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At least 19 recordsLinked to original sources
PubMed1979

Chicken liver amidophosphoribosyltransferase. Ligand-induced alterations in molecular properties.

A homogeneous amidophosphoribosyltransferase (EC 2.4.2.14) preparation, which was sensitive to purine nucleotide inhibitors, was obtained from chicken liver. From the result of sodium dodecyl sulfate polyacrylamide gel electrophoresis, the subunit weight was estimated to be approximately 58 000. In Tris-HCl buffer, the predominant form of the enzyme had an S20,w of 6.5, Strokes radius of 40 A, and estimated molecular weight of 110 000. Incubation with 5-phosphoribosyl 1-pyrophosphate or Pi resulted in an increase in the S20,w to 9.1--9.5, Strokes radius 50 A, and estimated molecular weight to 200 000. Incubation of the large form with AMP led to a decrease in the molecular wight of the enzyme. It is concluded that chicken liver amidophosphoribosyltransferase is an allosteric protein whose activity is regulated by a series of conformational changes induced by a number of ligands.

Adenosine Monophosphate↗
PubMed1975

Imbalance of purine metabolism in hepatomas of different growth rates as expressed in behavior of glutamine-phosphoribosylpyrophosphate amidotransferase (amidophosphoribosyltransferase, EC 2.4.2.14).

The behavior of glutamine-phosphoribosylpyrophosphate amidotransferase (amidophosphoribosyltransferase, EC 2.4.2.14) was determined in normal, differentiating, and regenerating liver and in a spectrum of hepatomas of widely different growth rates. The liver and tumor enzymes were measured in 100,000 x g supernatants prepared from 20% tissue homogenates containing 0.25 M sucrose and 1 mM MgC12. Kinetic studies were carried out on the amidotransferase in the curde supernatant from liver and rapidly growing hepatoma 3924A so that under optimum standard assay conditions only the enzyme amount would be the limiting factor. The kinetic results showed that certain properties of the amidotransferase from liver and hepatoma were similar. The liver and hepatoma enzyme exhibited apparent Km's for: glutamine, 1.7 and 2.3 mM; MgC12, 0.7 and 1.1 mM, and phosphoribosylpyrophosphate. S0.5 for 0.9 and 0.4 mM, respectively...

Amidophosphoribosyltransferase↗
PubMed1977

Purine biosynthesis in mutant mammalian cells.

De novo purine biosynthesis has been studied in lymphocyte cell lines established from Lesch-Nyhan patients deficient in hypoxanthine-guanine phosphoribosyltransferase (HGPRT), in in vitro differentiating erythroleukaemic cell lines cloned from cells charactistic of virus-induced murine leukaemia, and in mutant hamster cells deficient in amidophosphoribosyltransferase. The relationship between cellular phosphoribosylpyrophosphate (PP-ribose-P) metabolism and the activity of the enzymes which catalyse the early steps of de novo purine biosynthesis has been explored. It was found that hamster cells deficient in amidophosphoribosyltransferase did not accumulate PP-ribose-P as do HGPRT-deficient cells. In these model systems, an accelerated rate of de novo purine biosynthesis tended to be associated with an increase in cellular PP-ribose-P cotent, but decreases in this rate results from the reduction in the activity of amidophosphoribosyltransferase. Regulation of ammonia-dependent de novo purine biosynthesis was similar to that of glutamine-dependent purine biosynthesis.

Adenosine↗
PubMed1979

Effects of benzimidazole on the purine and pyrimidine metabolism of yeast.

Yeast cells inhibited by benzimidazole accumulate hypoxanthine with associated efflux of xanthine. Unlike control cells, inhibited cells contain no detectable free UMP and CMP. Benzimidazole decreases uptake of [8-14C]hypoxanthine into the intracellular pool of hypoxanthine and xanthine but causes radioactive xanthine to accumulate in the medium. In inhibited cultures there is a threefold increase in incorporation of [8-14C]hypoxanthine into the total (intracellular plus extracellular) xanthine. Uptake of [8-14C]hypoxanthine into free nucleotides and into bound adenine and guanine was inhibited by 70%. Uptake of [U-14C]glycine into IMP, AMP, GMP, DNA and RNA was also substantially decreased. Incorporation of [2-14C]uracil into the intracellular uracil pool was inhibited by 30% and into free uridine and cytidine by over 90%. Benzimidazole inhibited incorporation of [8-3H]IMP into AMP and GMP, and decreased substantially the activity of glutamine-amidophosphoribosyltransferase (EC 2.4.2.14). Yeast cultures were shown to N-ribotylate benzimidazole. Results are consistent with benzimidazole inhibiting yeast growth by competing for P-rib-PP and so depriving other ribotylation processes such as the 'salvage' pathways and de novo synthesis of purines and pyrimidines.

Amidophosphoribosyltransferase↗
PubMed1977

Effect of lowered intracellular ATP and GTP concentrations on purine ribonucleotide synthesis and interconversion.

The effects of lowered intracellular ATP and GTP concentrations on enzymes of purine ribonucleotide synthesis and intercoversion were studied using intact Ehrlich ascites tumor cells. The apparent rates of phosphoribosyl pyrophosphate synthetase (EC 2.7.6.1) and of inosinate dehydrogenase (EC 1.2.1.14) were increased in cells containing lowered purine nucleotide concentrations, but apparent activities of amidophosphoribosyltransferase (EC 2.4.2.14), the purine phosphoribosyltransferases, and other enzymes of purine ribonucleotide interconversion were not affected.

Adenine Phosphoribosyltransferase↗
PubMed1979

Glutamine phosphoribosylpyrophosphate amidotransferase from Escherichia coli. Purification and properties.

Glutamine 5-phosphoribosylamine:pyrophosphate phosphoribosyltransferase (amidophosphoribosyl-transferase) has been purified to homogeneity from Escherichia coli. The molecular weight of the native enzyme was 194,000 by sedimentation equilibrium centrifugation and 224,000 by gel filtration. A subunit Mr = 57,000 was estimated by gel electrophoresis in sodium dodecyl sulfate. Cross-linking experiments gave species of Mr = 57,000, 117,000, and 177,000. A trimer or tetramer of identical subunits is indicated for the native enzyme. Highly active E. coli amidophosphoribosyl-transferase lacks significant nonheme iron. Enzyme activity was not enhanced by addition of iron salts and sulfide. Amidophosphoribosyltransferase exhibited both NH3- and glutamine-dependent activities. Glutaminase activity was detected in the absence of other substrates. Both glutamine- and NH3-dependent activities were subject to end product inhibition by purine 5'-ribonucleotides. AMP and GMP, in combination, gave synergistic inhibition. AMP and GMP exhibited positive cooperativity. In addition, GMP promoted cooperativity for saturation by 5-phosphoribosyl-1-pyrophosphate. Glutamine utilization was inhibited by NH3, suggesting that the amide of glutamine is transferred to the NH3 site prior to amination of 5-phosphoribosyl-1-pyrophosphate. The glutamine-dependent activity was selectively inactivated by the glutamine analogs L-2-amino-4-oxo-5-chloropentanoic acid and 6-diazo-5-oxo L-norleucine (DON) and by iodoacetamide. Incorporation of 1 eq of DON/subunit (Mr = 57,000) caused complete inactivation of the glutamine-dependent activity, thus providing evidence for one glutamine site per monomer and for the functional identity of the subunits. Following alkylation with iodoacetamide, carboxymethylcysteine was the only modified amino acid isolated from an acid hydrolysate. The glutamine-dependent activity was sensitive to oxidation. Inactivation by exposure to air was reversed by incubation with high concentrations of dithiothreitol.

Amidophosphoribosyltransferase↗
PubMed1979

Rat liver glutamine 5-phosphoribosyl-1-pyrophosphate amidotransferase [EC 2.4.2.14]. Purification and properties.

Glutamine 5-phosphoribosyl-1-pyrophosphate (PRPP) amidotransferase (amidophosphoribosyltransferase), [EC 2.4.2.14] was purified 1,600-fold from rat liver. The preparation gave two protein bands on acrylamide gel electrophoresis, of which only the main band showed enzyme activity. The molecular weight of the enzyme was estimated to be 215,000, 200,000, and 195,000 by Sephadex G-150 gel filtration, polyacrylamide gel electrophoresis, and sucrose density grandient ultracentrifugation, respectively. The apparent Km values for glutamine and PRPP were 1.24 mM and 0.57 mM, respectively. The concentration-activity curve for PRPP changed from a hyperbolic to a sigmoidal form on addition of AMP or GMP, and this inhibition by AMP was prevented by increasing the PRPP concentration. In the presence of high concentrations of inorganic phosphate, the catalytic activity was decreased and the sensitivity to AMP inhibition was slightly increased. The molecular size of liver amidotransferase was not changed by the addition of PRPP, AMP, or 2-mercaptoethanol. The purified rat liver enzyme has a broad pH-range of activity between 6.5 and 8.5.

Amidophosphoribosyltransferase↗
PubMed1977

Superactivity of phosphoribosylpyrophosphate synthetase, due to feedback resistance, causing purine overproduction and gout.

A mutant feedback-resistant, physiologically superactive, phosphoribosylpyrophosphate (PP-ribose-P) synthetase was found in a family with purine overproduction, gout and uric acid lithiasis. In haemolysates and cultured fibroblasts from the propositus, the mutant enzyme exhibited resistance to feedback inhibition by normal cell constituents, such as ADP and GDP; normal affinity to substrates and to activator Pi was demonstrated in the haemolysate. In both erythrocytes and cultured fibroblasts, the superactivity of the mutant enzyme was manifest in increased PP-ribose-P content and availability for nucleotide synthesis, leading to an acceleration of the rate of purine synthesis de novo in the fibroblasts. The enzyme abnormality and the resulting increase in PP-ribose-P content and generation were demonstrated in the erythrocytes of one of the propositus' two siblings who was similarly affected but not in the propositus' father, his second brother and four sons, who were all clinically and biochemically normal, nor in the erythrocytes of the clinically normal hyperuricosuric mother. However, cultured fibroblasts from her skin exhibited variability in PP-ribose-P content and availability and in the rate of purine synthesis de novo, these parameters being increased in most cultures. The mother's fibroblast cultures were found to contain two cell populations, one with normal and the other with mutant PP-ribose-P synthetase, indicating an X-linked pattern of inheritance of the synthetase superactivity in this gouty family.

Adolescent↗
PubMed1976

Increased PRPP synthetase activity in cultured rat hepatoma cells containing mutations in the hypoxanthine-guanine phosphoribosyltransferase gene.

Nine independently derived clones of mutagenized rat hepatoma cells selected for resistance to 6-mercaptopurine (6-MP) or 6-thioguanine (6-ThioG) have been isolated. Each has severely reduced catalytic activity of hypoxanthine-guanine phosphoribosyltransferase (HPRT) and seven of them possess significantly increased activities of phosphoribosylpyrophosphate (PRPP) synthetase. The degrees of elevations of PRPP synthetase activities do not correlate with the degrees of deficiencies of HPRT activities. The cells from one of these clones, 1020/12, posses 40% of the normal HPRT catalytic activity and overproduce purines. We have extensively examined the cells from this clone. Immunotration studies of 1020/12 cells indicate that there is a mutation in the structural gene for HPRT. Although they possess increased specific catalytic activities of the enzyme. PRPP synthetase, the catalytic parameters, heat stability, and isoelectric pH of PRPP synthetase from 1020/12 cells are indistinguishable from those of the enzyme from wild-type cells. The cause of purine overproduction by 1020/12 cells appears to be the elevated PRPP synthetase activity, rather than a PRPP "sparing" effect stemming from reduced HPRT activity. Support for this idea is provided by the observation that the complete loss of HPRT activity in a clone derived from 1020/12 cells does not further enhance the levels of PRPP synthetase or purine overproduction. We propose that the elevated levels of PRPP synthetase activity in these HPRT deficient cells result from a mutational event in the structural gene for HPRT, and that this causes the disruption of a previously undescribed regulatory function of this gene on the expression of the PRPP synthetase gene.

Amidophosphoribosyltransferase↗