Phosphoribosylpyrophosphate Synthetase Research Articles

Inhibition of phosphoribosylpyrophosphate synthetase by 4-methoxy-(MRPP) and 4-amino-8-( d-ribofuranosylamino)pyrimido[5,4-d]pyrimidine (ARPP)

The basis for the antitumor activities of the exocyclic amino nucleosides 4-amino-(ARPP) and 4-methoxy-8-( d-ribofuranosylamino)pyrimido[5,4-d]pyrimidine (MRPP) was investigated. The primary target of these nucleosides appeared to be 5-phospho-α- d-ribofuranose-1-pyrophosphate (PRPP) synthetase. MRPP-5'-monophosphate was a competitive inhibitor ( K i = 40 μM) of the activation of this enzyme by the cofactor inorganic phosphate ( K a = 2.2 mM). Consequently, ARPP and MRPP treatment of WI-L2 cultures rapidly inhibited both de novo pyrimidine and purine synthesis as well as the nucleotide salvage reactions dependent on PRPP. ARPP or MRPP treatment completely prevented [ 14C]bicarbonate incorporation into acid-soluble pyrimidine and purine nucleotides. The rate of salvage of [8- 14C]hypoxanthine to form IMP was decreased by 85%. Treatment of cells with these agents caused a 50% reduction in the steady-state level of PRPP. When the capacity of the treated cells for sustained synthesis of PRPP was examined by adenine incorporation, the rate of adenine uptake was inhibited by > 50%. In vivo treatment of BDF 1 mice with a single dose of ARPP (173 mg/kg) or MRPP (62 mg/kg) extended the mean life span of the mice, which had been inoculated intraperitoneally 1 day earlier with 1 × 10 6 L1210 murine leukemia cells, by 62 and 82% respectively. These studies indicate that MRPP and ARPP inhibit PRPP synthetase, and that PRPP synthetase may be a viable target in the development of certain antitumor agents.

Impaired erythrocyte NAD synthesis: a metabolic abnormality in thalassemia.

We have recently demonstrated that phosphoribosylpyrophosphate (PRPP) synthetase activity is decreased in RBC from individuals with thalassemia minor. Because NAD biosynthesis requires PRPP, the product of the PRPP synthetase reaction, we have investigated NAD synthesis in thalassemic RBC. NAD synthesis was measured in intact RBC both by following the accumulation of unlabeled NAD and by following the incorporation of 14C-nicotinic acid into NAD. Using both assay systems, we demonstrate that NAD synthesis is decreased significantly in thalassemic RBC compared to either normal or high reticulocyte red cells. Although this suggested that NAD content should be decreased in thalassemic RBC, no significant difference in NAD content was found among thalassemic, normal, or high reticulocyte red cells. Mechanisms for the lack of a significant decrease in NAD content in thalassemic RBC are discussed. These results indicate that NAD synthesis is impaired in thalassemic RBC possibly as a result of their decrease in PRPP synthetase activity. Our data provide evidence that thalassemic RBC have secondary metabolic abnormalities in addition to their primary defect in hemoglobin synthesis.

Primary structure of the tms and prs genes of Bacillus subtilis

The nucleotide sequence was determined of a 3211 nucleotide pair EcoRI-PvuII DNA fragment containing the tms and prs genes as well as a part of the ctc gene of Bacillus subtilis. The prs gene encodes phosphoribosylpyrophosphate (PRPP) synthetase, whereas the functioning of the tms and ctc gene products remains to be established. The prs gene contains an open reading frame of 317 codons resulting in a subunit Mr of 34828. An open reading frame comprising the tms gene contained 456 codons resulting in a putative translation product with an Mr of 49,554. Comparison of the deduced B. subtilis PRPP synthetase amino acid sequence with PRPP synthetases from Escherichia coli and rat liver showed extensive similarity. The deduced Tms amino acid sequence was found to be 43% similar to the deduced amino acid sequence of ecourfl, a gene of E. coli with unknown function.

Characterization of the Escherichia coli prsA1-encoded mutant phosphoribosylpyrophosphate synthetase identifies a divalent cation-nucleotide binding site

The prsA1 allele, specifying a mutant Escherichia coli phosphoribosylpyrophosphate (PRPP) synthetase, has been cloned. The mutation was shown by nucleotide sequence analysis to result from substitution of Asp-128 (GAT) in the wild type by Ala (GCT) in prsA1. This alteration was confirmed by chemical determination of the amino acid sequence of a tryptic peptide derived from the purified mutant enzyme. The mutation lies at the N-terminal end of a 16 residue sequence that is highly conserved in E. coli, Bacillus subtilis, and rat PRPP synthetases and has the following consensus sequence: DLHAXQIQGFFDI/VPI/VD. There was little alteration in the Km for ribose 5-phosphate. The Km for ATP of the mutant enzyme was increased 27-fold when Mg2+ was the activating cation but only 5-fold when Mn2+ was used. Maximal velocities of the wild type and mutant enzymes were the same. The mutant enzyme has a 6-fold lower affinity for Ca2+, as judged by the ability of Ca2+ to inhibit the reaction in the presence of 10 mM Mg2+. Wild type PRPP synthetase is subject to product inhibition by AMP, but AMP inhibition of the prsA1 mutant enzyme could not be detected. It has been previously proposed that a divalent cation binds to PRPP synthetase and serves as a bridge to the alpha-phosphate of ATP and AMP at the active site. The prsA1 mutation appears to alter this divalent cation site.

Classic disease descriptions in the modern curriculum

The rising number of laboratory tests and procedures ordered by physicians have contributed to increasing health care costs [l]. An increasing dependence on laboratory tests appears to have replaced confidence in the interpretation of data obtained from history and physical examination. Clinical skills have been deemphasized in undergraduate medical education, and the acquisition of these skills may be less exciting to students and teachers than an understanding of newer diagnostic technologies [2]. This trend is reflected in the development of the modern medical textbook. The standard medical text expands with each edition as it attempts to keep pace with new and complex diagnostic and treatment modalities. At the same time, clinical descriptions of disease have been condensed and simplified. The premise of this essay is that the precise and elegant pictures of disease presented in the writings of the great clinicians of the past should be reintroduced into current curriculum to reestablish the primary role of listening to and examining patients. These clinicians, dependent on bedside skills, preserved their lifetimes of clinical wisdom in their writings. More importantly, these writings may inspire students to appreciate signs and symptoms of disease better than modern-day books or teachers. Clinical descriptions of disease from previous eras can supplement modern clinical texts. For example, gout is discussed in 10 pages of text in Cecil’s 18th edition published in 1988. Discussion of the disease includes genetics, prevalence in relation to serum uric acid, mechanisms of hyperuricemia, purine biosynthesis, crystal-phagocyte interaction, pathology of tophi, clinical manifestions, gouty nephropathy, gout with specific enzyme deficiencies including glycogen storage disease type I, hypoxanthine guanine phosphoribosyltransferase deficiency, and phosphoribosylpyrophosphate synthetase variants, diagnosis, and treatment. One paragraph is devoted to clinical symptoms and reads as follows:

Rat Liver Phosphoribosyl Pyrophosphate Synthetase: Existence of the Purified Enzyme as Heterogeneous Aggregates and Identification of the Catalytic Subunit1

Mammalian phosphoribosyl pyrophosphate (PRPP) synthetase has been extensively investigated. However, considerable ambiguity remains concerning its physical and regulatory properties. We purified PRPP synthetase from rat liver and studied some of the physical properties, in parallel with cloning experiments (Taira, M. et. al. [1987] J. Biol. Chem. 262, 14867-14870). 1) The enzyme was purified to a specific activity of 7,280 milliunits/mg, the highest value in the literature for a mammalian PRPP synthetase. The apparent molecular mass was over 1,000 kDa. 2) The final preparation contained 34-kDa, 38-kDa, and 40-kDa protein species, as analyzed by SDS gel electrophoresis. 3) Further attempts at separation using conventional procedures only led to a co-purification of the components. Thus, the purified enzyme appears to exist as complex aggregates composed of heterogeneous components. 4) Gel filtration of the enzyme in the presence of 1 M MgCl2 isolated part of the 34-kDa component, free of other species. The preparation was catalytically active, indicating that this component is the catalytic subunit. 5) The amino acid composition of the 34-kDa subunit and the amino acid sequences of its N-terminal region and of two tryptic peptides were determined. The results are in accord with the results of cDNA analyses.

Increase in phosphoribosyl pyrophosphate induced by ATP and P i depletion in hepatocytes

A series of compounds that induce depletion of ATP and Pi when added to isolated rat hepatocytes were found to cause a remarkable, although transient, elevation in the concentration of phosphoribosyl pyrophosphate (PRPP) in these cells. After the addition of 5 mM fructose, xylitol, tagatose, or D-xylulose, PRPP increased from a basal value of 6 +/- 1 nmol/g of cells to, respectively, 68 +/- 11, 42 +/- 11, 67 +/- 22, and 530 +/- 50 nmol/g of cells (means +/- SEM of 3-9 experiments). In each case, the increase in PRPP was preceded by a latency period of 5-10 min. PRPP reached maximal levels 15 min after the addition of fructose and 30 min after that of xylitol and D-xylulose, but continued to increase for as long as 60 min after the addition of tagatose. Most striking was that the increase in PRPP closely paralleled the restoration of intracellular Pi. Ribose 5-P increased about two- to fivefold after the addition of fructose, xylitol, and tagatose, and approximately 12-fold after D-xylulose. The mechanism by which ATP- and Pi-depleting compounds stimulate the activity of PRPP synthetase in isolated rat hepatocytes is discussed.

Tissue-differential expression of two distinct genes for phosphoribosyl pyrophosphate synthetase and existence of the testis-specific transcript

Cloning of cDNA coding for rat phosphoribosyl pyrophosphate (PPRibP) synthetase (EC 2.7.6.1) revealed two distinct types of subunit, referred to as PRS I and PRS II (Taira et al. (1987) J. Biol. Chem. 262, 14867–14870). Tissue-specific expression of PRS I and PRS II genes (designated PRPS1 and PRPS2, respectively), was shown for 16 rat organs, using Northern blot analysis. The 2.3 kb PRPS1 mRNA level was high in the brain and adrenal gland, whereas the 3.7 kb PRPS2 mRNA level prevailed in the lung and spleen. Both genes were highly expressed in the thymus, adipose tissue and testis. In other mammals (mouse, calf and human), these two types of mRNA were also detected in various tissues and cell lines. Thus, the expression of each gene is regulated in a tissue-specific manner and there may be functional differences between catalytic and/or regulatory properties of subunits PRS I and II of this enzyme. In the testis, an additional PRPS1-related transcript of 1.4 kb was noted in rats, mice and humans. This transcript may belong to a group of testis-specific gene expressions or functions.

Molecular cloning and sequencing of human cDNA for phosphoribosyl pyrophosphate synthetase subunit II

cDNA clones for human phosphoribosyl pyrophosphate synthetase subunit II (PRS II) were isolated. The five overlapping clones contained 2457 base pairs (bp) covering a 954-bp complete coding region for 318 amino acid residues. Homologies between human and rats PRS II were 99% of the amino acid and 88% of the nucleotides in the coding region. This amino acid homology seems to be the highest so far reported for enzymes involved in nucleotide metabolism and glycolysis. The highly conserved structure may be required for unique catalysis and rigid regulation of this enzyme.

Pyrimidine nucleotides impair phosphoribosylpyrophosphate (PRPP) synthetase subunit aggregation by sequestering magnesium. A mechanism for the decreased PRPP synthetase activity in hereditary erythrocyte pyrimidine 5′-nucleotidase deficiency

The activity of phosphoribosylpyrophosphate (PRPP) synthetase (ATP: D-ribose-5-phosphate pyrophosphotransferase, EC 2.7.6.1) is decreased in the erythrocyte in hereditary pyrimidine 5'-nucleotidase (P5N) deficiency. Given the increased pyrimidine nucleotide content of the P5N-deficient erythrocyte, we evaluated the effects of prototypic pyrimidine nucleotides on the activity of PRPP synthetase. In normal hemolysate a 1.0 mM combination of cytidine tri-, di- and monophosphate (CTP/CDP/CMP) inhibited PRPP synthetase activity and changed the ribose 5-phosphate (R5P) saturation curve from a hyperbola to a biphasic shape. Untreated crude hemolysate from P5N-deficient erythrocytes showed a biphasic R5P kinetic curve. Since the activity of PRPP synthetase is dependent on its state of subunit aggregation, we examined PRPP synthetase subunit aggregation using gel permeation chromatography. P5N-deficient erythrocytes had a decreased absolute amount of aggregated PRPP synthetase and almost a total loss of disaggregated PRPP synthetase. Using normal hemolysate, 1 mM CTP/CDP/CMP interfered with the ability of 1.0 mM ATP and 2.0 mM MgCl2 to promote PRPP synthetase subunit aggregation. Increasing the MgCl2 to 6.0 mM overcame the inhibitory effect of CTP/CDP/CMP. Thus, the decreased PRPP synthetase activity of the P5N-deficient erythrocyte is due, at least in part, to the ability of the accumulated pyrimidine nucleotides to sequester magnesium and to interfere with the subunit aggregation of PRPP synthetase.

Potent and specific inhibitors of mammalian phosphoribosylpyrophosphate (PRPP) synthetase

The monophosphates of the exocyclic amino ribonucleosides, 4-amino- and 4- methoxy-8-( d- ribofuranosylamino)pyrimido[5,4-d] pyrimidine , are potent and specific inhibitors of human erythrocyte and B-lymphoblast PRPP synthetase. The inhibition by MRPP monophosphate is competitive ( K i =35 μ m with the PRPP synthetase cofactor, P i ( K m=2 m m ). The nucleosides are phosphorylated to the active metabolite by adenosine kinase and these nucleoside monophosphates accumulate in the cell. β-ARPP is a substrate, albeit poor, for adenosine deaminase and solutions of the β-anomer of this nucleoside and its monophosphate anomerize over time to give α- and β-mixtures. β-MRPP is more resistant to adenosine deaminase and anomerization of the nucleoside and its monophosphate is negligible. The effect of treatment of cells with the nucleosides is a time-dependent and nearly universal reduction in the nucleotide content which appears to result from a reduction in the availability of PRPP for dependent metabolic pathways. In studies with the WI-L2 lymphoblasts, some of these pathways, de novo and salvage (hypoxanthine and guanine) synthesis of purine nucleotides, are more sensitive to a restriction of PRPP availability than others, i.e. de novo pyrimidine synthesis. The nucleosides have shown promise as therapeutic agents in a mouse leukemia evaluation system but may also have future use in unravelling the complex regulation of PRPP synthetase and the dependent nucleotide synthesis pathways.

Complete cDNA sequence of rat phosphoribosylpyrophosphate synthetase subunit II (PRS II)

Complete cDNA sequence of rat phosphoribosylpyrophosphate synthetase subunit II (PRS II)

Localization of human phosphoribosylpyrophosphate synthetase subunit I and II genes (PRPS1 and PRPS2) to different regions of the X chromosome and assignment of two PRPS1-related genes to autosomes

Complementary DNA clones for phosphoribosylpyrophosphate synthetase subunits I and II (PRS I and PRS II) were used to determine the chromosomal localization of the corresponding human genes. Southern blot analysis of genomic DNAs isolated from human placenta and a panel of human-mouse somatic cell hybrids revealed that the rat PRS I cDNA probe detected at least five human specific DNA segments (23, 20, 14.5, 6.7, and 4.3 kb) in BamHI digests. The 23-, 14.5-, and 6.7-kb DNA segments were detected only if the hybrids contained human chromosome X or translocation chromosome 7p+ (7qter greater than 7p22::Xq21 greater than Xqter), indicating the location of these segments to Xq21-qter (PRPS1). The 20- and 4.3-kb DNA segments did not cosegregate with the other three segments, and spot blot hybridization analysis using flow-sorted human chromosomes indicated that these are the PRPS1-related genes (PRPS1L1 and PRPS1L2) and could be assigned to chromosomes 7 and 9, respectively. The human-specific PRS II cDNA probe revealed a BamHI DNA segment (17 kb), which segregated condordantly with the X chromosome but not with the PRPS1 gene. We surmise that the gene for PRS II (PRPS2) is located at a different region of the X chromosome, namely Xpter-a21.

Biochemical pharmacology and antitumor properties of [formula omitted

1. 1. APP is activated by adenosine kinase to its 5′-phosphate (APP-MP). 2. 2. APP-MP inhibits PRPP synthetase, and depletes cellular PRPP and purine and pyrimidine nucleotides. 3. 3. APP inhibits synthesis of DNA and RNA, and blocks cells in G1 phase of the cell cycle. 4. 4. APP retains full activity against MDR cells. 5. 5. APP is equally active against quiescent and proliferating CHO cells. 6. 6. APP has only weak activity against L1210 leukemia in vivo, but has substantial activity against mammary carcinoma 16/c. 7. 7. In vitro, APP has a relatively high ratio of solid tumor: leukemia activity.

Chemiluminescent assays in the study of purine metabolism.

The discovery that in cells from patients with hypoxanthine guanine phosphoribosyltransferase (HGPRT) deficiency purine overproduction is associated with an elevated intracellular concentration of phosphoribosylpyro-phosphate (PRPP) (1) has led to a reevaluation of the importance of this compound in the regulation of purine biosynthesis de novo. The work which flowed from this discovery led to the identification of an inherited super-activity of PRPP synthetase in over a dozen families in which affected individuals showed gout and/or uric acid overproduction (2). However to date HGPRT deficiency and increased activity of PRPP synthetase are the only two well documented enzyme defects of purine synthesis and reutilization identified among hyperuricemic patients. These genetic aberrations account for less 10% of all hyperuricemic patients who overproduce uric acid.

Early mitogenic stimulation of metabolic flux through phosphoribosyl pyrophosphate into nucleotides in Swiss 3T3 cells and requirement of external Magnesium for the response

5-Phosphoribosyl 1-pyrophosphate (PRPP) is a common precursor for the synthesis of all nucleotides and also serves as a critical regulator for the synthesis. In spite of a number of studies in vitro on mammalian PRPP synthetase, our understanding of the regulation of PRPP synthesis in situ is very limited. Various mitogens are known to activate purine and pyrimidine de novo biosynthesis and purine base phosphoribosylation as an early response in quiescent mouse fibroblasts. We aimed at elucidation of the underlying mechanism for the possible increase in PRPP synthesis in mitogen-stimulated mouse fibroblasts in culture. In order to quantitatively follow metabolic flux through PRPP into nucleotides, [ribosyl- 14C]inosine was enzymatically prepared and used as a tracer to preferentially label intracellular ribose phosphate. The radioactivity incorporation into cellular nucleotides was measured. Evidence supported the validity of the method. Prior exposure of quiescent Swiss 3T3 cells in culture to epidermal growth factor (EGF) plus insulin for 45–60 min enhanced approximately 2-fold the radioactivity incorporation from [ribosyl- 14C]inosine into nucleotides, without increasing the specific radioactivity of intracellular free ribose 5-phosphate. [ 14C]Uracil incorporation into nucleotides, a measure for PRPP-independent ribose phosphate utilization for nucleotide synthesis, was not increased. These and other results indicate that EGF plus insulin stimulates the metabolic flux through PRPP. A similar stimulation was induced by bombesin and melittin in combination with insulin and by fibroblast growth factor alone. Quiescent Swiss 3T6 cells and human fetal fibroblasts showed a similar stimulation of nucleotide synthesis in response to exposure to serum. For characterization of intracellular signaling pathways, we examined effects of several inhibitors and agents on the stimulation. The divalent cation ionophore A23187 mimicked the response to EGF and insulin in Swiss 3T3 cells, thereby suggesting involvement of divalent cation mobilization in this increase. The effect of the ionophore was not additive to that of the growth factors. Omission of Ca 2+ from the incubation medium did not affect the response to EGF and insulin, whereas the omission of Mg 2+ did abolish the response. Furosemide, an inhibitor of Mg 2+ influx, partially inhibited the stimulated synthesis of nucleotides. Thus, the entry of external Mg 2+ into the cells may play a critical role in this signal transduction. These results provided an important access to elucidation of the intracellular mechanisms for the mitogen-induced increase in PRPP and nucleotide syntheses.

Evidence for Early Mitogenic Stimulation of Metabolic Flux through Phosphoribosyl Pyrophosphate into Nucleotides in Swiss 3T3 Cells1

Various mitogens activate purine and pyrimidine de novo biosynthesis and purine base phosphoribosylation as an early response in quiescent fibroblasts. Increased synthesis of 5-phosphoribosyl 1-pyrophosphate (PRPP) may precede or underlie these activations, but little direct evidence has been presented for this notion, due to lack of suitable analytical methods. To preferentially label intracellular ribose phosphate and quantitatively follow metabolic flux through PRPP into nucleotides, we prepared [ribosyl-14C]inosine and used it as a tracer. Evidence showed the validity of this method. Prior exposure of quiescent Swiss 3T3 cells in culture to epidermal growth factor plus insulin for 45-60 min enhanced approximately 2-fold the radioactivity incorporation from [ribosyl-14C]inosine into nucleotides, without increasing the specific radioactivity of intracellular free ribose 5-phosphate. [14C]Uracil incorporation into nucleotides, a measure of PRPP-independent ribose phosphate utilization for nucleotide synthesis, was not increased. These and other results indicate that epidermal growth factor plus insulin stimulates the metabolic flux through PRPP. Similar extents of stimulation were induced by bombesin and melittin in combination with insulin and by fibroblast growth factor alone, suggesting the presence of an unknown signaling pathway common to these mitogens. This system is highly useful for studies of the mechanisms that stimulate in situ activity of PRPP synthetase.

Inherited superactivity of phosphoribosylpyrophosphate synthetase: Association of uric acid overproduction and sensorineural deafness

Purpose: Superactivity of 5-phosphoribosyl 1-pyrophosphate (PP-Rib-P) synthetase, inherited as an X chromosome-linked trait, has been reported in nearly 20 families in which overproduction of uric acid is invariably present in hemizygous affected males. Clinical manifestations of PP-Rib-P synthetase superactivity are mainly limited to gout in early adulthood. Neurologic deficits, including sensorineural deafness, have rarely been described. We herein document the association of PP-Rib-P synthetase superactivity, gout with excessive uric acid synthesis, and sensorineural deafness in an additional family. Patients and methods: Two members of a Spanish family were studied: an eight-year-old boy (Patient I) with tophaceous gout, purine nucleotide and uric acid overproduction, and sensorineural deafness, and his 27-year-old mother (Patient 2), who had gout. Fibroblast cultures were initiated from skin biopsy specimens, and measurements of PP-Rib-P and purine nucleotide metabolism in the fibroblasts were performed. Results: A labile but superactive PP-Rib-P synthetase was demonstrated in the fibroblasts cultured from both Patients 1 and 2. The kinetic basis of PP-Rib-P synthetase superactivity in this family was resistance to purine nucleotide inhibition of enzyme activity. More severe derangements in the enzyme and in PP-Rib-P and purine synthesis in Patient 1's cells than in Patient 2's cells suggest that Patient 1 is hemizygous and Patient 2 is heterozygous for an X chromosome-linked genetic defect. Limited pedigree data support this view. Compared with affected members of seven other families with PP-Rib-P synthetase superactivity, these patients are intermediate in the range of clinical expression and in the severity of the enzyme defect as measured by the degree of aberration of PP-Rib-P and purine nucleotide synthesis in fibroblasts. Metabolic abnormalities were more severe in Patient 1's cells than in the cells of most male patients (in whom clinical expression is limited to early adult-onset gout) but were less severe than in the cells of two patients in whom more complex enzyme defects were associated with uric acid overproduction and neurodevelopmental abnormalities (including deafness) in male children and adult women. Conclusion: Certain defects resulting in PP-Rib-P synthetase superactivity may be causally related to neurologic impairment, most commonly sensorineural deafness.

Impaired erythrocyte phosphoribosylpyrophosphate formation in hemolytic anemia due to pyruvate kinase deficiency

RBCs from patients with hemolytic anemia due to pyruvate kinase (PK) deficiency are characterized by a decreased total adenine and pyridine nucleotide content. Because phosphoribosylpyrophosphate (PRPP) is a precursor of both adenine and pyridine nucleotides, we investigated the ability of intact PK-deficient RBCs to accumulate PRPP. The rate of PRPP formation in normal RBCs (n = 11) was 2.89 +/- 0.80 nmol/min.mL RBCs. In contrast, the rate of PRPP formation in PK-deficient RBCs (n = 4) was markedly impaired at 1.03 +/- 0.39 nmol/min.mL RBCs. Impaired PRPP formation in these cells was not due to the higher proportion of reticulocytes. To study the mechanism of impaired PRPP formation, PK deficiency was simulated by incubating normal RBCs with fluoride. In normal RBCs, fluoride inhibited PRPP formation, caused adenosine triphosphate (ATP) depletion, prevented 2,3-diphosphoglycerate (DPG) depletion, and inhibited pentose phosphate shunt (PPS) activity. These results together with other data suggest that impaired PRPP formation is mediated by changes in ATP and DPG concentration, which lead to decreased PPS and perhaps decreased hexokinase and PRPP synthetase activities. Impaired PRPP formation may be a mechanism for the decreased adenine and pyridine nucleotide content in PK-deficient RBCs.

Impaired Erythrocyte Phosphoribosylpyrophosphate Formation in Hemolytic Anemia due to Pyruvate Kinase Deficiency

RBCs from patients with hemolytic anemia due to pyruvate kinase (PK) deficiency are characterized by a decreased total adenine and pyridine nucleotide content. Because phosphoribosylpyrophosphate (PRPP) is a precursor of both adenine and pyridine nucleotides, we investigated the ability of intact PK-deficient RBCs to accumulate PRPP. The rate of PRPP formation in normal RBCs (n = 11) was 2.89 +/- 0.80 nmol/min.mL RBCs. In contrast, the rate of PRPP formation in PK-deficient RBCs (n = 4) was markedly impaired at 1.03 +/- 0.39 nmol/min.mL RBCs. Impaired PRPP formation in these cells was not due to the higher proportion of reticulocytes. To study the mechanism of impaired PRPP formation, PK deficiency was simulated by incubating normal RBCs with fluoride. In normal RBCs, fluoride inhibited PRPP formation, caused adenosine triphosphate (ATP) depletion, prevented 2,3-diphosphoglycerate (DPG) depletion, and inhibited pentose phosphate shunt (PPS) activity. These results together with other data suggest that impaired PRPP formation is mediated by changes in ATP and DPG concentration, which lead to decreased PPS and perhaps decreased hexokinase and PRPP synthetase activities. Impaired PRPP formation may be a mechanism for the decreased adenine and pyridine nucleotide content in PK-deficient RBCs.