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)

A medium is described which can be used for the selection of somatic cell hybrids between HPRT- and APRT- mutant cells. This medium, called GAMA, contains as its relevant constituents the following: the purines guanine and adenine and the purine biosynthetic pathway inhibitors mycophenolic adic, which blocks conversion of adenine ribonucleotides to guanine ribonucleotides, and azaserine, which blocks de novo purine synthesis.
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PMID:A selective medium (GAMA) for the isolation of somatic cell hybrids from HPRT- and APRT- mutant cells. 76 86

Erythrocytes, obtained from a normal adult male and from a patient with Lesch-Nyhan syndrome, were incubated with [8-14C]adenine and [8-14C]hypoxanthine (Table 1). The labeled adenine was utilized to about the same extent for the synthesis of AMP by the normal subject's and the patient's erythrocytes. Deamination of AMP to IMP occurred to about the same extent in both samples. In contrast, hypoxanthine was utilized extensively for IMP synthesis in the normal erythrocyte only. The amount of total label in the IMP was about 100 times that of the Lesch-Nyhan erythrocyte, a consequence of the deficiency of hypoxanthine-guanine phosphoribosyltransferase (HGPRT) activity in the syndrome. No significant labeling of the AMP occurred. When aliquots of erythrocytes from both sources were incubated with 4-amino-5-imidazolecarboxamide (AICA) and sodium [14C]formate, extensive labeling of the IMP occurred in normal and in Lesch-Nyhan erythrocytes. The data suggest that AICA serves as a substrate for the adenine phosphoribosyltransferase (APRT) of the Lesch-Nyhan erythrocyte and that the ribotide of AICA, 5'-phosphoribosyl-5-aminoimidazole-4-carboxamide (AICAR), undergoes formylation by labeled N10-formyl tetrahydrofolic acid formed from the reaction of sodium [14C]formate with the tetrahydrofolic acid of the cell. The formyl-AICAR undergoes ring closure to IMP by a series of reactions comparable to those described for the normal erythrocyte. When 5-amino-1-ribosyl-4-imidazolecarboxamide (rAICA) and sodium [14C]formate were incubated with erythrocyte suspensions, extensive utilization for IMP synthesis was also observed in normal erythrocytes and in erythrocytes from Lesch-Nyhan patients (Table 2). The reaction sequence is somewhat different from that of AICA. AICA is not a substrate for the purine nucleoside phosphorylase of rabbit or human erythrocytes. The mechanism of rAICA utilization is visualized as a direct phosphorylation of the ribosyl compound, possibly by the adenosine kinase of the human cell. The ribotide, AICAR, formed by this mechanism, undergoes formylation and ring closure, yielding IMP. The glutamine antagonist, diazooxonorleucine (DON), was added to aliquots of patients' cells incubated with rAICA and sodium [14C]formate. DON is an effective inhibitor of the conversion of IMP to GMP and its presence in an incubation suspension resulted in a somewhat greater radioactivity of the total cellular IMP. The extension of the current studies to Lesch-Nyhan cells in culture may serve to assist in the direct evaluation of the regulatory role of IMP in the de novo pathway of purine nucleotide biosynthesis. Because of the substrate requirements of the reactions, the metabolism of AICA and rAICA may also serve to differentiate the roles of purine nucleotides and of phosphoribosylpyrophosphate (PRPP) in the pathway regulation. The findings presented also offer a possible therapeutic approach to the early treatment of the disease in the afflicted neonate...
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PMID:Lesch-Nyhan syndrome: the synthesis of inosine 5'-phosphate in the hypoxanthine-guanine phosphoribosyltransferase-deficient erythrocyte by alternate biochemical pathways. 87 Aug 76

Adenine and adenosine metabolism has been studied in intact human erythrocytes in vitro using high performance liquid chromatography, isotopic labeling and electrophoresis. Their metabolism to nucleotides was controlled by phosphoribose diphosphate synthesis which was phosphate dependent. Adenosine formed hypoxanthine or IMP depending upon Pi concentration, but adenosine kinase and deaminase activities were not affected by P levels. Free [14C]adenine and [14C]hypoxanthine were found in cellular extracts. Rapid interconversions occurred to give a distribution for ATP : ADP : AMP of 10 : 1 : 0.1. Marked decomposition of ATP to ADP and AMP occurred during incubations in plasma and Earle's media in air on nitrogen, but ATP levels remained stable in phosphate buffers and in the presence of oxygen. At physiological Pi (1 mM) adenosine kinase activity grossly exceeded adenine phosphoribosyltransferase activity. The latter was approximately 7 fold that of hypoxanthine phosphoribosyltransferase activity. These differences decreased with increasing Pi levels. No significant increase in corresponding nucleotides was obtained by incubation with high levels (0.5 mM) of adenine, guanine or guanosine at physiological Ii, ATP increased by 10% independently of the substrate employed and significant amounts of IMP and GTP were formed adenosine and guanosine, respectively. The existence of a bound intracellular pool of ATP is suggested.
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PMID:Studies on adenine and adenosine metabolism by intact human erythrocytes using high performance liquid chromatography. 94 98

1. The hypoxanthine/guanine and adenine phosphoribosyltransferase activities in a wide variety of human tissues were studied during their growth and development from foetal life onward. A wide range of activities develop after birth, with especially high values in the central nervous system and testes. 2. Postnatal development of hypoxanthine/guanine phosphoribosyltransferase was also defined in the rat. Although there were increases in the central nervous system and testes, there was also a rise in activity in the liver, which was less marked in man. 3. A sensitive radiochemical assay method, using dTTP to inhibit 5'-nucleotidase activity, suitable for tissue extracts, was developed. 4. No definite evidence of the existence of tissue-specific isoenzymes of hypoxanthine/guanine or adenine phosphoribosyltransferase was found. Hypoxanthine/guanine phosphoribosyltransferase in testes, however, had a significantly different thermal-denaturation rate constant. 5. The findings are discussed in an attempt to relate activity of hypoxanthine/guanine phosphoribosyltransferase to biological function. Growth as well as some developmental changes appear to be related to increase in the activity of this enzyme.
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PMID:Developmental changes in purine phosphoribosyltransferases in human and rat tissues. 101 39

Mutants of Chinese hamster ovary cells deficient in glucose-6-phosphate dehydrogenase (D-glucose-6-phosphate: NADP 1-oxidoreducatse, EC 1.1.1.49) activity were isolated after mutagenesis with ethyl methane sulfonate. The mutants were induced at frequencies of about 10-4 and do not differ in growth properties from wild-type cells. They were isolated by means of a sib selection technique coupled with a histochemical stain of colonies for enzyme activity. The lack of enzyme activity is not due to a dissociable inhibitor, and is recessive in hybrid cells. Multiple mutants that lack hypoxanthine phosphoribosyltransferase activity (IMP:pyrophosphate phosphoribosyltransferase, EC 2.4.2.8) and adenine phosphoribosyltransferase activity (AMP:pyrophosphate phosphoribosyltransferase, EC 2.4.2.7) were isolated by further mutagenesis. By following segregation of wild-type phenotypes from heterozygous multiply marked hybrid cells, it was shown that the genes responsible for glucose-6-phosphate dehydrogenase activity and hypoxanthine phosphoribosyltransferase activity are linked in Chinese hamster cells, in agreement with the location of both on the X chromosome in humans. No linkage to adenosine phosphoribosyltransferase was found. The isolation of mutant cells carrying linked markers should prove useful for studying chromosomal events such as segregation, breakage, recombination, and X-chromosome reactivation.
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PMID:Isolation of mammalian cell mutants deficient in glucose-6-phosphate dehydrogenase activity: linkage to hypoxanthine phosphoribosyl transferase. 105 32

Evidence for derepression of the gene for hypoxanthine phosphoribosyltransferase (HPRT; IMP: pyrophosphate phosphoribosyltransferase, EC 2.4.2.8) on the human inactive X chromosome was obtained in hybrids of mouse and human cells. The mouse cells lacked HPRT and were also deficient in adenine phosphoribosyltransferase (APRT; AMP: pyrophosphate phosphoribosyltransferase; EC2.4.2.7). The human female fibroblasts were HPRT-deficient as a consequence of a mutation on the active X but contained a normal HPRT gene on the inactive X. The two human X chromosomes were further distinguished by differences in morphology: the inactive X was morphologically normal while the active X included most of the long arm of autosome no. 1 translocated to the distal end of the X long arm. Forty-one hybrid clones were first isolated by selection for the presence of APRT; when these clones were selected for HPRT, six of them yielded derivatives having human HPRT with incidences of about 1 in 10-6 APRT-selected hybrid cells. The HPRT-positive derivatives contained a normal-appearing X chromosome indistinguishable from the inactive X of the parental human fibroblasts. The active X with the translocation was not found in any of the HPRT-positive hybrid cells. Human phosphoglycerokinase (ATP:3-phospho-D-glycerate 1-phosphotransferase. EC 2.7.2.3) and glucose-6-phosphate dehydrogenase (D-glucose 6-phosphate: NADP 1-oxidoreductase, EC 1.1.1.49), which are specified by X-chromosomal loci, were not detected in the hybrids expressing HPRT even though they contained an apparently intact X chromosome. The observations are most simply explained by the infrequent, stable derepression of inactive X chromosome segments that include the HPRT locus but not the phosphoglycerokinase and glucose-6-phosphate dehydrogenase loci.
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PMID:Localized Derepression on the Human Inactive X Chromosone in Mouse-Human Cell Hybrids. 105 21

Permanent transfer of genetic information from chromosomes isolated from human diploid cells to recipient cells has been demonstrated. Human metaphase chromosomes were incubated with mouse A9 fibroblasts deficient in hypoxanthine phosphoribosyltransferase (IMP:pyrophosphate phosphoribosyltransferase, EC 2.4.2.8) and adenine phosphoribosyltransferase (AMP:pyrophosphate phosphoribosyltransferase, EC 2.4.2.7). Colonies of cells containing hypoxanthine phosphoribosyltransferase appeared during growth in a selective medium. The hypoxanthine phosphoribosyltransferase gene product in four independent colonies was identified as human donor species by both gel electrophoresis and isoelectric focusing; hence these colonies did not result from reversion of ta9 parental cells. Other X-linked human genes, glucose-6-phosphate dehydrogenase (D-glucose-6-phosphate:NAD(+) 1-oxidoreductase, EC 1.1.1.49) and phosphoglycerate kinase (ATP:3-phospho-D-glycerate 1-phosphotransferase, EC 2.7.2.3), were not expressed in these same colonies. Dissociation of expression of these X-linked genes probably results from chromosomal fragmentation during uptake, but other mechanisms have not been excluded.
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PMID:Human gene expression in rodent cells after uptake of isolated metaphase chromosomes. 105 70

To evaluate the regulation of adenine nucleotide metabolism in relation to purine enzyme activities in rat liver, human erythrocytes and cultured human skin fibroblasts, rapid and sensitive assays for the purine enzymes, adenosine deaminase (EC 2.5.4.4), adenosine kinase (EC 2.7.1.20), hyposanthine phosphoribosyltransferase (EC 2.4.28), adenine phosphoribosyltransferase (EC 2.4.2.7) and 5'-nucleotidase (EC 3.1.3.5) were standardized for these tissues. Adenosine deaminase was assayed by measuring the formation of product, inosine (plus traces of hypoxanthine), isolated chromatographically with 95% recovery of inosine. The other enzymes were assayed by isolating the labelled product or substrate nucleotides as lanthanum salts. Fibroblast enzymes were assayed using thin-layer chromatographic procedures because the high levels of 5'-nucleotidase present in this tissue interferred with the formation of LaCl3 salts. The lanthanum and the thin-layer chromatographic methods agreed within 10%. Liver cell sap had the highest activities of all purine enzymes except for 5'-nucleotidase and adenosine deaminase which were highest in fibroblasts. Erythrocytes had lowest activities of all except for hypoxanthine phosphoribosyltransferase which was intermediate between the liver and fibroblasts. Erhthrocytes were devoid of 5'-nucleotidase activity. Hepatic adenosine kinase activity was thought to control the rate of loss of adenine nucleotides in the tissue. Erythrocytes had excellent purine salvage capacity, but due to the relatively low activity of adenosine deaminase, deamination might be rate limiting in the formation of guanine nucleotides. Fibroblasts, with high levels of 5'-nucleotidase, have the potential to catabolize adenine nucleotides beyond the control od adenosine kinase. The purine salvage capacity in the three tissues was erythrocyte greater than liver greater than fibroblasts. Based on purine enzyme activities, erythrocytes offer a unique system to study adenine salvage; fibroblasts to study adenine degradation; and liver to study both salvage and degradation.
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PMID:Adenine nucleotide metabolism in relation to purine enzymes in liver, erythrocytes and cultured fibroblasts. 118 98

Male New Zealand White rabbits were immunized with human adenine phosphoribosyltransferase (APRT) and hypoxanthine-guanine phosphoribosyltransferase (HGPRT), which were purified about 2000-fold and 800-fold, respectively, from erythrocytes by DEAE-cellulose chromatography, ammonium sulfate precipitation and preparative polyacrylamide gel electrophoresis. Specific immunoprecipitations of APRT and HGPRT were achieved with the antisera that were obtained and by using polyethylene glycol as a substitute for goat anti-(rabbit) gamma globulin. The activities of the human forms of these enzymes, whether from red blood cells or from cultured cells, were almost completely eliminated under the conditions of immunoprecipitation used. Little or no reduction of APRT and HGPRT activities from mouse and Chinese hamster cells was observed. This discriminatory capacity of the antisera was successfully used for the identification of human APRT and HGPRT in human-mouse and human-hamster cell hybrids using the immunoprecipitation reaction.
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PMID:Adenine phosphoribosyltransferase and hypoxanthine-guanine phosphoribosyltransferase immunoprecipitation reactions in human-mouse and human-hamster cell hybrids. 118 4

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 muM. 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 degrees 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 isozymes. Isoelectric focusing of leukocyte extracts from the patient revealed the presence of the mutant enzyme. Cultured diploid fibroblasts from the propositus appeared to function normally, as shown by the inability to grow in 50-100 muM azaguanine and by the normal incorporation of [14C]hypoxanthine into nucleic acid. In contrast, erythrocytes from the patient displayed abnormal properties, including the increased synthesis of phosphoribosylphyrophosphate and elevated functional activity of orotate phosphoribosyltransferase and orotidylic decarboxylase. These unique kinetic, physical, and functional properties provide support for heterogeneous structural gene mutations in partial deficiencies of hypoxanthine-guanine phosphoribosyltransferase.
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PMID:Hypoxanthine-guanine phosphoribosyltransferase. Characterization of a mutant in a patient with gout. 118 48


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