PRPP Concentration Research Articles

Hydrophilic-interaction liquid chromatography-tandem mass spectrometric determination of erythrocyte 5-phosphoribosyl 1-pyrophosphate in patients with hypoxanthine-guanine phosphoribosyltransferase deficiency.

Mutations in the gene encoding hypoxanthine-guanine phosphoribosyltransferase (HPRT) cause Lesch-Nyhan disease (LND) and its variants (LNV). Due to the technical problems for measuring the HPRT activity in vitro, discordances between the residual HPRT activity and the clinical severity were found. 5-Phosphoribosyl 1-pyrophosphate (PRPP) is a substrate for HPRT. Since increased PRPP concentrations were observed in erythrocytes from patients with LND and LNV, we have turned our attention to erythrocyte PRPP as a biomarker for the phenotype classification. In the present work, a method for determination of PRPP concentration in erythrocyte was developed using liquid chromatography-tandem mass spectrometry (LC-MS/MS) with multiple reaction monitoring (MRM). Packed erythrocyte samples were deproteinized by heating and the supernatants were injected into the LC-MS/MS system. All measurement results showed good precision with RSD <6%. PRPP concentrations of nine normal male subjects, four male patents with LND and six male patients with LNV were compared. The PRPP concentrations in erythrocyte from patients with LND were markedly increased compared with those from normal subjects, and those from patients with LNV were also increased but the degree was smaller than those with LND. The increase pattern of PRPP concentration in erythrocyte from patients with HPRT deficiency was consistent with the respective phenotypes and was correlated with the disease severity. PRPP concentration was suggested to give us supportive information for the diagnosis and the phenotype classification of LND and LNV.

Identification and Active Expression of the Mycobacterium tuberculosis Gene Encoding 5-Phospho-α-D-ribose-1-diphosphate: Decaprenyl-phosphate 5-Phosphoribosyltransferase, the First Enzyme Committed to Decaprenylphosphoryl-D-arabinose Synthesis

Decaprenylphosphoryl-d-arabinose, the lipid donor of mycobacterial d-arabinofuranosyl residues, is synthesized from phosphoribose diphosphate rather than from a sugar nucleotide. The first committed step in the process is the transfer of a 5-phosphoribosyl residue from phosphoribose diphosphate to decaprenyl phosphate to form decaprenylphosphoryl-5-phosphoribose via a 5-phospho-alpha-d-ribose-1-diphosphate:decaprenyl-phosphate 5-phospho-ribosyltransferase. A candidate for the gene encoding this enzyme (Rv3806c) was identified in Mycobacterium tuberculosis, primarily via its homology to one of four genes responsible for d-arabinosylation of nodulation factor in Azorhizobium caulinodans. The resulting protein was predicted to contain eight or nine transmembrane domains. The gene was expressed in Escherichia coli, and membranes from the expression strain of E. coli but not from a control strain of E. coli were shown to convert phosphoribose diphosphate and decaprenyl phosphate into decaprenylphosphoryl-5-phosphoribose. Neither UDP-galactose nor GDP-mannose was active as a sugar donor. The enzyme favored polyprenyl phosphate with 50-60 carbon atoms, was unable to use C-20 polyprenyl phosphate, and used C-75 polyprenyl phosphate less efficiently than C-50 or C-60. It requires CHAPS detergent and Mg(2+) for activity. The Rv3806c gene encoding 5-phospho-alpha-d-ribose-1-diphosphate:decaprenyl-phosphate 5-phosphoribosyltransferase is known to be essential for the growth of M. tuberculosis, and the tuberculosis drug ethambutol inhibits other steps in arabinan biosynthesis. Thus the Rv3806c-encoded enzyme appears to be a good target for the development of new tuberculosis drugs.

Kelley-Seegmiller syndrome due to a unique variant of hypoxanthine-guanine phosphoribosyltransferase: reduced affinity for 5-phosphoribosyl-1-pyrophosphate manifested only at low, physiological substrate concentrations

A male child, who presented at the age of 3.5 years with acute renal failure, was diagnosed as having partial deficiency of hypoxanthine-guanine phosphoribosyltransferase (HPRT; EC 2.4.2.8). The underlying HPRT mutation was unique in that the specific activity of HPRT in erythrocyte and in fibroblast lysates was normal, but the rate of uptake of hypoxanthine into nucleotides of intact cultured fibroblasts was markedly reduced (23% of normal). The low functioning of HPRT in the intact fibroblasts was associated with decreased utilization of endogenously generated hypoxanthine and with decreased utilization of the cosubstrate 5-phosphoribosyl-1-pyrophosphate (PRPP). The non-utilized hypoxanthine was excreted into the incubation medium. The accumulation of PRPP was indicated by the 2.3-fold increase in the rate of uptake of adenine into intact cell nucleotides and by the 7.5-fold enhancement of the rate of de novo purine synthesis. Kinetic studies of HPRT activity in fibroblast lysates revealed reduced affinity of the enzyme for PRPP (apparent K m 500 μM in comparison to 25 μM in control lysates), manifested in low activity at low (physiological), but not at high PRPP concentrations. The apparent K m for hypoxanthine was normal (23 μM in comparison to 14.2 μM in control lysates). With allopurinol treatment, our patient has had no problems since presentation, and is developing normally at 5 years of age.

The Intracellular Formation of a Mononucleotide of the Anti-Influenza Agent 1,3,4-thiadiazol-2-ylcyanamide (LY217896)

LY217896 is a substituted thiadiazole compound with anti-influenza activity in vitro and in the mouse model of infection. LY297336 is a ribosylated (N-4) derivative of LY217896. A highly polar intracellular metabolite of LY217896 was isolated by HPLC, and mass spectral analysis and treatment of the metabolite with alkaline phosphatase showed that it was a monophosphate (LY307987) derived from LY217896. The formation of LY307987 was inhibited by 43 and 63% when 10 μm of LY217896 was incubated with 100 μM of 8-aminoguanosine (8AGuo) and guanine (Gua), respectively, whereas inosine (Ino) and hypoxanthine (Hx) had no effect on the formation of LY307987. LY217896 inhibited the incorporation of [14C]-Hx into nucleic acids in cells which metabolize LY217896; however, LY217896 did not inhibit the formation of inosine 5′-monophosphate (IMP) from Hx in a cell-free HGPRT (hypoxanthine-guanine phosphoribosyltransferase)-catalysed reaction. Incubation of MDCK cells with 10 μm of LY217896 resulted in an 8-fold increase in the level of intracellular IMP. At 100 μm, neither LY217896 nor LY297336 inhibited inosine 5′-monophosphate dehydrogenase (IMPDH) and only cellular extracts which contained intracellular metabolites of LY217896 inhibited IMPDH. Quantification of the 5-phosphorylribose pyrophosphate (PRPP) levels in BS-C-1, MDCK, and MCN cells showed a positive correlation between PRPP concentration and cellular metabolism of LY217896. Combination studies of LY217896 with 2′,3′-dideoxyinosine (ddlno) or 2′,3′-dideoxyguanosine (ddGuo) showed that LY217896 enhanced the antiretroviral activities of these dideoxynucleosides, which is consistent with an inhibitory effect on IMPDH.

Biochemical effect of three different inhibitors of purine/pyrimidine metabolism on differentiation in HL60 cells

The effects of three different nucleotide biosynthesis inhibitors were tested on differentiation and purine/pyrimidine metabolism in HL60 cells. Of the three nucleotide biosynthesis inhibitors, acivicin and mycophenolic acid were able to differentiate HL60 cells, while alanosine failed to do so. Differentiation of HL60 cells by acivicin and mycophenolic acid was associated with substantial decreases in both the guanylate and adenylate pools and appeared to be dependent on the state of depletion of intracellular GTP. Simultaneous addition of guanosine or guanine to mycophenolic acid-treated cells restored the GTP pool and prevented differentiation from occurring. Adenine or adenosine had no such effect, while hypoxanthine and inosine partially reversed the differentiation. In acivicin-treated cells, simultaneous addition of guanine caused partial prevention of differentiation. Even though treatment of HL60 cells with alanosine resulted in the depletion of guanylates, this effect was secondary to the depletion of adenylates and developed only upon prolonged exposure. In all the inhibitor-treated cells the activities of the key regulatory enzymes of de novo purine biosynthesis were affected. Even though the measurable activity of hypoxanthine/guanine phosphoribosyl transferase was enhanced in inhibitor-treated cells, the activity of the salvage pathway was inhibited in mycophenolic acid and alanosine-treated cells. Besides de novo purine nucleotide biosynthesis, de novo pyrimidine nucleotide biosynthesis was also inhibited in inhibitor-treated cells. The inhibition of purine and pyrimidine nucleotide biosynthesis in mycophenolic acid, acivicin and alanosine-treated cells resulted in an increase in the steadystate concentration of PRPP. Since purine and pyrimidine nucleotides play an important role in the synthesis of important macromolecules, it can be suggested that depletion of guanine ribonucleotide as a result of inhibition of early de novo purine biosynthesis, or due to specific inhibition of de novo guanine nucleotide biosynthesis, may be an obligatory step in the initiation of differentiation in mycophenolic acid and acivicin-treated HL60 cells.

On the biochemical modulation of 6-mercaptopurine by methotrexate in murine WEHI-3b leukemia cells in vitro

The chemicals 6-mercaptopurine (6-MP) and methotrexate (MTX) are the cornerstones in the maintenance treatment of acute lymphoblastic leukemia. The intracellular metabolism of 6-MP to 6-thioguanosine nucleotides (TGN) via 6-thioinosine 5′-monophosphate (TIMP) is crucial for its cytotoxic effect. MTX inhibits purine de novo synthesis and thereby increases the intracellular PRPP being a substrate for the phosphoribosylation of 6-MP to TIMP. Hypoxanthine has been shown to inhibit the uptake of 6-MP over the cell membrane and the phosphoribosylation of 6-MP to TIMP. We have previously shown that the conversion of TIMP to TGN decreases at 6-MP concentrations above 5 μM in vitro. The aim of the present study was therefore to investigate the effect of MTX increasing the PRPP and TIMP concentrations and of hypoxanthine decreasing the TIMP concentration on the formation of TGN from TIMP. Murine myelomonocytic leukemia cells (WEHI-3b) were treated with 6-MP in vitro. The drug concentration was kept constant by continuous addition of 6-MP during the experiment. With this technique, the concentration of TGN begins to decrease already at 6-MP concentrations above 2 μM. The addition of 0.2 μM MTX 6 h before 6-MP strongly inhibited the purine de novo synthesis, decreased the ATP, and increased the PRPP concentration 4-fold. The intracellular concentrations of TIMP and to a lesser extent TXMP also increased. The concentrations of the TGN were, however, basically unaffected by the preincubation with MTX. Simultaneous addition of 20–50 μM hypoxanthine and 6-MP decreased the accumulation of all cellular 6-MP metabolites. It is concluded that the synergistic cytotoxic effect of the combination of 6-MP and MTX is not based on biochemical modulation of the 6-MP metabolism by MTX.

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.

Stimulation of ribose-5-phosphate and 5-phosphoribosyl-1-pyrophosphate generation by pyrroline-5-carboxylate in mouse liver in vivo: Evidence for a regulatory role of ribose-5-phosphate availability in nucleotide synthesis

Pyrroline-5-carboxylase (P5C), a physiological stimulator of hexose-monophosphate-pentose pathway activity, was found before to increase 5-phosphoribosyl-1-pyrophosphate (PRPP) generation and nucleotide synthesis in human erythrocytes and cultured fibroblasts. We now report the stimulation of PRPP generation by P5C also in mouse liver in vivo. In addition we demonstrated a simultaneous elevation in ribose-5-phosphate (R5P) concentration, which was relatively smaller and transient. The demonstrated effect of P5C on liver R5P and PRPP content in vivo provides strong evidence for the physiological role of R5P availability in the regulation of PRPP and purine production.

Stimulation of Protein, RNA, and Nucleotide Synthesis in Lymphocytes After Abdominal Surgery Is Not Affected by Postoperative Amino Acid Supply

The effects of abdominal surgery on protein, RNA, and de novo purine nucleotide synthesis in lymphocytes, and modification of these changes by postoperative amino acid supply, were investigated in 24 patients undergoing cholecystectomy (n = 12) or removal of gastric cancer (n = 12). Mono-nuclear cells were isolated from the peripheral venous blood and incubated with radioactive tracers in vitro. Protein and RNA synthesis, as measured using [14C] glycine and [3H]uridine, respectively, increased postoperatively. Nucleotide synthesis determined by the incorporation of radioactivity from [14C] glycine into nucleotides increased simultaneously. The concentration of 5-phosphoribosyl 1-pyrophosphate (PRPP) estimated by the incorporation of [14C]adenine into nucleotides also increased. These changes were greater and of longer duration in patients with cancer operation than in those with cholecystectomy. In neither case were they affected by the amount of amino acid intake, or increases in energy intake. These results suggest that abdominal surgery stimulates protein and ribonucleic acid (RNA) synthesis in lymphocytes. Increased RNA synthesis may be ensured by increased synthesis of nucleotides, and increased PRPP concentrations appear to regulate the rate of nucleotide synthesis. The responses are apparently dependent upon the severity of surgery, but unrelated to the amount of amino acid supplied postoperatively.

Modulation of 5-fluorouracil efficacy

In clinical trials methotrexate (MTX) and folinic acid (FA) have been the most widely tested modulators of FU. MTX, if given prior to FU, has been shown to increase FUmediated cytotoxicity in vitro and in vivo. MTX increases intracellular concentration of PRPP (5-phosphoribosyl 1 -pyrophosphate) which is a cosubstrate for FU anabolism. Also, MTX polyglutamates may improve FdUMP binding to TS.

Inosine/pyruvate/phosphate medium but not adenosine/pyruvate/phosphate medium introduces millimolar amounts of 5-phosphoribosyl 1-pyrophosphate in human erythrocytes. A 31P-n.m.r. study

Incubation of human erythrocytes in medium containing inosine (10 mM), pyruvate (10 mM), phosphate (50 mM) and NaCl (75 mM) at pH 6.6 leads to a more than 1000-fold increase in the concentration of 5-phosphoribosyl 1-pyrophosphate (PRPP), as identified and quantified by 31P-n.m.r. spectroscopy. The accumulation is highly pH-dependent, with a maximum at extracellular pH 6.60, and the maximum value of 1.3-1.6 mmol/l of erythrocytes is attained within 1 h at 37 degrees C. PRPP was accumulated despite high concentrations of 2,3-bisphosphoglycerate (2,3-BPG), an inhibitor of PRPP synthetase. The concentration of PRPP correlated with the intracellular concentration of inorganic phosphate (Pi). Substitution of either adenosine or adenosine plus inosine for inosine in the medium did not lead to 31P-n.m.r.-detectable accumulation of PRPP. These results show that neither 2,3-BPG nor PRPP itself inhibits the synthesis of PRPP in the human erythrocyte. Adenosine, however, prevents the inosine-stimulated accumulation of PRPP.

13 INHERITED SUPERACTIVITY OF PRPP SYNTHETASE (PS): ASSOCIATION OF PURINE OVERPRODUCTION AND SENSORINEURAL DEAFNESS

In fibroblasts from an 8 year-old male (VRG) with tophaceous gout, uric acid overproduction, and sensorineural deafness, and from his gouty mother, superactivity of PS due to purine nucleotide inhibitor-resistance was found. Compared with normal cells, VRG fibroblasts showed increases in the following: PRPP concentration (3-fold) and generation (2-fold); purine synthesis de novo (2.5-fold); purine base salvage (2.6-fold); ATP and GTP concentrations (1.4-fold); and excretion of newly synthesized purines (5-fold). Derangements in PS and in PRPP and purine synthesis were attenuated in cells from the mother, suggesting that VRG is a hemizygote for an X chromosome-linked defect and his mother is a heterozygote. Comparison was made of affected males in 8 families with PS superactivity. VRG was intermediate in range of expression of PS superactivity and in severity of the enzyme defect as measured by the degree of aberration of PRPP and purine nucleotide synthesis in fibroblasts. Metabolic abnormalities were more severe in VRG than in most patients (in whom expression is limited to early adult-onset gout with uric acid overproduction), but less severe than in 2 patients with more complex defects in PS associated with uric acid over-production and neurodevelopmental abnormalities, including deafness in hemizygous male children and heterozygous women. Within the spectrum of defects resulting in PS superactivity, certain derangements may be causally related to neurological impairment, most commonly sensorineural deafness.

94 CHANNELING OF IMP INTO GUANYLATE SYNTHESIS IN THE GROWTH PROGRAM OF HEPATOMA 3924A CELLS

The purpose was to elucidate the relationship of IMP utilization for de novo guanylate and adenylate synthesis during the display of the proliferative program of hepatoma 3924A cells. Purine compounds in the cells were labeled with [14C] formate or L- [3-14C]serine in a serine-free assay medium, hydrolyzed to purine bases, separated and counted. Under these assay conditions labeling of adenine or guanine was proportionate with incubation time and with number of cells. During the transition of hepatoma cells from plateau phase into logarithmic proliferation the concentration of PRPP increased 14-fold, followed by an 8-fold rise in purine de novo synthesis. In plateau phase cells predominantly adenylates were produced. After replating the resting cells there was a sharp increase in the relative labeling of guanine with a concurrent marked decrease in that of adenine in the late lag phase. The subsequent decline of the relative labeling of guanine was accompanied with the restoration in that of adenine with the increase in the cell density. The striking redirection in the distribution of label from IMP utilization to the preferential synthesis of guanylates during the expression of the biochemical proliferative program of cancer cells underlines the potential significance of this pathway as a target of chemotherapy. (Supported by Outstanding Investigator Grant CA-42510 to G.W.)

12 PRPP AND PURINE NUCLEOTIDE METABOLISM IN HUMAN LYMPHOBLASTS WITH BOTH PRPP SYNTHETASE SUPERACTIVITY AND HGPRT DEFICIENCY

PRPP and purine nucleotide synthesis and culture growth in response to medium additions were compared in cloned human B lymphoblast lines selected for resistance to growth inhibition by 6-thioguanine (TG) and in the respective normal and PS-superactive parental cell lines. TG-resistant cell lines were severely deficient in HGPRT (<0.2% normal activity). Cells with both HGPRT deficiency and PS superactivity retained the kinetic defect in PS of the parental cells but had PRPP concentration 20-fold greater than normal cells and 6- and 5-fold greater, respectively, than the parental PS-superactive cells and cells defective in HGPRT alone. Nevertheless, PRPP generation in HGPRT-deficient lines was minimally increased (<8%) compared with parental cell lines and purine synthesis de novo in the cell line with both enzyme defects was comparable to that in the PS-superactive parental line.Regardless of the status of PS activity, all HGPRT-deficient cell lines showed growth patterns typical of HGPRT deficiency in HAT medium and in medium with 8-azaguanine. However, growth of HGPRT-defIcient cell lines, including the line with PS superactivity, was more sensitive than that of parental lines to inhibition of purine synthesis de novo by 6-methylthioinosine (MMPR), especially when adenine was provided, most likely reflecting compromised guanine nucleotide synthesis in HGPRT-deficient cells when purine synthesis de novo is blocked and adenine is the sole source of purine for salvage.

Properties of allosteric nicotinamide mononucleotide glycohydrolase from Azotobacter vinelandii: activation and inhibition.

In order to clarify the regulation mechanism of NMN glycohydrolase, a number of purine and pyrimidine nucleotides were tested as activators for the enzyme. Among naturally occurring nucleotides, pppGpp was shown to be the most potent activator (KA = 0.0087 mM). The effectiveness of these nucleotides estimated from Vmax/KA was in the order: pppGpp greater than ppGpp greater than pppppG greater than ppppG greater than pGpp greater than GTP greater than or equal to 2'-GMP greater than or equal to GppppG greater than dGTP greater than ITP greater than GDP = 2'-O-methylGTP. XTP, UTP, dTTP gave no effect. In contrast, ATP, CTP, and 7-methylGTP were inhibitory. A comparison of the data indicated that activation of the enzyme is specific for the base structure, guanine, and that effectiveness of nucleotides as activators results from the negatively charged phosphate moiety. Consistent with these results, inorganic polyphosphates (tri-, tetra-, and pentaphosphate) and 5-phosphoribose 1-pyrophosphate were shown to function as inhibitors of the enzyme. The inhibition mechanism was shown to be competitive with GTP. However, apparent Ki values for these inhibitors were dependent on the activator concentration. Furthermore, a shift of cooperativity type and progressive increase of positive cooperativity concomitant with increase of PRPP concentration were observed. These results indicate that this enzyme may be allosteric and an important regulatory component of pyridine nucleotide cycle metabolism.

Increased rate of de novo purine synthesis and its mechanism in regenerating rat liver

In regenerating rat liver at 12 h after a 70% hepatectomy, [14C]glycine incorporation to hepatic acid-soluble purines increased by 2.4-fold with a 26% increase of the mean rapidly miscible glycine pool size and its comparable turnover rate. Amidophosphoribosyltransferase (EC 2.4.2.14, ATase) activity increased 1.8-fold at 18 h and 5-phosphoribosyl-1-pyrophosphate (PRPP) concentration increased 3.0-fold at 12 h after surgery. Decreases in ATP and GTP concentrations of 13 and 15%, respectively, in regenerating rat liver at 12 h after surgery were counterbalanced by increased AMP and GMP concentrations of 13 and 44%, respectively, with comparable inhibitory potentials on ATase. These results suggest than an increased rate of de novo purine synthesis in regenerating rat liver is mediated by the increased ATase activity and PRPP concentrations.

Increased rate of purine biosynthesis in rat liver after bilateral adrenalectomy

In bilaterally adrenalectomized rat liver the increased rate of de novo purine synthesis was shown by the increased [14C]glycine incorporation into hepatic acid-soluble purines with unchanged rapidly miscible glycine pool size and its turnover rate and by the increased rate of chasing of radiolabeled purines. At 24 h after adrenalectomy, the rate of de novo purine synthesis increased by 70%, 5-phosphoribosyl-1-pyrophosphate (PRPP) content increased by 200%, the specific activity of amidophosphoribosyltransferase (EC 2.4.2. 14; ATase) did not change, ATP and GTP showed a 33 and 24% decrease, and AMP and ADP showed a 245 and 38% increase. Combined, the metabolic pool size data reflected an unchanged total inhibitory potential on ATase. Replacement with corticosterone acetate for 24 h partially restored some of these abnormalities. These results suggest that the increase in the rate of de novo purine synthesis in adrenalectomized rat liver is secondary to increased catabolism of purine ribonucleotides and mediated by increased PRPP concentrations.

In vivo inhibition of the rate of de novo purine synthesis in rat liver by glucagon

The rates of de novo purine and protein synthesis were assessed in vivo in rat liver after bolus administration of glucagon. The specific activity of hepatic purines and the specific activity ratio of hepatic purine/protein were used as an index of the rate of de novo purine synthesis and the rate relative to protein. Glucagon at doses of 0.01 mg to 0.1 mg 200 g body weight (BW), administered as an intravenous bolus, inhibited dose-dependently the rate of de novo purine synthesis and the rate relative to protein although it increased dose-dependently the hepatic concentration of 5-phosphoribosyl 1-pyrophosphate (PRPP). The inhibition of the rate of de novo purine synthesis recovered to control levels during the period between 60 and 90 minutes after glucagon administration. Dibutyryl cyclic AMP (Bt 2 cAMP) partially mimicked this effect of glucagon in that it did not increase the rate of de novo purine synthesis in spite of increased concentrations of PRPP. These results suggest that an intravenous bolus of glucagon inhibits the rate of de novo purine synthesis through increasing cAMP concentration. The data are consistent with inhibition of amidophosphoribosyltransferase (ATase) in spite of increased PRPP concentrations.

Phosphoribosylpyrophosphate synthetase superactivity. A study of five patients with catalytic defects in the enzyme.

Superactive phosphoribosylpyrophosphate (PRPP) synthetases were characterized in fibroblasts and erythrocytes from 5 unrelated men with gout and/or hyperuricemia and uric acid overproduction. The kinetic basis of enzyme superactivity in all patients was increased maximal reaction velocity. Affinities of the enzymes for substrates and activators and responsiveness to inhibitors were normal, and levels of immunoreactive enzyme in patient and control fibroblast and erythrocyte extracts were comparable. Enzymes purified to homogeneity from 2 patients confirmed the presence of isolated catalytic defects. Altered physical properties of certain of the superactive enzymes suggested the presence of several distinctive structural defects among the aberrant forms. Fibroblasts from each affected patient showed increased PRPP concentration and generation, as well as accelerated rates of all PRPP-requiring purine nucleotide synthetic pathways. These findings support the concept that enzyme superactivity results in uric acid overproduction as a consequence of increased rates of PRPP and purine nucleotide synthesis. Cultured cells from female relatives of 2 patients showed evidence for the heterozygous carrier state, as measured both by enzyme activities and by rates of PRPP and purine synthesis. The clinical phenotype in 4 patients was limited to early adult-onset gout and its consequences, whereas the fifth patient expressed a familial constellation of hyperuricemia, sensorineural deafness, ataxia, and renal insufficiency. The severity of the derangements in PRPP synthetase and in PRPP and purine synthesis in cells from the 5 patients, however, was comparable. The neurologic accompaniments of enzyme superactivity found in 1 family described here, and in 2 others described previously, thus may not necessarily be consequences of primary defects in PRPP synthetase.

HPRT‐Deficiency associated with normal PRPP concentration and APRT activity

Deficiencies of HPRT are usually associated with increased concentrations of PRPP and increased levels of APRT activity in erythrocytes. We report the case of a male with a partial deficiency of HPRT in whom these two parameters were normal. The clinical features of this patient were those associated with severe hyperuricaemia and gout. Studies of intact erythrocytes showed rates of incorporation of [14C]hypoxanthine and of [14C]adenine into purine nucleotides which were almost indistinguishable from normal. However, HPRT activity in erythrocyte lysates was only 9% of normal. In cell extracts of cultured lymphoblasts, the HPRT activity was 20% of control values and the APRT activity was normal. The PRPP concentration and the rate of de novo purine synthesis in cultured lymphoblasts were both intermediate between controls and lymphoblasts from patients with the Lesch-Nyhan syndrome.