Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.4.2.8 (hypoxanthine-guanine phosphoribosyltransferase)
2,527 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

IMP:pyrophosphate phosphoribosyltransferase (IPPase) (EC 2.4.2.8) has been purified over 7000-fold from human erythrocytes. The purified enzyme moved as a single band on disc electrophoresis. Antisera prepared in rabbits and rats against the purified enzyme precipitated and neutralized the enzyme, but had no effect on AMP-pyrophosphate phosphoribosyltransferase (EC 2.4.2.7) activity. Evidence was found for isozymes (enzyme variants) of IPPase in normal erythrocytes. Erythrocyte lysates of five patients with Lesch-Nyhan disease reacted with antisera against normal IPPase. Lysates from LN erythrocytes blocked the inactivation of normal enzyme by the antibody. LN erythrocytes had about the same concentration of enzyme protein as normal erythrocytes. The genetic defect in LN results in the production of essentially normal amounts of an immunologically identifiable but catalytically incompetent enzyme. Thus LN is apparently the result of a mutation in a structural gene and is not due to deletion of a structural gene or defect in a regulatory gene.
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PMID:Purification of IMP:pyrophosphate phosphoribosyltransferases, catalytically incompetent enzymes in Lesch-Nyhan disease. 432 3

We here report the establishment of a seemingly permanent hybrid cell line formed by fusion of the cells of two biochemically mutant human lymphocyte lines. One parental line (UM-1-6TGr) was deficient in hypoxanthine-guanine phosphoribosyl transferase (IMP: pyrophosphate phosphoribosyltransferase, EC 2.4.2.8), and had two marker chromosomes. The second parental line (UM-21-5) was a clonal derivative of a citrullinemic lymphocyte line, and was, like the line of origin, dificient in argininosuccinic acid synthetase [(L)-Citrulline: (L)-aspartate ligase (AMP-forming), EC 6.3.4.5]. This line also had a marker chromosome, which was a B5 with a very prominent secondary constriction. After trypsinization of both parental lines, followed by addition to the fusion mixture of beta-propiolactone-inactivated Sendai virus, the cells were placed in a doubly selective medium (hypoxanthine-aminopterin-thymidine-containing medium in which the arginine was replaced with citrulline) to prevent the proliferation of the mutant parents. Under selective conditions, 97-99% of cells were found to be tetraploid, containing the three marker chromosomes; and the specific activities of the hybrid line transferase and synthetase were intermediate between normal and mutant line values. Furthermore, the UM-1-6TGr and UM-21-5 lines were producers of gamma and mu heavy chains of immunoglobulin, and of kappa light chains, as determined by immunodiffusion and immunofluorescence, and the hybrid line continued to synthesize and to secrete detectable levels of these same immunoglobulins. These studies demonstrate the genic and cytogenetic stability of this hybridized lymphocyte cell line, and prove that hybridization per se does not extinguish the activity of either the regulatory of structural genes involved in immunoglobulin synthesis.
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PMID:Establishment of a tetraploid, immunoglobulin-producing cell line from the hybridization of two human lymphocyte lines. 436 96

Tissue culture fibroblasts derived from patients with Lesch-Nyhan disease (congenital hyperuricosuria) have a reduced IMP:pyrophosphate phosphoribosyltransferase (EC 2.4.2.8) activity and therefore incorporate, as detected by radioautography, much smaller amounts of tritiated hypoxanthine or guanine into cell nuclei and cytoplasm than do normal cells. However, Lesch-Nyhan cells grown in close contact with normal fibroblasts incorporate these purines. This phenomenon, which requires cell to cell contact for correction of the mutant phenotype, has been called metabolic cooperation. After separation of Lesch-Nyhan cells from normal cells, there is a prompt reversion to the mutant phenotype although the transferase is stable under these conditions for many hours.These results are most compatible with the transfer from normal to mutant fibroblasts of the product of the normal enzyme, a nucleotide or a nucleotide derivative, rather than the transfer of the transferase or informational macromolecules leading to the synthesis of the enzyme. Metabolic cooperation may provide a mechanism for maintaining normal cell function in the heterozygote in vivo. Evidence has been presented previously that selection of normal cells, presumably during embryogenesis, also provides a means for achieving normal function in the heterozygote.
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PMID:Evidence for transfer of enzyme product as the basis of metabolic cooperation between tissue culture fibroblasts of Lesch-Nyhan disease and normal cells. 527 81

1. The purine bases adenine, hypoxanthine and guanine were rapidly incorporated into the nucleotide fraction of Ehrlich ascites-tumour cells in vivo. 2. The reaction of 5'-phosphoribosyl pyrophosphate with adenine phosphoribosyltransferase from ascites-tumour cells (K(m) 6.5-11.9mum) was competitively inhibited by AMP, ADP, ATP and GMP (K(i) 7.5, 21.9, 395 and 118mum respectively). Similarly the reactions of 5'-phosphoribosyl pyrophosphate with both hypoxanthine phosphoribosyltransferase and guanine phosphoribosyltransferase (K(m) 18.4-31 and 37.6-44.2mum respectively) were competitively inhibited by IMP (K(i) 52 and 63.5mum) and by GMP (K(i) 36.5 and 5.9mum). 3. The nucleotides tested as inhibitors did not appreciably compete with the purine bases in the phosphoribosyltransferase reactions. 4. It was postulated that the purine phosphoribosyltransferases of Ehrlich ascites-tumour cells may be effectively separated from the adenine nucleotide pool of these cells.
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PMID:Inhibition of purine phosphoribosyltransferases from Ehrlich ascites-tumour cells by purine nucleotides. 596 81

1. The progress curves of adenine phosphoribosyltransferase and of hypoxanthine phosphoribosyltransferase activity plotted against 5-phosphoribosyl pyrophosphate concentration were hyperbolic in nature. The inhibition of the former enzyme by AMP and GMP and of the latter enzyme by IMP and GMP showed completely competitive characteristics. 2. The effect of temperature on the reaction of adenine phosphoribosyltransferase and of hypoxanthine phosphoribosyltransferase was examined. The energy of activation of the former enzyme decreased at temperatures greater than 27 degrees and that of the latter enzyme at temperatures greater than 23 degrees . For each enzyme, the change in the heat of formation of the 5-phosphoribosyl pyrophosphate-enzyme complex at the critical temperature was approximately equal to the change in the energy of activation but was in the opposite direction. The inhibitor constants with both enzymes in the presence of nucleotides varied in different ways with temperature from the Michaelis constants for 5-phosphoribosyl pyrophosphate indicating that different functional groups were involved in binding substrates and inhibitors. 3. ATP was found to stimulate adenine-phosphoribosyltransferase activity at concentrations less than about 250mum and to inhibit the enzyme at concentrations greater than 250mum. The stimulation was unaffected by 5-phosphoribosyl pyrophosphate concentration but the inhibitory effect could be overcome by increasing concentrations of this compound. At low concentrations ATP reversed the inhibition of adenine phosphoribosyltransferase by AMP and GMP to an extent dependent on their concentration. 4. The properties of adenine phosphoribosyltransferase changed markedly on purification. Crude extracts of ascites-tumour cells had Michaelis constants for 5-phosphoribosyl pyrophosphate and adenine 75 and six times as high respectively as those obtained with purified enzyme. ATP had no stimulatory effect on activity of the purified enzyme or on that of crude extracts heated 15min. or longer at 55 degrees . 5. It is suggested that at low concentrations ATP is bound to an ;activator' site which is separate from the substrate binding site of adenine phosphorytransferase and that at high concentrations ATP competes with 5-phosphoribosyl pyrophosphate at the active site of the enzyme.
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PMID:Studies on the nature of the regulation by purine nucleotides of adenine phosphoribosyltransferase and of hypoxanthine phosphoribosyltransferase from Ehrlich ascites-tumour cells. 606 4

To study the activation and cytotoxic mechanism of bredinin (4-carbamoyl-1-beta-D-ribofuranosylimidazolium-5-olate), a novel nucleoside antibiotic with potent cytotoxic and immunosuppressive effects, we isolated in a single-step manner five mutants resistant to 10 microM bredinin from cultured mouse mammary carcinoma FM3A cells mutagenized with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). Such resistant (Brdr) mutants were 15- to 19-fold less sensitive to the antibiotic than wild-type cells and maintained stably their resistant phenotypes in the absence of bredinin for more than 3 months. They were cross-resistant to tubercidin, an adenosine analog. Like wild-type cells, Brdr mutants were capable of incorporating radioactivity from ring-labeled adenosine into the acid-insoluble macromolecular fraction. However, hypoxanthine-guanine phosphoribosyltransferase-deficient (HGPRT-) mutants derived from the Brdr cells did not incorporate the radioactivity at all or at a markedly reduced rate, indicating that blockade of the pathway via adenosine deaminase present in the Brdr cells resulted in loss of their ability to utilize adenosine. Enzyme assays using cell-free extracts revealed that all the Brdr mutants had less than 3% of the adenosine kinase (AK) activity found in wild-type cells. These results demonstrate that the bredinin resistance is attributed to a defective AK activity and, therefore, that bredinin is metabolized by AK, which may phosphorylate it to a toxic nucleotide, bredinin 5'-monophosphate (Brd-MP), in sensitive cells. Among exogenously added purine bases, guanine was able to reverse the cytotoxic effect of bredinin on both wild-type cells and F5 cells carrying the vector pSV2-Escherichia coli xanthine-guanine phosphoribosyltransferase (XGPRT) gene, while xanthine was able to do so only in F5 cells because the base was metabolized to XMP by the cells. These results support the mechanism of bredinin cytotoxicity, that Brd-MP formed in sensitive cells exposed to the antibiotic blocks the conversion of IMP to XMP by inhibiting IMP dehydrogenase.
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PMID:Genetic and biochemical studies on the activation and cytotoxic mechanism of bredinin, a potent inhibitor of purine biosynthesis in mammalian cells. 614 13

2-Amino-6-chloro-1-deazapurine is of interest as a purine analog with demonstrated in vivo activity against mouse leukemia L1210. That the active form of this agent is a nucleotide and that the nucleotide is formed by the action of hypoxanthine (guanine) phosphoribosyltransferase were shown by the facts that (a) L1210 cells deficient in hypoxanthine phosphoribosyltransferase were insensitive to the analog; (b) hypoxanthine, but not adenine, prevented the formation of the analog nucleotide by enzyme preparations containing activities of both hypoxanthine and adenine phosphoribosyltransferases; and (c) the cytotoxicity of the analog was prevented by hypoxanthine. The ribonucleoside of this analog was not toxic to cell cultures and hence is not phosphorylated or cleaved to the base. In intact HEp-2 cells and L1210 cells, the analog was metabolized to the nucleoside 5'-phosphate which accumulated to concentrations as high as 1000 nmoles/10(9) cells; no di- or triphosphates were detected. In HEp-2 cells, the analog reduced the pools of purine nucleotides with some accumulation of IMP. The toxicity of minimal inhibitory concentrations of the analog to HEp-2 cells could be prevented or reversed by 4(5)-amino-5(4)-imidazolecarboxamide (AIC); the toxicity of higher concentrations could be prevented or reversed by a combination of adenine and guanosine but not by AIC. The analog inhibited the incorporation of formate into purine nucleotides and into macromolecules at concentrations that had no effect on utilization of hypoxanthine; at higher concentrations the incorporation of hypoxanthine was inhibited. Low concentrations also inhibited the utilization of uridine and thymidine. The incorporation of hypoxanthine and AIC into guanine nucleotides, but not adenine nucleotides, was inhibited. These results indicate two sites of inhibition of the biosynthesis of purine nucleotides, the more sensitive one being on an early step of the pathway and the less sensitive one on the IMP-GMP conversion. That the blockade of de novo synthesis probably was at the site of feedback inhibition was indicated by the fact that the analog inhibited the accumulation of formylglycinamide ribonucleotide in azaserine-treated cells but did not inhibit the synthesis of 5'-phosphoribosyl 1-pyrophosphate. Comparative studies were performed with the related analog, 2-amino-6-chloropurine, which has been reported to produce a similar dual blockade of the purine pathway. This purine was less toxic than its 1-deaza analog; it produced a modest decrease in adenine nucleotides but increased pools of guanine nucleotides.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Mode of action of 2-amino-6-chloro-1-deazapurine. 614 12

Tiazofurin, a C-nucleoside, was cytotoxic in hepatoma 3924A cells grown in culture with an LC50 = 7.5 microM. In the culture, a closely linked dose-related response of tumor cell-kill and depletion of GTP pools was observed after tiazofurin treatment. In rats carrying subcutaneously transplanted hepatoma 3924A solid tumors, a single intraperitoneal injection of tiazofurin (200 mg/kg) caused a rapid inhibition of IMP dehydrogenase (EC 1.2.1.14) activity and depleted GDP, GTP, and dGTP pools in the tumor; concurrently, the 5-phosphoribosyl 1-pyrophosphate (PRPP) and IMP pools expanded 8- and 15-fold, respectively. Tiazofurin decreased tumoral IMP dehydrogenase activity and dGTP pools in a dose-dependent manner over a range of 50-200 mg/kg; by contrast, the depletion of GTP and the accumulation of IMP and PRPP pools were near maximum at 50 mg/kg. The increase in PRPP pools may be attributed to an inhibition by IMP of the activity of hypoxanthine-guanine phosphoribosyltransferase (EC 2.4.2.8). The IMP dehydrogenase activity and the pools of ribonucleotides returned to the normal range by 24-48 h after the single injection of tiazofurin. However, the markedly depleted dGTP pools remained low for 72 h. Tiazofurin treatment resulted in significant anti-tumor activity in rats inoculated with hepatoma 3924A. The decrease in GTP levels and particularly the sustained depletion in the dGTP pools may explain, in part at least, the chemo-therapeutic action of tiazofurin on hepatoma 3924A. This is the first report showing that a marked therapeutic response was achieved against rapidly growing hepatoma 3924A by treatment with a single anti-metabolite.
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PMID:Modulation of IMP dehydrogenase activity and guanylate metabolism by tiazofurin (2-beta-D-ribofuranosylthiazole-4-carboxamide). 614 52

The isolation and characterization of a mutant mouse T-cell lymphoma (S49) with altered purine metabolism is described. This mutant, AU-100, was isolated from a mutagenized population of S49 cells by virtue of its resistance to 0.1 mM 6-azauridine in semisolid agarose. The AU-100 cells are resistant to adenosine mediated cytotoxicity but are extraordinarily sensitive to killing by guanosine. High performance liquid chromatography of AU-100 cell extracts has demonstrated that intracellular levels of GTP, IMP, and GMP are all elevated about 3-fold over those levels found in wild type cells. The AU-100 cells also contain an elevated intracellular level of pyrophosphoribosylphosphate (PPriboseP), which accounts for its resistance to adenosine. However AU-100 cells synthesize purines de novo at a rate less than 35% of that found in wild type cells. Furthermore, the intact cells of this mutant S49 cell line cannot efficiently incorporate labeled hypoxanthine into nucleotides since the salvage enzyme HGPRTase is inhibited in situ. The AU-100 cell line was found to be 80% deficient in adenylosuccinate synthetase, but these cells are not auxotrophic for adenosine or other purines. The significant alterations in the control of purine de novo and salvage metabolism caused by the defect in adenylosuccinate synthetase are mediated by the resulting increased levels of guanosine nucleotides.
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PMID:Abnormal regulation of purine metabolism in a cultured mouse T-cell lymphoma mutant partially deficient in adenylosuccinate synthetase. 615 49

Three cytidine analogs containing modifications in the 5-position of the cytosine ring (5-azacytidine, 5-aza-2'-deoxycytidine and pseudoisocytidine) induced the expression of human hypoxanthine/guanine phosphoribosyltransferase (IMP; pyrophosphate phosphoribosyltransferase, EC 2.4.2.8) gene (HPRT) from a structurally normal inactive human X chromosome retained in a mouse-human somatic cell hybrid. Between 0.1% and 8% of the cells surviving treatment with these analogs were able to form colonies in selective medium (hypoxanthine/aminopterin/thymidine/glycine medium), but two other analogs, 5-fluoro-2'-deoxycrytidine and 5.6-dihydro-5-azacytidine, did not induce HPRT expression. The inactive X chromosome present in the hybrid, was found to be late replicating, and experiments with synchronized cells showed that the induction of HPRT expression by 5-aza-2'-deoxycytidine occurred maximally in cells treated in the latter half of the S phase. Two division cycles were required after analog treatment for the highest frequency of expression of the induced gene. Because these analogs are powerful inhibitors of the methylation of cytosine residues in DNA, the results imply that demethylation of specific DNA sequences may be required for the reexpression of human HPRT.
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PMID:Cell cycle-specific reactivation of an inactive X-chromosome locus by 5-azadeoxycytidine. 617 64


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