The Influence of Ribose 5-Phosphate Availability on Purine Synthesis ...

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Effect of Mycophenolic Acid, Alanosine, and Hadacidin on de Novo Purine Synthesis during Glucose Starvation in Cul- tured Lymphoblasts-To eliminate the ...
Vol. 259, No. 5. Issue of March 10, pp. 2927-2935 1984 Printed in S. A.

THEJ O U R N A L OF BIOLOGICAL CHEMISTRY 0 1984 by The American Society of Biological Chemists, Inc.

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The Influence of Ribose 5-Phosphate Availability on Purine Synthesis of Cultured Human Lymphoblasts and Mitogen-stimulated Lymphocytes* (Received for publication, August 8, 1983)

Renate B. Pilzz, Randall C. Willis, and Gerry R. Boss4 With the technical assistance of Soha D. Idriss

From the Department of Medicine, University of California, Sun Diego, La Jolh, California 92037

purine synthesis (1-4). It serves as both substrate and activator of amidophosphoribosyltransferase (glutamine phosphoribosylpyrophosphate amidotransferase, EC 2.4.2.141, the first and presumed rate-determining enzyme of de nouo purine synthesis (5) (Fig. I).* In intactcells increased availability of PP-Rib-P correlates with enhanced de nouo purine synthesis (6-8) and decreased availability of PP-Rib-P with reduced de nouo purine synthesis (9-11). However, some workers have been unable to demonstate such correlations (12). Rib-5-P, the precursor of PP-Rib-P, hasalso been proposed to be an important factor controlling the rate of de nouo purine synthesis, but this has never been established (1-3, 13). That increased Rib-5-P availability leads to purine overproduction is suggested by three genetic disorders which cause hyperuricemia inman. The first is a mutantPP-Rib-P synthetase (ATP:a-~-ribose-5-phosphate pyrophosphotransferase, EC 2.7.6.1) with increased affinity for Rib-5-P; this effectively increases the kinetic availability of Rib-5-P (14). The othertwo are glucose 6-phosphatase deficiency (15) and increased activity of glutathione reductase (16); both mutations are thoughtto increase Rib-5-P generation by increasing the flux through the pentose phosphate pathway (3) (Fig. 1). The experimental decrease of the NADPH/NADP ratio induced by methylene blue or other electron acceptors significantly increases the intracellular concentrations of Rib-5-P and PP-Rib-P and stimulates de nouo purine synthesis (6-8). We chose the following experimental approaches to explore the influence of Rib-5-P availability on the rate of de m u 0 purine synthesis in cultured human Iymphoblasts: 1) starvation for glucose to limit the Rib-5-P generation through the pentose phosphate pathway; 2) incubation with purine nucleosides to serve as alternative Rib-5-P sources; and 3) PP-Rib-P’ plays a critical role in the regulation of de nouo inhibition of purine nucleoside phosphorylase (purine nucleoside:orthophosphate ribosyltransferase, EC 2.4.2.1) to prevent * This work was supported in part by United States Public Health endogenous ribose reutilization from intracellular nucleotide Service Grants GM 17702,AM 13622, HD 10847, and 2S07RR05665- breakdown (Fig. 1). The above studies were performed with 15 administered through the Academic Senate, University of California, San Diego, the Kroc Foundation, and the Clayton Foundation. de nouo purine synthesis under normal feedback control by The costs of publication of this article were defrayed in part by the purine nucleotides and with feedback inhibition released by a payment ofpage charges. This article must therefore be hereby combination of drugs that decrease the formation of adenylmarked “aduertisement” in accordance with 18 U.S.C. Section 1734 ates and guanylates from IMP. The latter condition allowed solely to indicate this fact. us to investigate theRib-5-P requirement for maximally $ Supported by the Deutsche Forschungsgemeinschaft. I Recipient of a New Investigator Award, AG 03156. To whom stimulated de m u 0 purine synthesis. We also used mitogenactivated peripheral blood lymphocytes as a model for stimreprint requests should be sent. The abbreviations used are: PP-Rib-P, a-5-phospho-D-ribosyl- ulated purine synthesis and measured the specific activities 1-pyrophosphate; Rib-5-P, a-~-ribose-5-phosphate; Rib-l-P, a - of PP-Rib-P synthetase and amidophosphoribosyltransferase, D-ribose-l-phosphate; PP-Rib-P synthetase, ATP:a-~-ribose-Band the intracellular concentrations of Rib-5-P, PP-Rib-P, phosphate pyrophosphotransferase (EC 2.7.6.1); HPR transferase, purine nucleotides, and inorganic phosphate. We found that inosinate-guany1ate:pyrophosphate phosphoribosyltransferase (EC 2.4.2.8); PHA, phytohemagglutinin; HEPES, 4-(2-hydroxyethyl)-l- the intracellular Rib-5-P concentration can be rate-determinpiperazineethanesulfonic acid. ing for de nouo purine synthesis. The intracelluar ribose 5-phosphate concentration was found to be an important determinant of rates of de novo purine synthesis. When ribose 5-phosphate production was reduced in cultured human lymphoblasts by glucose starvation, the intracellular phosphoribosylpyrophosphateconcentrationand rates of de nouo purinesynthesis decreased. Inosinate-guany1ate:pyrophosphate phosphoribosyltransferase (HPR transferase)-deficient cells were relatively more resistant to glucose starvation. To minimize the effect of purine nucleotide feedback inhibition on the de novo pathway, cells were treated with inhibitors of IMP dehydrogenase and adenylosuccinate synthetase. In normal lymphoblasts, purine synthesiswas stimulated only at glucose concentrations greater than 100 pM while in HPR transferase-deficient lymphoblasts, stimulation occurred even in the absence of glucose. The differences between the normal and HPR transferase-deficient cells were lost when ribose reutilization from endogenous nucleotide breakdown was impaired in the HPR transferase-deficient cells by incubation with 2‘-deoxyinosine. Endogenous ribose reutilization for purine synthesisis, therefore, important when either glucose availability is limited or synthesis is stimulated. In the absence of glucose,exogenous purine nucleotides restored the intracellular concentrations of ribose 5-phosphate, phosphoribosylpyrophosphate, and purinenucleotides to almost 100%and rates of purine synthesisto 50-75% of those at 10 mM glucose. When ribose 5-phosphate production was increased in peripheral blood lymphocytes by phytohemagglutinin activation, the intracellular phosphoribosylpyrophosphate concentration and ratesof de nouo purine synthesisincreased.

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EXPERIMENTAL PROCEDURES~ RESULTS

Effect of Glucose Starvation on de Novo Purine Synthesis in Cultured Lymphoblasts-When normal or HPR transferasedeficient lymphoblasts were transferred to glucose-deficient media, rates of de nouo purine synthesis decreased by 75% within the first 15 min. After 2 h of starvation, rates of de novo purinesynthesis stabilized in the normal andHPR transferase-deficient lymphoblasts at 7 f 2 and 14 -t 3%, respectively, of the control values measured in the presence of 10 mM glucose. Addition of 10 mM glucose to either of the cell types at any time during a6-h glucose starvation resulted in a rapid (within 15 min) and a complete restoration of de novo purine synthesis. Maximal rates of de novo purine synthesis occurred at 10 mM glucose in both cell types, but halfmaximal rates of de novo purine synthesis occurred at greater than 500 pM glucose in the normal cells and 100 PM glucose in the HPR transferase-deficient cells (Figs. 2 and 3, respectively, open circles). Effect of Mycophenolic Acid, Alanosine, and Hadacidin on de Novo Purine Synthesis during Glucose Starvation in Cultured Lymphoblasts-To eliminate the control of de novo purine synthesis by purine nucleotide feedback inhibition (35 ) , we incubated lymphoblasts with the combination of mycophenolic acid, alanosine, and hadacidin. We previously demonstrated (27) and further show below that these drugs decrease the intracellular concentration of purine nucleotides and cause a stimulation of de novo purine synthesis. In addition to release of feedback inhibition, the drugs also increase the reutilization of the ribose moiety of purine nucleotides (see below). Inthe normal lymphoblasts, de nouo purine synthesis was stimulated by the drugs only at glucose concentrations greater than 100 p M (Fig. 2, closed circles). IntheHPR transferase-deficient lymphoblasts, on the other hand, de rwvo purine synthesis was stimulated significantly ( p < 0.05, Student’s t test) by the drugs at all concentrations of glucose and even in its absence (Fig. 3, closed circles). In both cell types, the degree of stimulation increased as theglucose concentration increased; stimulation was maximal at 10 mM glucose in the normal lymphoblasts and at 1 mM glucose in the HPRtransferase-deficient lymphoblasts. Effect of Alternative Ribose Sources on de Novo Purine Synthesis inCultured Lymphoblasts-Since lymphoblasts lack ribokinase and are not readily permeable to ribose phosphates, we used purine nucleosides to function as alternative ribose phosphate source^.^ Inosine, guanosine, and theiranalogs are readily transported inside the cell and are then phosphohydrolyzed by purine nucleoside phosphorylase. This phosphorolysis yields the purine base and Rib-1-Pwhich is converted to Rib-5-Pby phosphoribomutase (Fig. 1). We used 8-aminoguanosine as thealternative ribose source for normal lymphoblasts. A disadvantage of 8-aminoguanosine is that this nucleoside is a relatively poor substrate of purine nucleoside phosphorylase (33). However, the advanPortions of this paper (including “Experimental Procedures,” parts of “Results,” Figs. 1 and 5-7, and Tables I-V) are presented in miniprint at the end of the paper. Miniprint is easily read with the aid of a standard magnifying glass. Full size photocopies are available from the Journal of Biological Chemistry, 9650 Rockville Pike, Bethesda, MD 20814. Request Document No. 83M-2281, cite the authors, and include a check or money order for $6.00 per set of photocopies. Full size photocopies are also included in the microfilm edition of the Journal that is available from Waverly Press. Pyrimidine nucleosides could not be used in this study since human lymphoblasts, like most cultured mammalian cells,do not have significant pyrimidine phosphorylase activity.

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GLUCOSE CONCENTRATION

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FIG. 2. Rates of de nouo purine synthesis in normal lymphoblasts: effect of glucose and 8-aminoguanosine under normal and feedback-released conditions. Normal lymphoblasts were transferred to glucose-deficientmedia and variable amounts of glucose were added (0).Half of the cultures received 20 p~ mycophenolic acid, 20 F M alanosine, and 500 pM hadacidin at time zero (0).Cultures were incubated for 2 h at 37 “C, followedby a 1-h labeling period with [“Clformate. Inset, effect of 8-aminoguanosine. Cultures were incubated in the absence of glucose, with 100PM glucose or with 10 mM glucose as described above. Q, no further additions; b plus 50 FM 8-aminoguanosine (added 15 min prior to theformate); c, plus 20 FM mycophenolicacid, 20 pM alanosine, and 500 f i u ~hadacidin (all three drugs added at time zero); d, plus 50 p~ 8-aminoguanosine (15 min prior to the formate), and 20 PM mycophenolic acid, 20 p~ alanosine, and 500 FM hadacidin (at time zero). The units of the ordinate are the same as for the main figure. All valuesrepresent the mean f S.D. of a t least four independent experiments, each done in duplicate.

tage of this compound is that theproduct, &aminoguanine, is not a substrate for HPR transferase and, therefore, cannot lead to the production of a nucleotide inhibitor of PP-Rib-P synthetase or amidophosphoribosyltransferase (34). In the absence of glucose, 50 phi 8-aminoguanosine increased de nouo purine synthesis of normal lymphoblasts almost &fold (compare bars a and b in the absence of glucose, inset, Fig. 2) and provided sufficient ribose to the lymphoblasts so that they further increased their rates of de novo purine synthesis in response to mycophenolic acid, alanosine, and hadacidin (compare bars c and d, inset, Fig. 2). In the presence of 100 p M glucose, 8-aminoguanosine increased de novo purine synthesis significantly only in the presence of mycophenolic acid, alanosine, and hadacidin (bar d at 100 FM glucose, inset, Fig. 2). At 10 mM glucose, the addition of 8-aminoguanosine had no significant effect. HPR transferase-deficient lymphoblasts are unable to salvage hypoxanthine and, therefore, afford the opportunity to provide Rib-5-P to thecell from the preferred purine nucleoside phosphorylase substrate, inosine, without producing purine nucleotides (Fig. 4). At concentrations below30 p ~ , inosine increased de nouo purine synthesis more than did glucose in the absence or presence of mycophenolic acid,

Ribose 5-Phosphate Synthesis and Purine 120

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10-5 10-4 10-3 10- 2 G LUCOSE CONCENTRATION (MI FIG. 3. Rates of de novo purine synthesis in HPR transferase-deficient lymphoblasts: effect of glucose and 2”deoxyinosine under normal andfeedback-releasedconditions. HPR transferase-deficient lymphoblasts were transferred to glucose-deficient media and variable amounts of glucose were added (0).Half of the cultures received 20 p~ mycophenolic acid, 20 p~ alanosine, and 500 p~ hadacidin at time zero (0).Cultures were incubated for 2 h followed by a 1-h labeling period with [“Clformate. Inset, effect of 2”deoxyinosine. Cultures were incubated in the absence of glucose, with 100 p~ glucose or with 10 m M glucose as described above. a, no further additions; b, plus 100 p~ Z‘-deoxyinosine (added 15 min prior to the formate label); c, plus 20 p~ mycophenolic acid, 20 FM alanosine, and 500 p~ hadacidin (all three drugs added at time zero); d, plus 100 G M Z’-deoxyinosine (15 min prior to [“Clformate), and 20 p~ mycophenolic acid, 20 p~ alanosine, and 500 WM hadacidin (at time zero). The units of the ordinate are the same as for the main figure. All values represent the mean f S.D. of a t least four independent experiments each done in duplicate.

alanosine, and hadacidin. The maximal rate of de novo purine synthesis was achieved with 30 p M to 1 mM inosine in the absence of the drugs and was 55% of the rate achieved with 1-10 mM glucose. However, in the presence of the drugs, the maximal rate of de novo purine synthesis was achieved with 1-3 mM inosine and was about 70% of the rate achieved with 1-10 mM glucose. These differences between glucose and inosine were still observed when the experimental protocol was modified and glucose and inosine were simultaneously added to parallel cultures. Effect of Purine Nucleoside Phosphorylase Inhibition on de Novo Purine Synthesis in Cultured Lymphoblasts-Inhibition of purine nucleoside phosphorylase activity should allow one to determine the contribution of endogenous ribose reutilization to the Rib-5-P and PP-Rib-P available for de novo purine synthesis (Fig. 1). We used 2‘-deoxyinosine to inhibit the phosphohydrolysis of inosine by purine nucleoside phosphorylase in HPR transferase-deficient lymphoblasts. While more potent purine nucleoside phosphorylase inhibitors are available, they all can either serve to some degree as a ribose source or function as inhibitors, of de novo purine synthesis (33). 2”Deoxyinosine is hydrolyzed by purine nucleoside phosphorylase, butthe resulting2’-deoxy-Rib-l-P cannot serve as a precursor of PP-Rib-P and as before, the HPR transferase-deficient cells cannot salvage the hypoxanthine.

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FIG. 4. Rates of de nouo purine synthesis in HPR transferase-deficient lymphoblasts: effect of inosine under normal and feedback-released conditions. HPR transferase-deficient lymphoblasts were transferred to glucose-deficientmedia (0).Half of the cultures received 20 FM mycophenolic acid, 20 pM alanosine, and 500 p~ hadacidin at time zero (0).After 1% h incubation, variable amounts of inosine were added; 15 min later, [’C]formate was added and thecells were incubated for 1 h. All values represent the mean & S.D.of at least four independent experiments, each done in duplicate.

In either the absence of glucose or in the presence of 100 PM glucose, [14C]formateincorporation was significantly reduced by 100 p~ 2’-deoxyinosine (compare bars a and b in the absence of glucose and at 100 pM glucose, inset, Fig. 3).

This suppression was due neither to 2‘-deoxy-Rib-1-P toxicity nor to nucleotide inhibition via residual HPR transferase activity, since it could be completely reversed by the addition of 100 p~ inosine. In the presence of 10 mM glucose, 2‘deoxyinosine had no effect. When de novo purine synthesis of HPR transferase-deficient lymphoblasts was stimulated by mycophenolic acid, alanosine, and hadacidin, the addition of Z‘-deoxyinosine significantly decreased the rate of [I4C]formate incorporation irrespective of the glucose concentration (compare bars c and d at all three glucose concentrations, inset, Fig. 3). The most pronounced effect of 2‘-deoxyinosine was seen in the absence of glucose where it prevented any stimulation of de novo purine synthesis by the drugs. These results indicate that the contribution of endogenous ribose reutilization totheRib-5-P available for de nova purine synthesis is significant when 1) the supply of Rib-5-P from the pentose phosphate pathway is reduced because of glucose starvation, or 2)denovo purine synthesis is stimulated by mycophenolic acid, alanosine, and hadacidin. Effect of Glucose Starvation on the Concentration of Purine Nucleotides in Cultured Lymphoblasts-When normal lymphoblasts were incubated in the glucose-deficient media for 2% h, the intracellular GTP, ATP, and ADP concentrations decreased by 43, 40, and 30%, respectively, as compared to control values measured in the presence of 10 mM glucose; the GDP concentration remained unchanged (Table 1). We

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Ribose 5-Phosphate and Purine Synthesis

were unable to detect major changes, greater than 2-fold, in the intracellularconcentrations of AMP, GMP, and IMP during glucose starvation (datanot shown). Results obtained with HPR transferase-deficient lymphoblasts were virtually identical. When inosine was added to glucose-starved HPR transferase-deficient cells the intracellular concentrations of purine nucleotides were similar to those at 10 mM glucose (data not shown). Effect of Mycophenolic Acid, Alanosine, and Hadacidin on the Concentration of Purine Nucleotides in Cultured Lymphoblasts-When normal lymphoblasts were incubated for 2% h in the presence of 10 mM glucose with mycophenolic acid, alanosine, and hadacidin, the intracellular concentration of purine nucleotides decreased (Table I). Compared to control cultures without the drugs, the intracellular ATP and ADP concentrations decreased by about 21 and 18%, respectively, while the intracellular GTP and GDP concentrations decreased by about 74 and 44%, respectively. When the drugs were added to glucose-starved lymphoblasts, they did not decrease the concentration of adenylates any further, while GTP and GDP decreased by 28 and 17%, as compared to glucose-starved cultures without the drugs. When the drugs were added to cells in 0.01, 0.1, and 1mM glucose, intermediate changes between those in the absence of glucose and 10 mM glucose wereseen in the intracellular concentrationsof purine nucleotides. Similar results were obtained in the HPR transferase-deficient lymphoblasts. Effect of Glucose and Purine Nucleosides on Rib-5-P and PP-Rib-P Concentrations and [I4C]Adenine Incorporationin Cultured Lymphoblasts-In the absence of exogenously added glucose, the intracellular Rib-5-P concentration was 70% of that at500 p M glucose and 42% of that at 10 mM glucose (the difference between no added glucose and 10 mM glucose is statistically significant at the 0.05 value, Student's t test). Likewise, the [14C]adenineuptake, which as ameasure of PPRib-P availability includes the cellular Rib-5-P content,significantly increased from the no added glucose condition more than 4-fold at 500 p~ glucose and more than 8-fold at 10 mM glucose. 8-Aminoguanosine at 50 p~ increased the PP-Rib-P concentration of normal, glucose-starved lymphoblasts to a value similar to that observed with 10 mM glucose. The Rib-5-P concentration inthe presence of 50 p M 8-aminoguanosine was about 50% and the [l4C]adenine uptake about 75% of that measured in the presence of 10 mM glucose. As shown above, 8-aminoguanosine stimulated the rate of de nouo purine synthesis significantly, but only to 55% of the rateobserved with 10 mM glucose. In thepresence of 10 mM glucose, the intracellular PP-RibP concentration in HPR transferase-deficient lymphoblasts was 2.1-fold higher than in normal lymphoblasts (Tables IT and 111). In the absence of glucose, this difference was only 1.2-fold. The [l4C]adenine incorporationof HPR transferasedeficient cells was about twice that of normal cells in the presence or absence of glucose. The Rib-5-P concentration of HPR transferase-deficient lymphoblasts was only slightly higher than that of normal lymphoblasts at all glucose concentrations. When 100 WM inosine was added to glucose-starved HPR transferase-deficient lymphoblasts, the Rib-5-P and PP-RibP concentrations were similar to those observed at 10 mM glucose,while the [14C]adenine incorporation was slightly lower. Inosine restored the rateof de nouo purine synthesisto only 55% of the rateobserved with 10 mM glucose. The purine nucleoside phosphorylase inhibitor 2"deoxyinosine did not reduce the low Rib-5-P and PP-Rib-Pconcentrations of the

glucose-starved HPR transferase-deficient lymphoblasts noticeably, but decreased the [14C]adenineincorporation. This latter value correlated with the decreased rate of de nouo purine synthesis caused by 2'-deoxyinosine. Effect of Mycophenolic Acid, Alanosine, and Hadacidin on Rib-5-P and PP-Rib-P Concentrations and [14C)Adenine Incorporation i n CulturedLymphoblasts-When normal lymphoblasts were incubated in the absence of any added ribose source, mycophenolic acid, alanosine, and hadacidin only marginally increased the intracellular Rib-5-P and PP-Rib-P concentrations and the[l4C]adenineincorporation (Table 11). These results correlated with the lack of stimulation of de nouo purine synthesis by the drugs under these conditions. When HPR transferase-deficient lymphoblasts were incubated under the same conditions, the drugs caused a significant increase in the Rib-5-P and PP-Rib-P concentrations and [14C]adenine incorporation; 2'-deoxyinosine prevented this increase (Table 111).Again, these results correlated with the rates of de novo purine synthesis seen in glucose-starved HPR transferase-deficient lymphoblasts: mycophenolic acid, alanosine, and hadacidin caused a 2.4-fold stimulation which was prevented by 2'-deoxyinosine. When normal and HPRtransferase-deficient lymphoblasts were then incubated at glucose concentrations that allowed half-maximal rates of de nouo purine synthesis,mycophenolic acid, alanosine, and hadacidin increased the Rib-5-P and PPRib-P concentrations and ['*C]adenine incorporation to values that were markedly above the values measured with 10 mM glucose in the absence of the drugs. Finally, when lymphoblasts were incubated at 10 mM glucose, the drugs caused a more than 4-fold increase in the Rib-5-P and PP-Rib-P concentrations and [l4C]adenine incorporation in both cell types. However, the stimulation of de novo purine synthesis under these conditions was only 2-2.5-fold. DISCUSSION

In this study, we demonstrated that the intracellular concentration of Rib-5-P, the precursor of PP-Rib-P, can be limiting for the rate of de nouo purine synthesis. When the intracellular Rib-5-P concentration was decreased in cultured lymphoblasts by glucosestarvation, the intracellular PP-RibP concentration and rates of de novo purine synthesis decreased. Moreover, when the intracellular Rib-5-P concentration was increased in peripheral blood lymphocytes by mitogen activation, the intracellular PP-Rib-P concentration and rates of de novo purine synthesis increased. This latter increase of purinesynthesis could not be attributed to an increase in the specific activities of either PP-Rib-P synthetase or amidophosphoribosyltransferase or to changes in the intracellular concentration of phosphate or purine nucleotides. When HPR transferase-deficient lymphoblasts were compared to normal lymphoblasts, the deficient cellsshowed increased resistance to glucose starvation: they maintained higher rates of de novo synthesis in the absence of glucose and required less glucose for half-maximal rates. The difference between the two cell types may beexplained by increased PP-Rib-P availability in the HPR transferase-deficient cells which is documented by an increased intracellular PP-Rib-P concentration and increased [l4C]adenineincorporation. This increased PP-Rib-P availability in the HPR transferase-deficient cells is because of ribose salvage from endogenous nucleotide breakdown in the absence of hypoxanthine salvage, i.e. unilateral ribose reutilization (42, 45). The contribution of this ribose reutilization to Rib-5-P availability for de novo purine synthesiswas studied in the HPRtransferase-deficient

Ribose 5-Phosphate and Purine Synthesis cells by treating them with X‘-deoxyinosine, an alternative substrate for purine nucleoside phosphorylase that does not yield Rib-5-P (33). We found that the reutilization of ribose from endogenous nucleotide breakdown becomes a significant factor for the maintenance of de novo purine synthesis at low glucose concentrations (5100 @hi), whereas in thepresence of higher glucose concentrations, ribose reutilization does not seem to be necessary for normal rates of de novo purine synthesis. We determined the degree to which purine nucleosides can replace glucose as a ribose source for de novo purine synthesis. On a molar basis, low concentrations of 8-aminoguanosine in normal lymphoblasts and inosine in HPR transferase-deficient lymphoblasts were more efficient than glucose in increasing Rib-5-P and PP-Rib-P concentrations, as well as rates of de novo purine synthesis. However, higher concentrations of nucleosides were less effective and neither nucleoside could fully replace glucose; maximal rates of de muo purine synthesis achieved in the presence of 8-aminoguanosine or inosine were about 55% of those observed in the presence of 10 mM glucose. Comparably high intracellular Rib-5-P and PP-Rib-P concentrations were established in thepresence of inosine and glucose. This eliminates the possibility that the rate of nucleoside transport or the conversion by purine nucleoside phosphorylase and phosphoribomutase might have been rate-limiting under these conditions. Inhibition of de novo purine synthesis by residual HPR transferase activity and consequent nucleotide formation is also excluded since the concentration of purine nucleotides in the presence of 1 mM inosine were the same as in the presence of 10 mM glucose4 and maximal, feedback-released de novo purine synthesis in the presence of mycophenolic acid, alanosine, and hadacidin with 1 mM inosine was still only 70% of that observed with 10 mM glucose in the presence of the drugs. We cannot exclude the possibility of some other unidentified side effect of high purine nucleoside concentrations on the de novo pathway. A more plausible explanation is that glucose metabolism provides a factor in addition to Rib-5-P that is necessary for maximal rates of de novo purine synthesis. The control of de novo purine synthesis is complex and feedback inhibition by purine nucleotides is a major determinant (3-5, 43). The intracellular concentrations of GTP, ATP, and ADP decreased during glucose starvation without major changes in thepurine nucleoside monophosphate pools. A partial release of feedback inhibition on PP-Rib-P synthetase and amidophosphoribosyltransferase in glucose-starved cells is, therefore, possible (5, 43). We felt it necessary to eliminate the control of de novo purine synthesis by feedback inhibition in order to fully evaluate the influence of Rib-5-P andPP-Rib-P availability on therate of de novo purine synthesis. The combination of mycophenolic acid, alanosine, and hadacidin prevents the synthesis of guanylates and adenylates from IMP, thereby depleting the intracellular nucleotide pools and releasing feedback inhibition (22, 24, 27). We found that in normal lymphoblasts de novo purine synthesis could be stimulated by the drugs only at glucose concentrations greater than 100 PM, whereas in HPR transferasedeficient lymphoblasts, stimulation was observed even in the absence of glucose. The changes in the concentration of purine nucleotides during glucose starvation andincubation with the drugs were virtually identical in both cell types. Again, the difference between the two cell types can be explained by the increased PP-Rib-P availability in the HPR transferase-deficient cells because of unilateral ribose reutilization (42, 45). R. B. Pilz, R. C. Willis, and G. R. Boss, unpublished results.

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In addition to release of feedback inhibition, mycophenolic acid, alanosine, and hadacidin increase ribose reutilization. Under normal conditions, cytoplasmic 5’-nucleotidase competes with IMP-dehydrogenase and adenylosuccinate synthetase for the substrate IMP(Fig. 1).Previous studies indicate that as much as 30-50% of newly synthesized IMP is subject to degradation and participates in the “inosinate cycle” (44, 45). This cycle is composed of cytoplasmic 5’-nucleotidase, purine nucleoside phosphorylase, phosphoribomutase, PPRib-P synthetase, and HPRtransferase (Fig. 1).In the presence of inhibitors of IMP-dehydrogenase and adenylosuccinate synthetase, the only possible fate of the accumulating IMP is degradation. Thus, the flux through the inosinate cycle is greatly enhanced. Hypoxanthinethat is generated can be excreted into the media, whereas the ribose moiety is salvaged as ribose phosphate, retained within the cell, and reutilized. Indeed, in the presence of mycophenolic acid, alanosine, and hadacidin, greater than 90% of the total de novo synthesized purines were found in the culture media and the intracellular Rib-5-PandPP-Rib-P concentrations were markedly increased. Because mycophenolic acid, alanosine, and hadacidin increase the flux through the inosinate cycle and increase Rib5-P reutilization, de novo purine synthesis in the presence of the drugs may require less ribose from the pentose phosphate pathway than without the drugs. We should note that we observed ribose reutilization for purine synthesis, but we do not know how much ribose may also be used for synthesis of other compounds, for example, lactic acid. The importance of ribose reutilization for the stimulation of de novo purine synthesis by mycophenolic acid, alanosine, and hadacidin was shown in HPR transferase-deficient cells, since 2“deoxyinosine prevented the stimulation at glucose concentrations of 100 p~ or less. Again, 100 P M glucose seems to be a critical concentration for de novo purine synthesis. Below this concentration, de novo purine synthesis cannot be increased in response to released feedback inhibition unless additional Rib-5-P and PP-Rib-P are provided by endogenous ribose reutilization in the absence of hypoxanthine salvage (HPR transferase deficiency) or by an alternative exogenous ribose source. Even at 10 mM glucose, stimulation of de novo purine synthesis by the drugs was reduced in the presence of 2‘deoxyinosine. The importance of ribose reutilization for the stimulation of de novo purine synthesisby mycophenolic acid, alanosine, and hadacidin also has been shown in studies with purine nucleoside phosphorylase-deficient lymphoblasts (46). Acknnwledgments-We gratefully appreciate the generous support of Dr. J. E. Seegmiller, in whose laboratory most of this work was performed. We are also thankful for the advice and technical assistance of Dr. L. Mahan, and the helpful discussions with Drs. L. F. Thompson and S. S. Matsumoto. Weacknowledge D. Lundemo’s skillful preparation of this manuscript. REFERENCES 1. Becker, M. A., Raivio, K. O., and Seegmiller, J. E. (1979) Ado. Enzymol. 49, 281-306 2. Wyngaarden, J. B., and Kelley, W. N. (1983) in The Metabolic

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Fredrickson, D. S., Goldstein, J. L., and Brown, M. S., e&) 5th ed., pp. 1043-1114, McGraw-Hill, New York Kelley, W. N., Holmes, E. W., and Van der Weyden, M. B. (1975) Arthritis Rheum. 18,673-680 Holmes, E. W., McDonald, J. A., McCord, J. M., Wyngaarden, J. B., and Kelley, W. N. (1973) J. Biol. Chem. 248, 144-150 Holmes, E. W., Wyngaarden, J. B., and Kelley, W. N. (1973) J. Biol. C h m . 248,6035-6040 Brosh, S., Boer, P., Kupfer, B.,de Vries, A., and Sperling, 0. (1976) J. Clin. Invest. 58.289-297

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7. Bashkin, P., and Sperling, 0.(1978) Biochim. Biophys. Acta538, 505-511 . ~. .~

-

8. Becker, M. A. (1976) Biochim. Biophys. Acta 435, 132-144 9. Kelley, W. N., Fox, I. H., and Wyngaarden, J. B. (1970) Biochim. Biophys. Acta 215,512-516 10. Boyle, J. A., Raivio, K. O., Becker, M.A., and Seegmiller, J. E. (1972) Biochim. Biophys. Acta 269,179-183 11. Bagnara, A. S., Letter, A. A., andHenderson, J. F. (1974)Biochim. Biophys. Acta 3 7 4 , 259-270 12. Hershfield, M. S., and Seegmiller, J. E. (1977) J. Biol. Chem. 252,6002-6010 13. Becker, M. A. (1976) J. Clin. Invest. 57, 308-318 14. Becker, M. A., and Seegmiller, J. E. (1975)Arthritis Rheum. 18, (suppl.) 687-693 15. Howell, R.R., Ashton, D. M., and Wyngaarden, J. B. (1962) Pediatrics 29,553-559 16. Long, W. K. (1962) Science (Wash. D.C.) 138,991-993 17. Levy, J . A., Buell, D. N., Creech, C., Hirshaut, Y., and Silverberg, H. (1971) J. Natl. Cancer Inst. 46,647-652 18. Pilz, R. B., Willis, R.C., and Seegmiller, J . E. (1982) J. Biol. Chem. 257,13544-13549 19. Iscove, N. N., and Melchers, I. (1978) J. Exp. Med. 147, 923933 20. Spieker-Polet, H.,and Polet, H. (1981) J. Zmmunol. 126, 949954 21. Sweeney,M. J., Hoffman, D. H., and Esterman, M. A. (1972) Cancer Res. 32, 1803-1809 22. Lowe. J. K.. Brox., L... and Henderson, J. F. (1977) Cancer Res. 37; 736-743 23. Hershfield, M. S., and Seegmiller, J. E. (1976) J. Biol. Chem. 25 1.7348-7354 24. Graff, J. C., and Plagemann, P. G. W. (1976) Cancer Res. 3 6 , 1428-1440 25. Hurlbert, R. B., Carrington, D. B., and Moore, E. C. (1977) Proc. Am. Assoc. Cancer Res. 18, 234 26. Shigeura, H. T., and Gordon, C. N. (1962) J. Biol. Chem. 237, 1932-1936

27. Willis, R. C., and Seegmiller, J. E. (1979) Adu. Enp. Med. Biol. 122B. 237-241 28. Henderson, J. F., and Khoo, M. K. Y. (1965)J. Biol. Chem. 240, 2358-2362 29. Skaper, S. D.,Willis, R. C., and Seegmiller, J. E. (1976) Science (Wash. D.C.) 193, 587-588 30. Boss, G.R., Idriss, S. D., Willis, R.C., and Seegmiller, J. E. (1983)Anal. Biochem. 1 3 0 , 283-286 31. Lowry, 0.H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. (1951)J. Biol. Chem. 193, 265-275 32. Lanzetta, P. A., Alvarez, L. J., Reinach, P. S., and Candia, 0.A. (1979) Anal. Biochem. 100,95-97 33. Willis, R.C., Robins, R. K., and Seegmiller, J. E. (1980) Mol. Pharrnacol. 18, 287-295 34. Stoeckler, J. D., Cambor, C., Kuhns, V., Chu, S. H., and Parks, R. E., Jr. (1982) Biochem. Pharmacol. 31, 163-171 35. Thompson, L. F., Willis, R. C., Stoop, J. W., and Seegmiller, J. E. (1978) Proc. Natl. Acad. Sci. U. S. A. 75, 3722-3726 36. Snyder, F. F., Mendelsohn, J., and Seegmiller, J. E. (1976) J. Clin. Invest. 58, 654-666 37. Peters, G. J., Oosterhof, A., and Veerkamp, J. H. (1981) Int. J. Bwchem. 13,577-583 38. Allsop, J., and Watts, R. W. E. (1978) J. Mol. Med. 3, 105-110 39. McCairns, E., Fahey, D., Sauer, D., and Rowe, P. B. (1983) J. Biol. Chem. 258,1851-1856 40. Meyer, L. J., and Becker, M. A. (1977) J. Biol. Chem. 252,39193925 41. Cross, M. E., and Ord, M. G. (1971) Biochem. J. 124,241-248 42. Gordon, R. B., Thompson, L., Johnson, L. A., and Emmerson, B. T. (1979) Biochim. Biophys. Acta 5 6 2 , 162-176 43. Fox, I. H., and Kelley, W. N. (1972) J. Biol. Chern. 247, 21262131 44. Barankiewicz, J., Gelfand, E. W., Issekutz, A., and Cohen, A. (1982) J. Biol. Chem. 257, 11597-11600 45. Willis, R. C., Kaufman, A. H., and Seegmiller, J. E. (1984) J . Biol. Chem. 259 in press 46. Boss, G. R. (1984) J. Biol. Chem. 259, 2936-2941

Ribose 5-Phosphate and Synthesis Purine

2933

YJPPLEMENTAL MTERIAL TO: THE INFLUENCE OF RIBOSE-5-PHOSPMTE AVAILABILITY OM PURINESYNTHESIS OF CULTUIEO HWAW LYllPHOBUSTS AND MITOGEN-STIWLATEO LYMPHOCYTES by Renate 8. P l l x ,R a n d a l l

c. u i l l i r .

6erry

R. BOIS

W a s u r m n t ofRlb5P

and PPRibP

EXPERIMEHTAl PROCEDURES h - i q i n and C u l t u r e o f M h o b l a r t r

The human 5 - l m h o b l a r t o l d c e l l l i n e YIL-2 (I71 was used I n t h i s study.A hmxanthine phorphorlbasyltranrferare (loorinatc-guanyl4te:pyophophate p h o s p b r l b r y l t r a n r referase. E.C. 2.4.2.8. HPRT) d e f i c i e n t d e r i w t l w of YIL-2 1.1 selectedby6-thioguanine IistdnCe; i t hadlessthan 0.51 o f m m a l HPRT a c t l v i t y and a 14-16 h d o u b l i n g time as cmpared t o a 15-20 h doubling m ti e O f t h ep a r e n t a l i n e .C e l l s *ere c u l t u r e d as described p r e v i o u s l y (181. guanine

Preparation O f Glucose-Deflclent Y d i u

Electronic C e l l Yolum P n a l y r i s

Hearurenrnt Treatment o f W h o b l a s t s Yith Wcophenolic k l d . A l M o r i n e and Hadacidin

Of

n w o x a n t h l n e i n the Lmphocyte Culture W d l u n

2934

Synthesis PurineSi-Phosphate andRibose I n t r a c e l l u l a r RlbSP and PPRibP Concentrations and 114ClMenine Incorporation I n H P R T - k f i c l e n t L m h a b l a r t r

@proximately 5 x 106 c e l l s *re washed in 2 m1 O f theprevlouIlymentioned HEPES buffer. The c e l lp e l l e t s *re e x t r a c t e d in 135 " 1o f 1 dl NaEOTA pH 7.4. Plus 15 y l o f 4 N pePChloliCacld.centrifugedfor 3 nin It 1.WO x 9. and 125 y l ofthesupernatants rere I m d i a t e l y n e u t r a l l z d w i t h 13 u l of 4 N KOH I n 0.4 M a r p h o l l n e e t h l n e w l f m i c a c i d . A l l procedures e r e performed as q u i c k l y IS p a s s i b l ea t 4% s i n c el a b i l e OrgMiC phosphateert e n hydrolyze i n theacld. increasing thevalues. The m u n t of inorganic phosphate I n 1 W vlof theneutralizedsupernatant #as d e t e m l n e db yt h er e t h c do fL a n z e t t ae t a1 0 2 ) using malachite greenlannanim molybdate In 4 N El. In t h i s ass&Y, c a l o r d e v e l o w n t frm inorganic phosphate Is c m p l e t e w i t h i n 1 min; furtherincrease i n c o l w by h y d r o l y s i s Of organic phosphateestevs i s prevented by t h e a d d l t i o n Of c i t r a t e .

glucose-deficient media. v a r i a b l e anlOUnt5 HPRT-deficient l y n p h o b l a r t l *re t r a n s f e r r e d to of glucose and/or 20 "I4 mycophenallcacld 20 "ll alanosine and 500 u M hadacid7n were added a t tire 0 15 i n d i c a t e d . 100 uH inosine 0; 100 Y M 2'-deoxYinorlne were added a f t e r1 - 3 4 h of incubation i n t h e absence o f glucose. Otherwise. theexperiment vas prformed d l des c r i b e d in thelegend

t o Table l .

Uycophenolic AODXTIOHALRESULTS

c14q~entne

Acid. Alanasine Ribore Source

Table I I n t r a c e l l u l a r C o n c e n t r a t i o n of Purlne NUcleOtides in

and HadaCidln

in t h e Media

mmal LniQhoblartr

were t r a n s f e r r e d t o g l u c o r e - d e f l c l e n w t dia. Varlable m o u n t 5 O f 91"lbnnal1yIphobIaSts c o w and/or 20 yH lRyCophenolicacid, 20 yM a l a n o r l n e and 5W hadacidin r r e added a t time 0 as i n d i c a t e d . 50 8-minoguanostne was added a f t e r 1 4 4 h o fI n c u b a t i o n i n t h e absence o f glucose. A f t e r a t o t a lo f 24 h o fi n c u b a t i o n .c e l l s =re harvested. wasned. e x t r a c t e d and t h e i n t r a c e l l u l a r concentration o f p u r i n e n u c l e o t i d s r were determined I S descrtbedunder Experimental PTocedures. A11 valuesWPrelentthe man o f 3independenterperimntr:for sake of C l e r l t y t h e S.0. we not I h P n b u t sere g e n e r a l l y < 15X o f t h e mean.

m e

cells) (HI

ATP

AOP

6TP

+

1.55

0.31

0.46

0. I8

1.59

0.33

0.33

0.15 1 m "M Inosine

1.61

0.44

0.53

0.21

1.73

0.45

0.33

0.16

0.43

0.62

0.19 0.14

1.86

10-4

10-3

M.(H_12.7

44.7:19.5 54.1+16.1

16.1+2.0

66.7r24.1

21.625.2

203.9237.4

10 nll Glucose

10-5

o la C e l l s

8.7rl.l

175.9:22.1

54.5216.1

122.3234.5

105.6234.9

38.827.1

478.9251.9 90.2:15.2

449.8:49.3

60P

(nnOl/l06 and Hadacidln

0

pmo1/ain/

83.7~21.9

69.7222.6

l w 31 G l w o s e Acid. Alanorine

PPRibP

p n O o / l aC e l l s

W o p h e n oGlllvcc o r e Concentration

Uptake RibSP

1.83

0.42

0.27

2.18

0.48

0.69 0.19

2.07

0.30

0.31

2.56

0.45

0.81

0.18

2.02

0.37

0.29

0.10

1w "M Deaylnarine

41.228.8

133.6221.4

111.7225.0

32.52?.2

367.6269.8

411.4202.5

62.6218.5

4.620.6

39.5215.4 42.7~14.4

42.529.1

0.el.l

0.11

10-2

Ould be detected ar-early aS h a f t e the a d d i t t o n of PHA. b y 36h t h e c e l l volume had lnre thandoubled t o 0.37 d / l o c e l l s . and at 72 h i t reache&0.45 &/1& cells.

54.3214.9

5W VH Glucose

10 dl G l ~ o s e

225.8149.5 41.75.9

38.8214.5

18.723.0

276.551.2

91.1221.0

E0.5~28.2

29.25.5

421.1214.1

426.2~75.0

78.5$11.2

46.5r16.1

62.2223.6

22.1$.2

8

Ribose 5-Phosphate and Synthesis Purine

2935

PPRibP Synthetase and I h l l d o p h o l ~ h ~ r i b ~ ~ y l t r l n l f e r a r e k t i v i t l e il n PHA-Stimulated L P -

~ n z m ek t i v i t f e s and I n t r a c e l l u l a r PhomhateConcentration o f PHA-Stimulated and Won-Stiwlated W h o c y t e r PPRibP smthetase activity, glutamine amidepholphoriboryltranrferare a c t i v i t y and the i n t r a cellularconcentration O f inorganic phosphate *ere determined as described under Experiwnt a l Procedures a t 0. 2, 4. 8. 12, 24, 48 m d 72 h ofculture. Since there was no change i o valuesobtainedduringthe time course. thedata were averaged fo1thestimullted and nomstimulatedcells.valuer represent t h e mean S.O. o f at least 3 independent experiments.

:

INU164TW TLE

Ih I

~

0-72 h

2

1.57 f .56

1.64

Inorganic Phosphate

me i n t r a c e l l u l a r PPRibP concentration i n unstieulated I m h o c y t e r #as 63.6 + 11.6 W t h i n 2 h a f t e r additTon of and decreased s l i g h t l y during tie i n c u l t m e (Fig, VIrB). mitogen theconcentration increased t o 96.5 + 20.6 in the s t i m u l a t e dc e l l s .M t e rt h a t increased f u r t h e r u n t i l 12 Ii and rslained elevated until 48 h; it return& the c&ntration t oc o n t r o l values by 72 h Of c u l t u r e h p r e s ed m I per c e l lb a s i st h e PPRibP content 1 the Ynftimulated c e l l s was 11.0 + 2.b c e l l s i n d increased io 56.6 11.1 pl011108 c e l l s a t 48 hafteraddition of FHA. There r e s u l t s are in c l m e a g r e a r n t i i i t h t h e values reportedby Snyder e t a1 (361 and byPeters e t a1 (37).

+ PHA

PHA

0-72 h

2.77

2

.37

.46

2.34

2

2.41

:.37

.53

% Intracellular Concentration

mild

""I

I A

Rsb5P

Of

PurineNucleotides

i n PHA-Stimulated L m h o c y t e r

The i n t r a c e l l u l wc o n c e n t r a t i o n of purine nucleotides remained unchanged during the first 24 h a f t e r PHR s t i m u l a t i o n (Table V I . At 48 h a mdest but significant (P 0.05 was mre prminent in thenucleoside mno- and di: student t t e s t ) increaseMCurmdthat phosphates than i n thenucleosidetripharphater. vhen thedata were ezpressed as m011106 m a l l increases were seen as e a r l y as 8 h post r t i n u l a t i o nb u t thesedifferences were not me Concentration o f r e f l e c t e d i n the concentration because of the increase i n c e l l volume Ivp WI mIs t prokablyoverestimated and was. themfare.expressed as ierr than t h e c a l c u l a t es are s i m i l a r t o those o f cultured human ed nudmr. me values fm t h e s t i m u l a t e d l m h o c l m h o b l d f t s (canpare t o Table1 a t 10 mI4 glucosef!