Phosphoribosylpyrophosphate Synthetase Research Articles

Purine metabolism in promyelocytic HL60 and dimethylsulphoxide-differentiated HL60 cells

Purine metabolism was studied in the human promyelocytic leukaemic cell line HL60 both before and after differentiation by dimethylsulphoxide (DMSO) treatment in vitro. DMSO-induced differentiation had no effect on the steady-state concentration of 5-phosphoribosylpyrophosphate (PRPP), even though the activity of the oxidative segment of the pentose phosphate pathway (OPP) increased two-fold and the activities of glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase were enhanced 3–6 times. [ 14C]-formate incorporation, on the other hand, showed that undifferentiated HL60 cells produced purines by the de novo pathway at six-fold the rate of the differentiated cells and at three times the rate measured in polymorphonuclear leukocytes (PMNs) separated from human blood. This is consistent with significantly higher activities (two to five-fold) of the key regulatory enzymes of the de novo purine synthesis pathway (PRPP synthetase, amidophosphoribosyl transferase and adenylosuccinate synthase) in the undifferentiated cells. Labelling the cells with [ 14C]-hypoxanthine showed that there was an active salvage pathway in the HL60 cells which remained unaltered by differentiation. The activities of the salvage enzymes were also the same, although hypoxanthine was taken up at twice the rate by the undifferentiated cells. Human PMNs had an active salvage pathway, but their rate was five times lower than that of the HL60 cells, consistent with much lower activities (three to eightfold) of the salvage pathway enzymes. These observations suggest that regulation of the synthesis and activities of the purine nucleotide synthesis enzymes may play an important role in the regulation of differentiation of myeloid cells.

A novel 39-kDa phosphoribosylpyrophosphate synthetase-associated protein of rat liver. Cloning, high sequence similarity to the catalytic subunits, and a negative regulatory role.

The rat liver phosphoribosylpyrophosphate (PRPP) synthetase exists as complex aggregates composed of the 34-kDa catalytic subunits (PRS I and II) and other 39- and 41-kDa proteins (Kita, K., Otsuki, T., Ishizuka, T., and Tatibana, M. (1989) J. Biochem. (Tokyo) 105, 736-741), which are termed here PRPP synthetase-associated proteins (PAPs). We have cloned the cDNA for the major one of 39 kDa (PAP39) from a rat liver cDNA library. Nucleotide sequencing showed that the clone encoded 356 amino acids containing sequences of all five peptides derived from PAP39. Surprisingly, the deduced amino acid sequence is markedly similar to those of the 34-kDa catalytic subunits. Excluding two regions (about 45 residues in total), PAP39 has a 48% identity with PRS I. Northern analysis detected a major 1.9-kilobase transcript in all 16 rat tissues examined, and the relative amounts of PAP39 mRNA to PRS I mRNA varied with tissues. Covalent cross-linking experiments gave definitive evidence for molecular interaction of PAP39 with the catalytic subunits. Immunoprecipitation experiments revealed that all the catalytic subunits existed as complexes containing PAP39. When PAPs were eliminated from the rat liver enzyme complex by gel filtration in the presence of 1 m MgCl2, a lyotrope, or by mild tryptic treatment, the enzyme activity of the remaining catalytic subunits increased. Based on these results, we propose that PAP39, the major component of PAPs, plays a negative regulatory role in PRPP synthesis and hence is an important factor controlling nucleotide syntheses in general.

A simplified method for the determination of phosphoribosylpyrophosphate synthetase activity in hemolysates

Methods currently employed for measurement of phosphoribosylpyrophosphate synthetase (ribophosphate pyrophosphokinase; PRPPs) activity are cumbersome and expensive, requiring an auxiliary enzyme reaction, a radioactive nucleobase and multiple incubations, or do not allow a complete kinetic study. The aim of the present study is to describe a simplified, single step, non-isotopic method for determination of PRPPs in hemolysate, appropriate for a complete screening of PRPPs activity disorders. Briefly, the charcoal-treated hemolysate is incubated with saturating amounts of substrates and P 1 P 5 di(adenosine 5') pentaphosphate (Ap5A), an inhibitor of human adenylate kinase, to prevent conversion of AMP to ADP. AMP generated in this reaction is then measured by HPLC. Adenylate kinase activity was fully inhibited by Ap5A, allowing the accurate determination of AMP. The method was sensitive and reproducible and mean and variance values compared closely with those reported using other, more complicated, assay procedures. The hyperbolic curve relating Pi concentration to initial reaction velocity was shifted to sigmoidal by addition of 0.02 mmol/l GDP which inhibited PRPPs activities only at inorganic phosphate concentrations < 2 mmol/l. This suggests that this method should provide sensitive and accurate screening for regulatory, as well as catalytic, defects underlying PRPPs superactivity.

Human X-linked phosphoribosylpyrophosphate synthetase superactivity is associated with distinct point mutations in the PRPS1 gene.

Superactivity of phosphoribosylpyrophosphate synthetase (PRS) is an X chromosome-linked disorder of purine metabolism, characterized by gout with uric acid overproduction and, in some families, neurodevelopmental impairment. Two highly homologous isoforms of PRS (PRS1 and PRS2), each encoded by a distinct X chromosome-linked locus, have been identified, and PRS1 and 2 cDNAs have been cloned. The entire 954-base pair translated regions of PRS1 and 2 cDNAs derived from cultured lymphoblasts and fibroblasts from two patients in whom purine nucleotide feedback resistance of PRS is associated with enzyme superactivity and neurodevelopmental defects were examined by direct sequencing after polymerase chain reaction amplification of PRS transcripts. Nucleotide sequences of PRS2 cDNAs from the patients and normal individuals were identical. In contrast, PRS1 cDNAs from the patients differ from normal PRS1 cDNA, each by a single base substitution. PRS1 cDNA from patient N. B. showed an A to G transition at nucleotide 341, corresponding to an asparagine to serine change at amino acid residue 113 of mature PRS1. A G to C transversion at nucleotide 547, indicating an aspartic acid to histidine change at amino acid 182, was found for PRS1 cDNA from patient S. M. Point mutations at the sites identified in the PRS1 cDNAs of the two patients were confirmed by the results of RNase mapping analysis. Normal, N. B., and S. M. PRS1 cDNAs were introduced into Escherichia coli BL21 (DE3)/pLyS, and recombinant N. B. and S. M. PRS1s showed the purine nucleotide feedback resistance phenotypes characteristic of PRS from patients' cells.

HPLC determination of oxidized and reduced pyridine coenzymes in human erythrocytes

The nucleotide concentrations in acid and alkaline erythrocyte extracts have been measured by RP-HPLC in healthy controls and in patients bearing different inherited disorders, with altered erythrocyte NAD(P) levels. The objective was the simultaneous determination of the nucleotide profile and of the oxidative state of pyridine coenzymes by the most suitable extraction method. Both alkaline and acid extractions were necessary to obtain the complete pattern, due to defective recovery of the oxidized or reduced coenzymes, respectively, during the extraction procedures. Purine nucleotide quantification seemed to be reliable by all methods. High NADP + levels were confirmed in two glucose-6-phosphate dehydrogenase deficient patients, coupled with raised NAD levels, lowered NADPH/NADP + ratio and increased NADH/NAD + ratio. Higher NAD + and normal or lower NADH/NAD + ratios were found in two hypoxanthine-phosphoribosyltransferase deficient patients, while a patient with superactive phosphoribosylpyrophosphate synthetase showed a decreased NADH level in addition to the low NAD + level previously found.

Cooperation of the prs and dnaA gene products for initiation of chromosome replication in Escherichia coli.

A new Escherichia coli mutant allele, named dnaR, that causes thermosensitive initiation of chromosome replication has been identified to be an allele of the prs gene, the gene for phosphoribosylpyrophosphate synthetase (Y. Sakakibara, J. Mol. Biol. 226:979-987, 1992; Y. Sakakibara, J. Mol. Biol. 226:989-996, 1992). The dnaR mutant became temperature resistant by acquisition of a mutation in the dnaA gene that did not affect the intrinsic activity for the initiation of replication. The suppressor mutant was capable of initiating replication from oriC at a high temperature restrictive for the dnaR single mutant. The thermoresistant DNA synthesis was inhibited by the presence of the wild-type dnaA allele at a high but not a low copy number. The synthesis was also inhibited by an elevated dose of a mutant dnaR allele retaining dnaR activity. Therefore, thermoresistant DNA synthesis in the suppressor mutant was dependent on both the dnaA and the dnaR functions. On the basis of these results, I conclude that the initiation of chromosome replication requires cooperation of the prs and dnaA products.

Molecular characterization of phosphoribosylpyrophosphate synthetase from Leishmania donovani

The phosphoribosylpyrophosphate synthetase (PRS) enzyme from parasitic protozoa plays a critical role in the acquisition of exogenous purine bases by providing the phosphoribosylpyrophosphate substrate for phosphoribosylation. To characterize a PRS enzyme from parasitic protozoa, the prs gene was isolated from a genomic library of Leishmania donovani DNA. A 1936-bp Sa/I fragment was sequenced that encompassed an open reading frame of 1113 nucleotides encoding a polypeptide of 371 amino acids and 40 787 Da. After gap alignment, the leishmanial PRS exhibited 40-2% amino acid identity with a variety of mammalian and prokaryotic PRSs. L. donovani PRS also contained an approx. 20-amino acid stretch that was highly homologous to the phosphoribosylpyrophosphate binding domains of mammalian phosphoribosyltransferase enzymes. Two prs-specific transcripts of 2.6 and 2.1 kb were detected by Northern analysis, and Southern blots of genomic DNA implied that the prs locus was not tandemly repeated in the L. donovani genome. PRS activity was detected in L. donovani extracts, and apparent K m values of approx. 30 μM and approx. 1 mM were calculated for ribose-5-phosphate and ATP, respectively. PRS was sensitive to inhibition by AMP and ADP but refractory to IMP, GMP, GTP, CTP, and UTP. The high apparent K m value of the parasite enzyme for ATP and its insensitivity to inhibition by many nucleotides suggested that kinetic differences between the L. donovani and human PRSs could provide an avenue for rational therapeutic manipulation of parasitic disease. The isolation of the L. donovani prs gene now provides an opportunity to genetically dissect the determinants responsible for the function and regulation of this indispensable enzyme of purine and pyrimidine metabolism in a genus of parasitic protozoa.

Fibroblast growth factor-dependent metabolism of hypoxanthine via the salvage pathway for purine synthesis in porcine aortic endothelial cells

In this study we examined the metabolism of hypoxanthine in fibroblast growth factor (FGF)-stimulated porcine aortic endothelial cells (PAEC). Our previous report indicated that hypoxanthine in fetal bovine serum (FBS) was an essential component for both basal and FGF-dependent growth of PAEC (Hayashi et al., Exp Cell Res 185: 217–228, 1989). Besides hypoxanthine, the addition of various purine bases and purine nucleosides, but not xanthine, xanthosine or any pyrimidine metabolites, restored the limited growth of PAEC cultured in medium containing 10% dialyzed FBS in the presence or absence of FGF. The metabolism of [ 14C]hypoxanthine was compared in PAEC treated with and without FGF. Treatment of PAEC with FGF for 24 hr enhanced the radioactivity incorporation from [ 14C]hypoxanthine into both the acid-soluble and -insoluble fractions approximately 2-fold. Upon Chromatographie analyses of hypoxanthine metabolites in the acid-soluble nucleotide fraction, it was found that in control PAEC hypoxanthine was largely metabolized to IMP, adenine nucleotides and uric acid, whereas in FGF-treated cells it was converted to ATP, ADP, GTP, xanthine and uric acid. The radioactivity of IMP was lowered in FGF-stimulated cells. The addition of FGF to PAEC increased phosphoribosyl pyrophosphate (PRPP) synthetase activity by approximately 8-fold and the PRPP content by approximately 2-fold, but it did not increase hypoxanthine-guanine phosphoribosyltransferase (HGPRT) activity or hypoxanthine transport. On the other hand, methotrexate, an inhibitor of de novo synthesis of purine, did not affect the growth of PAEC. Analyses of the rate of [ 14C]formate incorporation into total purine compounds showed that PAEC had a low capacity to synthesize purines de novo, which was not stimulated by FGF. These data indicate that FGF stimulates the synthesis of PRPP necessary for the salvage synthesis of purine nucleotides in conjunction with purine bases, e.g. hypoxanthine.

Gout, uric acid and purine metabolism in paediatric nephrology

Although gout and hyperuricaemia are usually thought of as conditions of indulgent male middle age, in addition to the well-known uricosuria of the newborn, there is much of importance for the paediatric nephrologist in this field. Children and infants may present chronically with stones or acutely with renal failure from crystal nephropathy, as a result of inherited deficiencies of the purine salvage enzymes hypoxanthine-guanine phosphoribosyltransferase (HPRT) and adenine phosphoribosyltransferase (APRT) or of the catabolic enzyme xanthine dehydrogenase (XDH). Genetic purine overproduction in phosphoribosylpyrophosphate synthetase superactivity, or secondary to glycogen storage disease, can also present in infancy with renal complications. Children with APRT deficiency may be difficult to distinguish from those with HPRT deficiency because the insoluble product excreted, 2,8-dihydroxyadenine (2,8-DHA), is chemically very similar to uric acid. Moreover, because of the high uric acid clearance prior to puberty, hyperuricosuria rather than hyperuricaemia may provide the only clue to purine overproduction in childhood. Hyperuricaemic renal failure may be seen also in treated childhood leukaemia and lymphoma, and iatrogenic xanthine nephropathy is a potential complication of allopurinol therapy in these conditions. The latter is also an under-recognised complication of treatment in the Lesch-Nyhan syndrome or partial HPRT deficiency. The possibility of renal complications in these three situations is enhanced by infection, the use of uricosuric antibiotics and dehydration consequent upon fever, vomiting or diarrhoea. Disorders of urate transport in the renal tubule may also present in childhood. A kindred with X-linked hereditary nephrolithiasis, renal urate wasting and renal failure has been identified, but in general, the various rare types of net tubular wasting of urate into the urine are recessive and relatively benign, being found incidentally or presenting as colic from crystalluria. However, the opposite condition of a dominantly inherited increase in net urate reabsorption is far from benign, presenting as familial renal failure, with hyperuricaemia either preceding renal dysfunction or disproportionate to it. Paediatricians need to be aware of the lower plasma urate concentrations in children compared with adults when assessing plasma urate concentrations in childhood and infancy, so that early hyperuricosuria is not missed. This is of importance because most of the conditions mentioned above can be treated successfully using carefully controlled doses of allopurinol or means to render urate more soluble in the urine. Xanthine and 2,8-DHA are extremely insoluble at any pH. Whilst 2,8-DHA formation can also be controlled by allopurinol, alkali is contraindicated. A high fluid, low purine intake is the only possible therapy for XDH deficiency.

Characterization of the hemA-prs region of the Escherichia coli and Salmonella typhimurium chromosomes: identification of two open reading frames and implications for prs expression.

The prs gene, encoding phosphoribosylpyrophosphate synthetase, is preceded by a leader, which is 302 bp long in Escherichia coli and 417 bp in Salmonella typhimurium. A potential open reading frame (ORF) extends across the prs promoter and into the leader. The region between the prs coding region and an upstream gene (hemA) in E. coli and S. typhimurium was cloned, sequenced and shown to encode two ORFs of unknown function. ORF 1 encodes a 23 kDa protein and ORF 2 a 31 kDa protein, as observed by denaturing PAGE of extracts of cells bearing plasmids encoding the ORFs. Both ORFs are transcribed in the same direction as the prs gene with ORF 2 extending into the prs leader. Northern blot analysis showed that the prs message in E. coli was on 1.3 and 2.7 kb transcripts. The shorter transcript encoded the prs gene only, while the longer transcript also encoded the two ORFs. Thus, the prs gene is transcribed from two promoters, the first promoter (P1) originating upstream of ORF 1, and expressing the prs gene in a tricistronic operon and a second promoter (P2), located within the ORF 2 coding frame, which transcribes the prs gene only. The transcripts encoding prs only were 20 times as abundant as the tricistronic transcripts under all conditions examined. This was the case whether cells containing plasmid-encoded or only chromosomally encoded copies of the hemA-prs region were probed for these transcripts. Derepression of the prs gene upon pyrimidine starvation was shown to be due to an increase in the amount of message originating from the promoter P2.

A cyanobacterial gene encoding a protein with extensive homology to mammalian phosphoribosylpyrophosphate synthetase.

Phosphoribosylpyrophosphate (PRibPP) is the primary precursor in the biosynthesis of purine and pyrimidine nucleotides. The synthesis of PRibPP is catalysed by a ubiquitous PRibPP synthetase in many organisms. We provide evidence that a cyanobacterium, Synechococcus sp., has a gene encoding a protein with striking homology to mammalian PRibPP synthetases.

DnaR function of the prs gene of Escherichia coli in initiation of chromosome replication

A new Escherichia coli mutant named dnaR, which was temperature sensitive in initiation of DNA replication, has been characterized through identification of the mutant gene. A 1.65 x 10(3) base-pair chromosomal DNA fragment isolated from wild-type cells, but not the corresponding fragment from the dnaR mutant, exhibited an activity that reversed temperature-sensitive growth of the mutant. The DNA fragment was found to include the entire prs-coding sequence and specify a 34,000 M(r) protein with phosphoribosylpyrophosphate synthetase activity. The dnaR mutation resided within the prs-coding segment and made the synthetase thermolabile. The coding segment for the dnaR product was determined, by introduction of various mutations into the cloned fragment, to be the same as that for the synthetase. The dnaR function of the prs gene product in DNA replication is discussed on the basis of an observation that thermal treatment of the dnaR mutant caused a delay in initiation of chromosome replication after the downshift, despite the presence of the synthetase activity at the preheat level.

Regulation of the purine salvage pathway in rat liver.

The regulation of purine metabolism in rat liver has been examined under conditions that alter the flux through the pathway. Rats were given intraperitoneal injections of ethanol, sodium acetate, or sodium phosphate to attain body water concentrations of approximately 70, 20, and 10 mM, respectively. The livers were freeze-clamped after 30 min, and extracts were made for the analysis of metabolites, cofactors, purine bases, and nucleosides; homogenates were made for the measurement of the activities and kinetic parameters of seven enzymes that participate in purine salvage. The values of the equilibrium constants of nine reactions were determined in vitro and compared with the ratios of the reactants measured in liver. The changes in phosphoribosylpyrophosphate (PRPP), a key intermediate in both the de novo and salvage pathways of purine metabolism, were directly correlated with the changes in ribose 5-phosphate (ribose-5-P); ([PRPP] = 1.7[ribose-5-P] - 7.4 mumol/kg). Ribose-5-P concentrations in turn could be predicted from the liver content of fructose 6-phosphate and glyceraldehyde 3-phosphate by calculation from the known equilibria. The maximum velocities in the tissue of the seven enzymes measured were calculated from the measured substrate values in the liver and with consideration of other effectors of enzyme activity. PRPP synthetase was the least active of the enzymes measured, indicating a possible rate-limiting step. The delta G of the enzyme steps differed from equilibrium values by factors ranging from 4 (nucleoside phosphorylase) to 10(5) (PRPP synthetase and purine transferase reactions). The regulation of purine salvage appeared to depend on the levels of PRPP and ribose-5-P.

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.

Physical mapping of loci in the distal half of the short arm of the human X chromosome: implications for the spreading of X-chromosome inactivation.

The relative order of 11 loci in the distal half of the short arm of the human X chromosome was examined using a panel of somatic cell hybrids containing structurally rearranged X chromosomes. The results show that the gene for phosphoribosylpyrophosphate synthetase 2 (PRPS2) is located between ZFX (zinc finger protein, X-linked) and STS (steroid sulfatase). The results also confirm the localization of ZFX distal to POLA (alpha-DNA polymerase). Previous studies have shown that STS and ZFX escape X-inactivation whereas POLA undergoes inactivation. Evaluation of PRPS2 expression in somatic cell hybrids containing inactive human X chromosomes showed that PRPS2 undergoes X-inactivation. These results provide further evidence for interspersion of loci that do and do not undergo X-inactivation on the human X chromosome.

Promoter regions of the human X-linked housekeeping genes PRPS1 and PRPS2 encoding phosphoribosylpyrophosphate synthetase subunit I and II isoforms

The 5′ regions of the human phosphoribosylpyrophosphate synthetase subunit I and II genes ( PRPS1 and PRPS2, respectively) were isolated and sequenced. A comparison of the nucleotide sequences between human and rat PRPS1 genes revealed that the sequences around the transcription initiation sites were conserved over 56 nucleotides, and that a TATA-like sequence, a CCAAT box and three putative Sp1 binding sites were present at almost the same positions in the GC-rich sequences. Two major transcription initiation sites were localized in the human PRPS1, one of the two was located 27 nucleotides downstream from the TATA-like sequence, while the upstream initiation site was in the TATA-like sequence. The promoter region of the human PRPS2 gene was also GC-rich and contained a TATA-like sequence, four Sp1 binding sites and a homopyrimidine stretch. The initiation sites were localized at 90 nucleotides upstream from the ATG initiation codon. Chloramphenicol acetyltransferase (CAT)/promoter fusion assays showed that a 2.0 kb region (human PRPS1) and a 1.1 kb region (human PRPS2) possessed the promoter activities in four cell lines. The CAT activities in the three human cell lines tended to correlate with the steady-state mRNA levels of the PRPS1 and PRPS2 genes. These results suggest that the 5′ flanking regions cloned contribute to the cell-differential expression of these two genes.

Studies on phosphoribosylpyrophosphate synthetase from Giardia intestinalis

A substantial degree of purification, up to 3200-fold, with recoveries of 8–11% of phosphoribosylpyrophosphate (P-Rib-PP) synthetase from Giardia intestinalis extracts was achieved by the high resolution techniques of anion exchange chromatography and chromatofocusing columns on a fast protein liquid chromatography system. A Mono P chromatofocusing column gave rise to an enzyme peak eluting from the column at pH 4.5, indicating that the enzyme has an isoelectric point (pI) at approximately this value. The molecular weight of the enzyme was found to be 150,000 from a Sephacryl S-200 column. Sodium dodecyl sulphatepolyacrylamide gel electrophoresis of the purified enzyme gave a single protein band with a subunit molecular weight of 38,000, indicating that the enzyme existed as a tetramer. The properties of G. intestinalis P-Rib-PP synthetase in terms of pH optimum, isoelectric point, subunit structure, phosphate requirement, metal and nucleotide specificity, appear to be very similar to those of the enzyme from other sources.

Phosphoribosylpyrophosphate synthetase in human erythrocytes: Assay and kinetic studies using high-performance liquid chromatography

A method using high-performance liquid chromatography (HPLC) for determination of phosphoribosylpyrophosphate (PRPP) synthetase activity in human erythrocytes has been developed and PRPP synthetase activity on purine and pyrimidine metabolic disorders has been studied. Kinetic properties of erythrocyte PRPP synthetase of patients with gout and of a patient with pyrimidine 5'-nucleotidase deficiency were compared with those of healthy subjects. The mean of PRPP synthetase activity of gouty patients was a little higher ( P < 0.01) than that of healthy subjects. The response of the enzyme for ATP of gouty patients was different from that of healthy subjects. The shapes of activation curve of the enzyme for inorganic phosphate were hyperbolic in gouty patients and in a patient with pyrimidine 5'-nucleotidase deficiency.

Acceleration of purine synthesis in mouse liver by glycogenolytic hormones

Administration (ip) into fed mice of glucagon, epinephrine, vasopressin, oxytocin, angiotensin II, and dibutyryl cyclic AMP (dbcAMP) resulted in a rapid (within 2.5 to 15 min) elevation of PRPP content (two- to threefold) and in acceleration of the rate of de novo purine synthesis (twofold). Inhibition of the epinephrine-stimulated glycogenolysis by 2,5-anhydromannitol diminished markedly the acceleration effect of the hormone on the rate of purine synthesis. Administration of the hormones caused a rapid rise in the liver content of glucose 6-phosphate (G6P) by 15-70% but did not increase the ribose 5-phosphate (R5P) content. Liver ATP content was not affected. The hormones did not cause direct activation of PRPP synthetase, as gauged by the specific activity of the enzyme, its Km for substrates R5P and ATP, and its sensitivity to inhibition by ADP and GDP. The hormones did not increase the liver content of the enzyme activators Pi and Mg2+. The results suggest that the glycogenolytic hormones accelerate purine synthesis by a metabolic mechanism associated with the enhancement of glycogenolysis. PRPP synthesis is probably enhanced by the glycogenolysis-induced alterations in the cellular content of some metabolites other than R5P.

Expression of rat phosphoribosylpyrophosphate synthetase subunits I and II in Escherichia coli. Isolation and characterization of the recombinant isoforms

The 34-kDa subunit of rat liver phosphoribosylpyrophosphate synthetase is a mixture of the two highly homologous isoforms, PRS I and PRS II. Heretofore, it was not possible to separate the two. We now describe isolation and characterization of the recombinant isoforms, named rPRS I and rPRS II. The respective rat cDNAs were inserted into vectors constructed from pKK233-2 by replacing its replication origin with that of pGEM-1 and expressed in Escherichia coli. The rPRS I and rPRS II were purified to apparent homogeneity with specific activities of 33,400 and 46,200 milliunits/mg, respectively; these values were at least 2.5-fold higher than the highest value for the mammalian enzyme so far reported. Both isoforms showed a similar dependency on Pi as an absolute activator. Sulfate partially substituted for Pi. The maximal activities of rPRS I and rPRS II with sulfate were 43 and 7%, respectively, of those seen with Pi. The two isoforms differed in sensitivity to inhibition by ADP and GDP. Inhibition of rPRS I and rPRS II by 0.3 mM ADP was 87 and 54%, respectively, and inhibition by 1 mM GDP was 93 and 24%, respectively. rPRS II was 180-fold more sensitive than rPRS I to heat inactivation at 49 degrees C.