Accelerated Turnover of Phosphoribosylpyrophosphate, a Purine Nucleotide Precursor, in Certain Gouty Subjects*

Jones, Oliver W.; Ashton, Doris M.; Wyngaarden, James Β.

doi:10.1172/jci104638

Cited by 41 publications

(8 citation statements)

References 29 publications

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“…(b) The presence of normal intracellular PP-ribose-P levels and increased synthesis of this compound in the erythrocytes of the propositus demonstrates a more subtle abnormality of PP-ribose-P metabolism. Increased turnover of this compound has been described in the erythrocytes from gouty patients (56) and has been associated with overproduction of uric acid (57). A direct inverse relationship between enzyme deficiency and erythrocyte PP-ribose-P levels has been demonstrated (58) and accounts for the normal concentration of this compound in the erythrocytes from the propositus.…”

Section: Discussionmentioning

confidence: 97%

Hypoxanthine-guanine phosphoribosyltransferase. Characterization of a mutant in a patient with gout.

Fox¹,

Dwosh²,

Marchant³

et al. 1975

J. Clin. Invest.

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ABSTRA CT The mutation in a young gouty male with a partial deficiency of hypoxanthine-guanine phosphoribosyltransferase has been evaluated. The serum uric acid was 11.8 mg/100 ml, and the urinary uric acid excretion was 1,279 mg/24 h. Erythrocyte hypoxanthine-guanine phosphoribosyltransferase was 34.2 nmol/h/mg, adenine phosphoribosyltransferase was 36.5 nmol/h/mg and phosphoribosylpyrophosphate was 2.6 AM. Hypoxanthine-guanine phosphoribosyltransferase from peripheral leukocytes and cultured diploid skin fibroblasts was within the normal range, but enzyme activity in rectal mucosa was below the normal range.Initial velocity studies of the normal enzyme and the mutant enzyme from erythrocytes with the substrates hypoxanthine, guanine, or phosphoribosylpyrophosphate showed that the Michaelis constants were similar. Product inhibition studies distinguished the mutant enzyme from the normal enzyme. Hyperbolic kinetics with increasing phosphoribosylpyrophosphate were converted to sigmoid kinetics by 0.2 mM GMP with the mutant enzyme but not with the normal enzyme.The mutant erythrocyte hypoxanthine-guanine phosphoribosyltransferase was inactivated normally at 80'C and had a normal half-life in the peripheral circulation. The mol wt of 48,000 was similar to the normal enzyme mol wt of 47,000. With isoelectric focusing, the mutant erythrocyte enzyme had two major peaks with isoelectric pH's of 5.50 and 5.70, in contrast to the isoelectric pH's of 5.76, 5.82, and 6.02 of the normal isoAn abstract of this work was published in Clin. Res. 22: 389a, 1974. Dr.

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Section: Discussionmentioning

confidence: 97%

Hypoxanthine-guanine phosphoribosyltransferase. Characterization of a mutant in a patient with gout.

Fox¹,

Dwosh²,

Marchant³

et al. 1975

J. Clin. Invest.

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“…The most widely discussed hypothesis regards the antagonistic effects of PP-ribose-P and purine ribonucleotides on the quaternary structure and thus the activity of the enzyme PP-ribose-P amidotransferase (EC 2.4.2.14) as the molecular basis of this regulation (33). Although no evidence of diminished purine ribonucleotide concentrations as a cause of purine overproduction in a disease state in man has yet been presented and although a recent study (34) has questioned the significance in vivo of purine nucleotide feedback inhibition of PP-ribose-P amidotransferase activity, a stimulatory effect of PP-ribose-P on purine synthesis de novo has been demonstrated in extensive pharmacological (18, [35][36][37][38][39][40][41], clinical (5,6,14,15,24,(42)(43)(44)(45), and kinetic studies (24,33,39,46). For this reason, determination of fibroblast PP-ribose-P concentration is a critical measurement for a study of the biochemical correlates of purine overproduction.…”

Section: Discussionmentioning

confidence: 99%

Patterns of phosphoribosylpyrophosphate and ribose-5-phosphate concentration and generation in fibroblasts from patients with gout and purine overproduction.

Becker¹

1976

J. Clin. Invest.

102

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A B S T R A C T In the majority of patients with gout and excessive uric acid production, underlying enzyme abnormalities have not been identified. In the present study, measurement of both the rate of generation and concentration of phosphoribosylpyrophosphate (PP-ribose-P) and the concentration of ribose-5-phosphate in cultured cells were undertaken to establish a classification of purine overproducers to direct study of additional enzyme defects. Fibroblasts were cultured from 24 individuals assigned to 4 groups: group 1, 5 normal controls; group 2, 5 patients with gout and normal daily urinary uric acid excretion (gouty controls); group 3, 7 patients with well-defined enzyme abnormalities and excessive urinary uric acid excretion (4 with hypoxanthine-guanine phosphoribosyltransferase deficiency and 3 with excessive PP-ribose-P synthetase activity); and group 4, 7 patients with gout and excessive uric acid excretion but without grossly abnormal activities of the above enzymes in erythrocyte lysates.In all 14 fibroblast strains from patients showing excessive production of uric acid (groups 3 and 4), rates of purine synthesis de novo and PP-ribose-P concentrations exceeded values for cells from control groups. Cells from group 3 patients with hypoxanthine-guanine phosphoribosyltransferase deficiency showed normal PPribose-P generation, while those with excessive PP-ri-

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“…The biochemically delineated causes of overproduction hyperuricemia in man are all associated with altered PRPP metabolism (35,36,38,39). Two of these reported biochemical diseases directly involve the enzyme PRPP synthetase (34,37).…”

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confidence: 99%

Characterization of a Feedback-Resistant Phosphoribosylpyrophosphate Synthetase from Cultured, Mutagenized Hepatoma Cells That Overproduce Purines

Green

Martin

1973

Proc. Natl. Acad. Sci. U.S.A.

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A clone of cells in which the regulation of purine metabolism is genetically altered was selected and isolated from chemically mutagenized HTC cells (a line of rat hepatoma cells in continuous culture). The clone, designated MAU V, was selected for increased ability to salvage exogenous purines by isolating it in medium containing methylmercaptopurine ribonucleoside, adenine, and uridine, in which medium wild-type cells cannot divide. We have characterized these cells as having an increased rate of de novo purine biosynthesis, apparently as the result of an altered phosphoribosylpyrophosphate (PRPP) synthetase. The altered enzyme has normal catalytic properties but an altered sensitivity to feedback inhibition by purine and pyrimidine nucleotides. The types of inhibitions (competitive and uncompetitive) exerted by AMP, ADP, and TDP on the wild-type enzyme have been maintained in the altered enzyme, but values for Ki have been increased by factors of 10, 17.5, and 5, respectively. The specific catalytic activities of AMP: pyrophosphate phosphoribosyltransferase and IMP:pyrophosphate phosphoribosyltransferase are normal. The mutant cell may serve as a model for a specific human disease, one type of dominantly inherited overproduction hyperuricemia.Purine biosynthesis in mammalian cells is regulated at numerous sites along the pathway (1). The relative importance of each of these sites in maintenance of the physiologic state is unclear. Aberrant regulation can result in human disease, most notably overproduction hyperuricemia leading to gout (2). We have been examining the regulation of purine biosynthesis in a line of rat hepatoma cells in continuous culture (HTC cells) (3), which we consider to be a model for the mammalian hepatocyte. Because of recent advances in the techniques for studying the genetics of somatic mammalian cells (4-6), and because genetic approaches have been so profitably employed in studies of procaryotic regulatory mechanisms (7) Scintillation counting fluid consisted of 4 g of Omnifluor, 700 ml of toluene, and 250 ml of Triton X-100, except that the fluid used when counting Hyamine papers contained 300 ml of 95% ethanol instead of the Triton.HTC cells were grown as suspension cultures or as monolayers in Swim's 77 medium (3) containing 2 mM L-glutamine and 10% (vol/vol) calf serum (growth medium). Cells in monolayer culture were mutagenized for 2 hr with 30 AM N-methyl-N-nitroso-N'-nitroguanidine, conditions under which approximately 50% of the population survive, and were allowed to recover for 3 days before being selected for their ability to grow in the simultaneous presence of methylmercaptopurine ribonucleoside (MMPR) (0.2 mM), adenine (0.2 mM), and uridine (0.5 mM). Cells of the two surviving colonies from eight T-flasks were dispersed into single-cell suspensions in separate spinner flasks; an aliquot was then cloned in "cloning medium" (Swim's 77 medium containing 10% calf serum and 10% fetal-calf serum, supplemented with 2 mM glutamine and 1 mM pyruvate) that contained 0.3 ...

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Accelerated Turnover of Phosphoribosylpyrophosphate, a Purine Nucleotide Precursor, in Certain Gouty Subjects*

Cited by 41 publications

References 29 publications

Hypoxanthine-guanine phosphoribosyltransferase. Characterization of a mutant in a patient with gout.

Hypoxanthine-guanine phosphoribosyltransferase. Characterization of a mutant in a patient with gout.

Patterns of phosphoribosylpyrophosphate and ribose-5-phosphate concentration and generation in fibroblasts from patients with gout and purine overproduction.

Characterization of a Feedback-Resistant Phosphoribosylpyrophosphate Synthetase from Cultured, Mutagenized Hepatoma Cells That Overproduce Purines

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