Regulation of purine nucleotide synthesis in human B lymphoblasts with both hypoxanthine-guanine phosphoribosyltransferase deficiency and phosphoribosylpyrophosphate synthetase superactivity.

Human B lymphoblast lines severely deficient in hypoxanthine-guanine phosphoribosyltransferase (HGPRT) were selected for resistance to 6-thioguanine from cloned normal and phosphoribosylpyrophosphate (PP-Rib-P) synthetase-superactive cell lines and were compared with their respective parental cell lines with regard to growth and PP-Rib-P and purine nucleotide metabolism. During blockade of purine synthesis de novo with 6-methylthioinosine or aminopterin, inhibition of growth of all HGPRT-deficient cell lines was refractory to addition of Ade at concentrations which restored substantial growth to parental cell lines. Ade-resistant inhibition of growth of parental lines by 6-methylthioinosine, however, occurred during Ado deaminase inhibition. Insufficient generation of IMP (and ultimately guanylates) to support growth of lymphoblasts lacking HGPRT activity and blocked in purine synthesis de novo best explained these findings, implying that a major route of interconversion of AMP to IMP involves the reaction sequence: AMP----Ado----Ino----Hyp----IMP. PP-Rib-P generation and purine nucleoside triphosphate pools were unchanged by introduction of HGPRT deficiency into normal lymphoblast lines, in agreement with the view that accelerated purine synthesis de novo in this deficiency results from increased availability of PP-Rib-P for the pathway. Cell lines with dual enzyme defects did not differ from PP-Rib-P synthetase-superactive parental lines in rates of PP-Rib-P and purine synthesis despite 5-6-fold increases in PP-Rib-P concentrations, excretion of nearly 50% of newly synthesized purines, and diminished GTP concentrations. Fixed rates of purine synthesis de novo in PP-Rib-P synthetase-superactive cells appeared to reflect saturation of the rate-limiting amidophosphoribosyltransferase reaction for PP-Rib-P. In combination with accelerated purine excretion, increased channeling of newly formed purines into adenylates, and impaired conversion of AMP to IMP, fixed rates of purine synthesis de novo may condition cell lines with defects in HGPRT and PP-Rib-P synthetase to depletion of GTP with consequent growth retardation.     

Purine deoxynucleosides and adenosine dialdehyde decrease 5-amino-4-imidazolecarboxamide (Z-base)-dependent purine nucleotide synthesis in cultured T and B lymphoblasts.

Deoxyadenosine (dAdo) and deoxyguanosine (dGuo) decrease methionine synthesis from homocysteine in cultured lymphoblasts; because of the possible trapping of 5-methyltetrahydrofolate this could lead to decreased purine nucleotide synthesis. Since purine deoxynucleosides could also inhibit purine synthesis de novo at an early step not involving folate metabolism, we measured in azaserine-treated cells 5-amino-4-imidazolecarboxamide (Z-base)-dependent purine nucleotide synthesis using [14C]formate. In the T lymphoblasts, Z-base-dependent purine nucleotide synthesis was decreased 26% by 0.3 microM-dAdo, 21% by 1 microM-dGuo and 28% by 1 microM-adenosine dialdehyde, a potent S-adenosylhomocysteine hydrolase inhibitor; homocysteine fully reversed the inhibitions. The B lymphoblasts were considerably less sensitive to the deoxynucleoside-induced decrease in Z-base-dependent purine nucleotide synthesis, with 100 microM-dAdo required for significant inhibition and no inhibition by dGuo at this concentration; homocysteine partly reversed the inhibition by dAdo. The observed decrease in Z-base-dependent purine nucleotide synthesis could not be attributed either to dUMP depletion changing the folate pools or to decreased ATP availability because dUrd was without effect and during the experimental period the intracellular ATP concentration did not change significantly. Cells with 5,10-methylenetetrahydrofolate reductase deficiency were relatively resistant to inhibition of Z-base-dependent purine nucleotide synthesis by dAdo and adenosine dialdehyde. Our results suggest that deoxynucleosides decrease purine nucleotide synthesis by trapping 5-methyltetrahydrofolate.     

Alteration of purine metabolism by AICA-riboside in human B lymphoblasts.

The effect of 5-amino-4-imidazole-carboximide (AI-CA)-riboside on different pathways of purine metabolism (biosynthesis de novo, salvage pathways, adenosine metabolism, ATP catabolism) was studied in human B lymphoblasts (WI-L2). AICA-Riboside markedly decreased intracellular levels of 5-phosphoribosyl-1-pyrophosphate and in consequence affected purine biosynthesis de novo and purine salvage pathways. AICA-riboside inhibited incorporation of glycine into purine nucleotides, but when formate was used as the precursor of purine biosynthesis de novo, a biphasic effect was observed. The incorporation of formate into purine nucleotides was increased by AICA-riboside at concentrations up to 2 mM but decreased at higher concentrations. Salvage of the purine bases adenine, hypoxanthine, and guanine was markedly inhibited and utilization of extracellular adenosine in B lymphoblasts was reduced by AICA-riboside. AICA-riboside increased ribose 1-phosphate concentrations and increased degradation of prelabeled ATP. No effect on the intracellular levels of orthophosphate was found. Proliferation of WI-L2 lymphoblasts was only slightly affected at concentrations of AICA-riboside below 500 microM but markedly inhibited by higher concentrations.     

Purine nucleotide reutilization by human lymphoblast lines with aberrations of the inosinate cycle

A purine nucleotide (inosinate) cycle is demonstrated with human lymphoblasts. The lymphoblast requires approximately 50 nmol of purine/10(6) cell increment. When the inosinate cycle is interrupted by the genetic, severe deficiency of either or both purine nucleoside phosphorylase (PNP) or hypoxanthine phosphoribosyltransferase (HPRT), purine accumulates in the culture medium as inosine, guanosine, deoxyinosine, and deoxyguanosine (PNP deficiency or PNP, HPRT deficiency) or hypoxanthine and guanine (HPRT deficiency). This accumulation represents an additional 25 to 32 nmol of purine which must be synthesized per 10(6) cell increment. PNP-deficient lymphoblasts have PPRibP contents characteristic of normal lymphoblasts, about 20 to 25 pmol/10(6) cells. HPRT-deficient lymphoblasts have four times higher PPRibP contents. The lymphoblast deficient for both PNP and HPRT has only a marginal elevation of PPRibP content, 1.5 times normal values. The elevated PPRibP content of HPRT-deficient cells reflects the efficient, unilateral reutilization of the ribose moiety of purine ribonucleotides and is not a cause of purine overproduction. Purine overproduction characterizing PNP-deficient lymphoblasts appears similar to overproduction from deficiency of HPRT, i.e. a break in the inosinate cycle rather than overactive de novo purine synthesis.     

Evidence for direct inhibition of de novo purine synthesis in human MCF-7 breast cells as a principal mode of metabolic inhibition by methotrexate

We have investigated the role of dihydrofolate (H2PteGlu) accumulation in the inhibition of de novo purine synthesis by methotrexate (MTX) in human MCF-7 breast cancer cells. Previous studies have shown that cytotoxic concentrations of MTX that inhibit dihydrofolate reductase produce only minimal depletion of the reduced folate cofactor, 10-formyltetrahydrofolate, required for purine synthesis. At the same time, de novo purine synthesis is totally inhibited. In these studies, we show that 10 microM MTX causes inhibition of purine synthesis at the step of phosphoribosylaminoimidazolecarboxamide (AICAR) transformylase, as reflected in a 2-3-fold expansion of the intracellular AICAR pool. The inhibition of purine synthesis coincides with the rapid intracellular accumulation of H2PteGlu, a known inhibitor of AICAR transformylase. When the generation of H2PteGlu is blocked by pretreatment with 50 microM 5-fluorodeoxyuridine (FdUrd), an inhibitor of thymidylate synthase, MTX no longer causes inhibition of purine synthesis. Intermediate levels of H2PteGlu produced in the presence of lower (0.1-10 microM) concentrations of FdUrd led to proportional inhibition of purine biosynthesis, and the exogenous addition of H2PteGlu to breast cells in culture re-established the block in purine synthesis in the presence of FdUrd and MTX. The early phases of inhibition of purine biosynthesis could be ascribed only to H2PteGlu accumulation. MTX polyglutamates, also known to inhibit AICAR transformylase, were present in breast cells only after 6 h of incubation with the parent compounds and were not formed in cells preincubated with FdUrd. The lipid-soluble antifolate trimetrexate, which does not form polyglutamates, produced modest 10-formyltetrahydrofolate depletion, but caused marked H2PteGlu accumulation and a parallel inhibition of purine biosynthesis. This evidence leads to the conclusion that MTX and the lipid-soluble analog trimetrexate cause inhibition of purine biosynthesis through the accumulation of H2PteGlu behind the blocked dihydrofolate reductase reaction.     

Helicobacter pylori relies primarily on the purine salvage pathway for purine nucleotide biosynthesis

Helicobacter pylori is a chronic colonizer of the gastric epithelium and plays a major role in the development of gastritis, peptic ulcer disease, and gastric cancer. In its coevolution with humans, the streamlining of the H. pylori genome has resulted in a significant reduction in metabolic pathways, one being purine nucleotide biosynthesis. Bioinformatic analysis has revealed that H. pylori lacks the enzymatic machinery for de novo production of IMP, the first purine nucleotide formed during GTP and ATP biosynthesis. This suggests that H. pylori must rely heavily on salvage of purines from the environment. In this study, we deleted several genes putatively involved in purine salvage and processing. The growth and survival of these mutants were analyzed in both nutrient-rich and minimal media, and the results confirmed the presence of a robust purine salvage pathway in H. pylori. Of the two phosphoribosyltransferase genes found in the H. pylori genome, only gpt appears to be essential, and an Δapt mutant strain was still capable of growth on adenine, suggesting that adenine processing via Apt is not essential. Deletion of the putative nucleoside phosphorylase gene deoD resulted in an inability of H. pylori to grow on purine nucleosides or the purine base adenine. Our results suggest a purine requirement for growth of H. pylori in standard media, indicating that H. pylori possesses the ability to utilize purines and nucleosides from the environment in the absence of a de novo purine nucleotide biosynthesis pathway.     

Regulation of de novo purine biosynthesis in Chinese hamster cells

Regulation of de novo purine biosynthesis was examined in two Chinese hamster cell lines, CHO and V79. De novo purine biosynthesis is inhibited at low concentrations of adenine. The mechanism of inhibition was studied using the RNA and protein synthesis inhibitors actinomycin D, cycloheximide, and azacytidine. Although all three inhibitors rapidly inhibited de novo purine biosynthesis in vivo, neither adenine nor the RNA and protein synthesis inhibitors could be found to have an effect in vitro on either phosphoribosylpyrophosphate (PRPP) synthetase or amido phosphoribosyltransferase, the first enzymes of the de novo pathway. However, in the presence of actinomycin D, cycloheximide, and azacytidine, there was a 50% or greater reduction in PRPP concentrations. This reduction in PRPP levels is correlated with a 2-fold increase in purine nucleotides in the acid-soluble pool. It is proposed that in the presence of the metabolic inhibitors there is an increase in nucleotide pools due to degradation of RNA, with a resulting feedback inhibition on de novo purine biosynthesis. In contrast to a previous report (Martin, D. W., Jr., and Owen, N. T. (1972) J. Biol. Chem. 247, 5477-5485), we could find no evidence for a repressor type mechanism in these cells.     

A new folate antimetabolite, 5,10-dideaza-5,6,7,8-tetrahydrofolate is a potent inhibitor of de novo purine synthesis

5,10-Dideazatetrahydrofolate (DDATHF) is a new antimetabolite designed as an inhibitor of folate metabolism at sites other than dihydrofolate reductase. DDATHF was found to inhibit the growth of L1210 and CCRF-CEM cells in culture at concentrations in the range of 10-30 nM. The inhibitory effect of DDATHF on the growth of L1210 and CCRF-CEM cells was reversed by either hypoxanthine or aminoimidazole carboxamide. Growth inhibition by DDATHF was prevented by addition of both thymidine and hypoxanthine, but not by thymidine alone. 5-Formyltetrahydrofolate reversed the effects of DDATHF in a dose-dependent manner. DDATHF had no appreciable inhibitory activity against either dihydrofolate reductase or thymidylate synthase in vitro, but was found to be an excellent substrate for folylpolyglutamate synthetase. DDATHF had little or no effect on incorporation of either deoxyuridine or thymidine into DNA, in distinct contrast to the effects of the classical dihydrofolate reductase inhibitor, methotrexate. DDATHF was found to deplete cellular ATP and GTP over the same concentrations as those inhibitory to leukemic cell growth, suggesting that the locus of DDATHF action was in the de novo purine biosynthesis pathway. The synthesis of formylglycinamide ribonucleotide in intact L1210 cells was inhibited by DDATHF with the same concentration dependence as inhibition of growth. This suggested that DDATHF inhibited glycinamide ribonucleotide transformylase, the first folate-dependent enzyme of de novo purine synthesis. DDATHF is a potent folate analog which suppresses purine synthesis through direct or indirect inhibition of glycinamide ribonucleotide transformylase.     

Major determinants of purine excretion from human lymphoblasts

WI-L2 B lymphoblasts deficient in hypoxanthine-guanine phosphoribosyltransferase (HGPRT) excreted amounts of hypoxanthine two to three times larger than CEM T lymphoblasts deficient in HGPRT, despite similar growth rates. ATP consumption occurred at a higher rate in WI-L2 cells than in CEM cells when cultivated in a glucose-free buffer, because of higher RNA synthesis in WI-L2 cells. The introduction of actinomycin D and azaserine resulted in lower hypoxanthine excretion in WI-L2 cells than in CEM cells, not in parallel with changes of the adenylate pool size. When the energy charge was high, de novo purine synthesis was a major determinant for purine excretion. The adenylate pool ratio (AMP/ATP) change caused by the introduction of oligomycin was greater during ATP depletion and recovery in WI-L2 cells than in CEM cells. WI-L2 cells were observed to have AMP deaminase activity three to four times higher than CEM cells. The major component of AMP deaminase in these cells was liver type. The higher rate of RNA synthesis caused greater changes of (AMP/ATP) and required higher AMP deaminase activity for recovery. When the energy charge was low, AMP deaminase was a major determinant for purine excretion.     

Mechanisms of deoxyguanosine lymphotoxicity. Human thymocytes, but not peripheral blood lymphocytes accumulate deoxy-GTP in conditions simulating purine nucleoside phosphorylase deficiency

This study was designed to simulate purine nucleoside phosphorylase (PNP) deficiency by preincubating with guanosine (Guo) to minimize PNP activity while investigating the metabolism of [14C] deoxyguanosine (dGuo) at physiologic concentrations (10 microM) by unstimulated thymocytes, tonsil-derived T and B lymphocytes, and peripheral blood cells over short time periods. GTP was the principal metabolite formed from dGuo by all cell types with functional PNP and hypoxanthine-guanine phosphoribosyltransferase, confirming formation via degradation to guanine with subsequent salvage by hypoxanthine-guanine phosphoribosyltransferase. Thymocytes also formed a small amount of deoxyguanosine triphosphate (dGTP), presumably through direct phosphorylation by deoxycytidine kinase. Incorporation of dGuo into GTP was effectively inhibited in all instances under PNP deficiency conditions and dGTP levels increased up to 10-fold in thymocytes, but tonsil-derived B or T lymphocytes and unfractionated PBL still accumulated no detectable dGTP. E and platelets formed low amounts of dGTP under these conditions. Preincubation with adenine (50 microM) to reverse any Guo-induced toxicity reduced the incorporation of dGuo into GTP without inhibitor in all cell types with intact adenine phosphoribosyltransferase, but had no effect on dGTP accumulation in thymocytes, with or without inhibitor, thus excluding any indirect formation of dGTP via the de novo route. The rapid metabolism of dGuo to GTP, in the absence of PNP inhibition and subsequent effects of the altered GTP concentrations on cellular metabolism, may account for the differing responses reported by investigators with the use of low dGuo concentrations (enhancing), compared with high (inhibitory), concentrations in mitogen-stimulated lymphocyte studies. The exclusive ability of thymocytes to accumulate significant amounts of dGTP, and inability of B cells to do so, provides a logical explanation for the selective T cell immunodeficiency in PNP deficiency.     

Pyrimidine synthesis in Burkholderia cepacia ATCC 25416

K. LI AND T.P. WEST. 1995. Pyrimidine synthesis in the food spoilage agent Burkholderia cepacia ATCC 25416 was investigated. The five de novo pathway enzymes of pyrimidine biosynthesis were found to be active in B. cepacia ATCC 25416 and growth of this strain on uracil had an effect on the de novo enzyme activities. The in vitro regulation of aspartate transcarbamoylase activity in B. cepacia ATCC 25416 was studied and its activity was inhibited by PP_i, ATP, GTP, CTP and UTP. The enzymes cytidine deaminase, uridine phosphorylase and cytosine deaminase were found to be active in the salvage of pyrimidines in ATCC 25416. Overall, de novo pyrimidine synthesis in B. cepacia ATCC 25416 was regulated at the level of enzyme activity and its pyrimidine salvage enzymes differed from those found in B. cepacia ATCC 17759.     

Decreased purine synthesis during amino acid starvation of human lymphoblasts

Normal human lymphoblasts starved for each of several essential, but not essential, amino acids had decreased DNA and RNA synthesis but no change in free intracellular purine nucleotides. The rates of purine nucleotide synthesis via the de novo and salvage pathways were measured by incorporating [14C]formate and [14C]hypoxanthine labels, respectively, into lymphoblasts starved for an amino acid or treated with a protein synthesis inhibitor. After 3 h of starvation, purine synthesis via the de novo pathway decreased 90% and via the salvage pathway decreased 60%. Cycloheximide and puromycin each reduced de novo synthesis by 96% and salvage synthesis by 72%. The decrease in purine synthesis de novo after removal of the amino acid was of first order kinetics and was fully and rapidly reversed by reconstitution with the amino acid. The synthesis of alpha-N-formylglycinamide ribonucleotide declined 97% after amino acid starvation; the synthesis of purines from 5-aminoimidazole-4-carboxamide riboside decreased 41%. The synthesis of guanylates decreased more than the synthesis of adenylates during amino acid starvation.     

Metabolic consequences of DNA damage: alteration in purine metabolism following poly(ADP ribosyl)ation in human T-lymphoblasts

The effect of DNA damage caused by N-methyl-N'-nitro-nitrosoguanidine (MNNG) on poly(ADP-ribose) synthesis, NAD levels, and purine nucleotide metabolism was studied in human T-lymphoblasts. Excessive DNA breaks caused by MNNG activated poly(ADP-ribose) polymerase and rapidly consumed intracellular NAD. NAD depletion was followed by rapid catabolism of ATP as well as induction of total purine nucleotide catabolism leading to excretion of purine catabolic products. MNNG-treated cells were not able to replenish the intracellular nucleotide pools due to the depletion of intracellular ATP and phosphoribosylpyrophosphate pools which are required for de novo purine biosynthesis. Inhibition of poly(ADP-ribose) polymerase by 3-aminobenzamide prevented both the depletion of NAD pools and the associated changes in purine nucleotide metabolism.     

Differential control of cell cycle,proliferation, and survival of primary T lymphocytes by purine and pyrimidine nucleotides

Purine and pyrimidine nucleotides play critical roles in DNA and RNA synthesis as well as in membrane lipid biosynthesis and protein glycosylation. They are necessary for the development and survival of mature T lymphocytes. Activation of T lymphocytes is associated with an increase of purine and pyrimidine pools. However, the question of how purine vs pyrimidine nucleotides regulate proliferation, cell cycle, and survival of primary T lymphocytes following activation has not yet been specifically addressed. This was investigated in the present study by using well-known purine (mycophenolic acid, 6-mercaptopurine) and pyrimidine (methotrexate, 5-fluorouracil) inhibitors, which are used in neoplastic diseases or as immunosuppressive agents. The effect of these inhibitors was analyzed according to their time of addition with respect to the initiation of mitogenic activation. We showed that synthesis of both purine and pyrimidine nucleotides is required for T cell proliferation. However, purine and pyrimidine nucleotides differentially regulate the cell cycle since purines control both G(1) to S phase transition and progression through the S phase, whereas pyrimidines only control progression from early to intermediate S phase. Furthermore, inhibition of pyrimidine synthesis induces apoptosis whatever the time of inhibitor addition whereas inhibition of purine nucleotides induces apoptosis only when applied to already cycling T cells, suggesting that both purine and pyrimidine nucleotides are required for survival of cells committed into S phase. These findings reveal a hitherto unknown role of purine and pyrimidine de novo synthesis in regulating cell cycle progression and maintaining survival of activated T lymphocytes.     

The PRPP synthetase spectrum: what does it demonstrate about nucleotide syndromes?

Defects in X-linked phosphoribosylpyrophosphate synthetase 1 (PRPS1) manifest as follows: (1) PRS-I enzyme "superactivity" (gain-of-function mutations affecting allosteric regions); (2) PRS-I overexpression (which may be linked to miRNA mutation); (3) severe PRS-I deficiency/Arts syndrome (missense mutations producing loss-of-function); (4) moderate PRS-I deficiency/Charcot-Marie-Tooth disease-5 (less severe loss-of-function mutations); and (5) mild PRS-I deficiency/Deafness-2 (mutations producing slight destabilization). Similar to Lesch-Nyhan disease, PRPS1-related disorders arise from phosphoribosyl-pyrophosphate (PRPP)-dependent nucleotide "depletion" of purine nucleotides (e.g., ATP, GTP). S-adenosylmethionine (SAMe) appears to partially alleviate purine depletion via a PRPP-independent path. Synthesis of pyrimidine nucleotides is PRPP dependent, with uridine monophosphate synthase deficiency producing pyrimidine nucleotide depletion. But pyrimidine salvage from uridine does not require PRPP, and this nucleoside is transported freely to pyrimidine-depleted tissues. Regulation of nicotinamide nucleotides is less clear; synthesis from pyridine nucleobases is PRPP dependent. Nucleotide "depletion" contrasts with nucleotide "toxicity," exemplified by the purine disorders adenosine deaminase (ADA) and purine nucleoside phosphorylase (PNP) deficiencies or by pyrimidine nucleotidase deficiency. These are characterized by the accumulation of one or more abnormal nucleotides such as succinyl- or deoxy-nucleotides or their metabolites, which interrupt other nucleotide or related pathways or are toxic to specific cell types. Theoretically, purine toxicity disorders would not be ameliorated by SAMe therapy, and this was confirmed for one adenylosuccinate lyase-deficient child. Nucleotide defects may also be seen as an aspect of mitochondrial disease, with SAMe-based mitochondrial therapy perhaps meriting further investigation.     

The purine nucleoside phosphorylase from Trichomonas vaginalis is a homologue of the bacterial enzyme

Trichomonas vaginalis is a parasitic protozoan and the causative agent of trichomoniasis. Its primary purine salvage system, consisting of a purine nucleoside phosphorylase (PNP) and a purine nucleoside kinase, presents potential targets for designing selective inhibitors as antitrichomonial drugs because of lack of de novo synthesis of purine nucleotides in this organism. cDNA encoding T. vaginalis PNP was isolated by complementation of an Escherichia coli strain deficient in PNP and expressed, and the recombinant enzyme was purified to apparent homogeneity. It bears only 28% sequence identity with that of human PNP but 57% identity with the E. coli enzyme. Gel filtration showed the enzyme in a hexameric form, similar to the bacterial PNPs. Steady-state kinetic analysis of T. vaginalis PNP-catalyzed reactions gave K(m)'s of 31.5, 59.7, and 6.1 microM for inosine, guanosine, and adenosine in the nucleosidase reaction and 45.6, 35.9, and 12.3 microM for hypoxanthine, guanine, and adenine in the direction of nucleoside synthesis. This substrate specificity appears to be similar to that of bacterial PNPs. The catalytic efficiency of this enzyme with adenine as substrate is 58-fold higher than that with either hypoxanthine or guanine, representing a distinct disparity with the mammalian PNPs, which have negligible activity with either adenine or adenosine. The kinetic mechanism of T. vaginalis PNP-catalyzed reactions, determined by product inhibition and equilibrium isotope exchange, was by random binding of substrates (purine base and ribose 1-phosphate) with ordered release of the purine nucleoside first, followed by inorganic phosphate. Formycin A, an analogue of adenosine known as an inhibitor of E. coli PNP without any effect on mammalian PNPs, was shown to inhibit T. vaginalis PNP with a K(is) of 2.3 microM by competing with adenosine. T. vaginalis PNP thus belongs to the family of bacterial PNPs and constitutes a target for antitrichomonial chemotherapy.     

The role of PNP enzyme in autologous rosette-forming cells

Since purine nucleoside phosphorylase has been associated with suppressor function in lymphocytes, enzyme activities were studied in autologous rosette-forming cells, a subset showing suppressor properties. Levels of this enzyme were higher in these cells than in other T cells. Con A induction of autologous red cell receptors and suppressor activity of T cells were both inhibited in dose-dependent fashion by Formycin B, a well known inhibitor of purine nucleoside phosphorylase. Inhibition of autologous rosette-forming cells was obtained after pulse treatment of cells with Formycin B for as little as 1 hr, whereas cell proliferation was only inhibited when Formycin B was present throughout culture; this confirms the independence of cell proliferation, and development of red cell receptors and suppressor activity. This study indicates a crucial role for purine nucleoside phosphorylase enzyme in induction of T cell suppressor activity.