation, approx'i- micromole of an unidentified fraction. The total mately 10 per cent of the total 04 recovered was C1' recovery in this experiment was 75 per cent. adenylic acid, 10 per cent was inosinic acid, and The synthesis of C"4-labeled nucleotides from C1' 80 per cent was a mixture of ADP and ATP. adenosine could go via inosine and hypoxanthine with condensation of the latter with PRPP, as in- Dephosphorylation of adenylic acid dicated in Table III. Since, in experiment 4, a A final experiment was done to demonstrate that pool of non-radioactive hypoxanthine did not de- an enzyme is present in heart muscle extract which crease the C1' incorporated into nucleotides, it may can liberate phosphate from adenylic acid. Six be concluded that hypoxanthine is not an inter- and three-tenths micromoles of adenosine-5-phos- mediate in the conversion of adenosine- to a nu- phate labeled with p12 were incubated with 5 cleotide. Non-radioactive inosine and adenine micromoles of MgCl2, 20 micromoles of Tris buf- gave similar results. fer pH 7.0, and 5.7 mg. of dialyzed enzyme in a The radioactive products of the reaction be- volume of 0.8 ml. for 60 minuted The TCA re- tween C14-adenosine and ATP were identified by action filtrates of the zero time contrl and the paper chromatography of the nucleotides in sol- incubation mixture were then chromatographed, in

TABLE v Synsthsis of nucotides from Cl adenosine and A TP *

Cm-labeled com eed Experi- Hypo- ment Conditions Time Nucleotide Inosine .anthine minutes micromoks 1 Adenosine + ATP (2.0) 15 0.092 Adlenosine (3.9)(3.9)f + ATP (2.0) 30 0.104 Adenosine (3.9) + ATP (2.0) 60 0.134 2 Adenosine (1.8) 60 0 0.99 0.57 Adenosine- (1,8) + ATP (0.8) 60 0.048 1.08 0.51 3 Adenosine (3.9) + ATP (0.8) 60 0.12 Adenosine (3.9) + ATP (2,.0) 60 0.15 1.90 0.62 4 Adenosine (1.8) + ATP (2.0) 60 0.067 Adenosine (1.8) + ATP (2.0) + hypoxanthine (5) 60 0.075 Adenosine (1,8) +ATP (2.0) + inosine (5) 60 0.067 Adenosine (1.8) + ATP (2.0) + adenine (5) 60 0.056 * The reaction vesels contained additions as indictd, plus MgCle, 2.5 mitromoles; Tris.buffer pH 7.0, 20 micro- moles; PGA, 14 micromoles; and dialyzed enzyme, 5.7 mg. in a final volume of 0.8 ml. Incubations were at 370 C. t The figures in parentheses represent micrmoles. 1576 DAVID A.. GOLDTHWAIT solvent C, and sections of this paper were eluted readily in cardiac muscle extracts. In experiment and counted. During the incubation radioactivity 3,,;Table II, 0.164 micromole of nucleotide was was liberated which, on chromatography, migrated synthesized in one hour. The synthesis of PRPP in the area corresponding to inorganic phosphate. may; be liniiting as only 0.047 micromole of nu- The amount recovered was the equivalent of 0.22 cleotide was. sjnthesized when ribose-5-phos- micromole of AMP82 split in one hour. plikte and ATP- rather' than PRPP were em- ployed. This activity, however, is almost ten DISCUSSION times the rate of glycine incorporation under some- The experimental results indicate that in this what comparable conditions. This enzymatic extract of heart muscle and under the conditions mechanism which has been shown to be present employed, the synthesis of purine nucleotides via in skeletal muscle (22) would require either ade- the de nova pathway (reactions 1 to 3, etc.) was nine or hypoxanthine. The demonstration of less efficient than synthesis via utilization of the both of these purine bases in the urine of normal free base (reaction 4) or via phosphorylation of humans (23) suggests that they probably are adenosine (reaction 5). The incorporation of available via the blood stream to cells of the glycine-1-C14 into glycinamide ribotide requires body. Indeed, hypoxanthine has been identified at least three enzymatic steps when ribose-5-phos- in plasma (24). That these free bases are utilized phate, ATP, glutamine and glycine are supplied. for synthesis by tissues such as liver At least two steps are required when PRPP is and intestine has been demonstrated by in tivo utilized and probably one when PRA is employed experiments (25, 26). It should be noted that (reactions 1 to 3). The stimulation of glycine- hypoxanthine is not utilized by extracts of bone 1_C14 incorporation in the presence of these sub- marrow for the synthesis of purine nucleotides strates suggests that the incorporation noted repre- (27). No experiments on purine utilization by sents purine precursor synthesis in spite of the cardiac muscle are reported and similar experi- fact that neither glycinamide ribotide nor formyl- ments on skeletal muscle are conflicting. The ob- glycinamide ribotide were isolated. If the glycine servation in the rat that the amino group of muscle incorporation does represent de novo synthesis, adenylic acid was easily labeled with N15H8 while then 0.006 micromole of purine precursors was the ring nitrogens were not (28) suggested that synthesized in one hour under these conditions only the amino group was in a dynamic equilib- when ribose-5-phosphate and glutamine were used. rium. However, when a high level of uniformly Spilman (21) has provided isotopic evidence labeled N'5-adenine was fed, muscle ATP be- that the acid soluble adenine nucleotides of dog came labeled (24). Comparable levels of labeled hieart muscle are in a dynamic state. Ninety min- hypoxanthine did not result in labeling of the utes after injection with C14-formate the dog was ATP (29). These results suggest that enzymes sacrificed and the acid soluble nucleotides were are present in skeletal muscle for the incorporation isolated and counted (counts per minute per 5 mg. of adenine, but that the turnover rate in this of purine). Activities were: gastrointestinal tract, tissue is slow. 680; heart, 467; kidney, 289; liver, 34; while the The conversion of the nucleoside adenosine to a urine was 3,150. The remarkably high nucleotide, in the presence of ATP and the muscle activity of the heart could represent exchange of extract (reaction 5), is indicated in Table V. As- the 2 carbon, de novo synthesis, or possibly syn- suming the rate of synthesis in the first 15 minutes thesis from hypoxanthine made in the liver and was maintained for one hour, 0.37 micromole of transported through the blood. From the ex- adenylic acid would be synthesized. This appears periment it is impossible to conclude which of these to be the most efficient mechanism. The mechanisms may be operative, but the high activity adenosine needed for this reaction might arise does indicate a dynamic state. from adenine via reaction 8. However, the pres- The condensation of a free purine base, either ence of a strong adenosine deaminase, as indicated adenine or hypoxanthine, with PRPP to form the by the recovery of inosine and hypoxanthine in corresponding nucleotide (reaction 4) occurs experiments 2 and 3, Table V, makes the sig- NUCLEOTIDE METABOLISM IN HEART MUSCLE EXTRACTS 1577 nificance of the adenosine kinase difficult to inter- SUMMARY pret, but differential cellular localization of these 1. Mechanisms of synthesis and degradation of enzymes cannot be ruled out. It is of interest that purine nucleotides in an extract of pig heart muscle Lowy, Davoll, and Brown (30) have found con- have been explored. siderable incorporation of adenosine-8-'4 and 2. In this extract the more active synthetic path- inosine-8-C" into the ATP of skeletal muscle. ways are via the phosphorylation of adenosine and Thus the more effective pathways for synthesis the condensation of adenine or hypoxanthine with of nucleotides in heart muscle extracts appear to PRPP. De novo synthesis appears less active. be the utilization of the free purine base and of 3. Evidence for the dephosphorylation of ade- adenosine. Which of the three pathways is nylic acid as well as for the enzymes adenylic acid utilized predominantly in tivo remains to be deaminase, adenosine deaminase, and nucleoside determined. phosphorylase is presented. With a dynamic state of the nucleotides in heart muscle, one would also expect to find degradative mechanisms operative. An enzyme responsible ACKNOWLEDGMENTS for deamination of adenylic acid to form ammonia The author is indebted to Mrs. Jean Tsau and Miss Betty Einsel for very capable technical assistance, to Dr and inosinic acid (reaction 6) was originally Robert Berne for many helpful suggestions, and to Drs. described in skeletal muscle (10), but has also Harland G. Wood and G. Robert Greenberg for their been isolated from heart muscle (31). It is support and the excellent facilities that were available. most reasonable to suspect that this enzyme was responsible for the recovery of inosinic acid (Table REFERENCES IV). The dephosphorylation of adenylic acid has 1. Szent-Gyorgyi, A., Chemical Physiology of Contrac- also been described in heart muscle (31), and evi- tion in Body and Heart Muscle. New York, Aca- dence is presented in this paper which supports demic Press, 1953. this finding. The enzyme nucleoside phosphory- 2. Mommaerts, W. F. H. 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