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: UNIPROT:P00492 (hypoxanthine-guanine phosphoribosyltransferase)
2,385 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Human erythrocyte lysate proteins were resolved into over 250 discrete spots by two-dimensional electrophoresis using isoelectric focusing in the first dimension and electrophoresis in the presence of sodium dodecyl sulfate, (SDS) in the second. The overwhelming excess of hemoglobin has made such analyses difficult in the past. However, with the ISO-DALT two-dimensional electrophoresis system, large numbers of red cell proteins can be mapped in the presence of hemoglobin. When hemoglobin and several other major proteins are removed by adsorption to DEAE-cellulose, additional minor components are seen, giving a total of over 275. With the use of purified preparations, the map positions of five cell enzymes or their subunits were determined: pyruvate kinase, catalase, glucose-6-phosphate dehydrogenase, hypoxanthine phosphoribosyltransferase, and carbonic anhydrase. The mapping techniques described complement and extend those traditionally used to find human red cell protein variants.
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PMID:Red cell proteins. I. Two-dimensional mapping of human erythrocyte lysate proteins. 10 31

Hypoxanthine phosphoribosyltransferase (HPRT, IMP:pyrophosphate phosphoribosyltransferase, EC 2.4.2.8) can be purified 5-to 10,000-fold from extracts of HeLa (human) cells by a three-step procedure consisting of high-speed centrifugation, adsorption to Sepharose-conjugated HPRT antibody, and sodium dodecyl sulfate/polyacrylamide gel electrophoresis. Purified enzyme labeled in vivo with radioactive lysine, arginine, or methionine was digested with trypsin and the tryptic peptides were separated by column chromatography on Bio-Rad cation exchanger Aminex A-5. Less than 50 ng (2 pmol) of HPRT is required to produce a tryptic peptide pattern. A methionine-labeled peptide was identified as the COOH-terminus because it was not labeled with either lysine or arginine. We have compared the tryptic peptide patterns of normal HeLaHPRT and a crossreacting HPRT protein lacking enzyme activity from HeLa mutant H23 [Milman et al. (1976) Proc. Natl. Acad. Sci. USA 73, 4589--4593]. The mutant protein has a new lysine-labeled peptide, but the chromatography patterns of arginine- or methionine-labeled peptides appear identical to those of the normal protein. The appearance in the H23 mutant HPRT protein of a new tryptic peptide provides strong evidence for a mutation in the HPRT structural gene. The tryptic peptide patterns were used to determine the total number of residues of labeled amino acid in the protein, and the values are reasonably consistent with those determined by conventional amino acid analysis pf erythrocyte HPRT.
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PMID:Tryptic peptide analysis of normal and mutant forms of hypoxanthine phosphoribosyltransferase from HeLa cells. 26 86

1. Hypoxanthine-guanine phosphoribosyltransferase (EC 2.4.2.8) from Saccharomyces cerevisiae was purified 9400-fold by affinity chromatography giving rise to an electrophoretically homogeneous preparation. 2. The molecular weight of the enzyme was determined by gel filtration with Sephadex G-100 and by sodium dodecylsulfate gel electrophoresis. Both methods reveal a molecular weight of 51,000. 3. The enzyme requires Mg2+ and has its pH optimum at 8.5. 4. Isoelectric focussing as well as gel electrophoresis of the purified extract reveals a single band which exhibits enzyme activity. The isoelectric point of the enzyme is 5.1. 5. The enzyme displays Michaelis-Menten kinetics with apparent Michaelis constants for hypoxanthine, guanine and phosphoribosylpyrophosphate of 23 microns, 18 microns, and 50 microns respectively.
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PMID:Purification and characterization of the hypoxanthine-guanine phosphoribosyltransferase from Saccharomyces cerevisiae. 37 63

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

Three major approaches to the complete purification of hypoxanthine phosphoribosyltransferase from human erythrocytes and rat brain are described. Preparative isoelectric focusing which has been used for the isolation of the human enzyme was not fully successful in the case of rat brain. Preparative polyacrylamide-gel electrophoresis in gel blocks yields enzyme samples of high purity as judged by analytical gel electrophoresis, but with a comparatively low specific enzyme activity. The most rapid and convenient method, a modification of the affinity chromatography on GMP agarose first described by Hughes[5] gives hypoxanthine phosphoribosyltransferase which is superior to the other preparations in its homogeneity and its specific activity. All three methods produce an identical enzyme protein detected by polyacrylamide electrophoresis on nondenaturing and sodium dodecylsulfate gels. Molecular data of hypoxanthine phosphoribosyltransferase derived from these studies are: Isoelectric points of 5.60; 5.85 and 5.90 for three isozyme peaks of the rat brain enzyme; and a molecular weight of 72000 for the native rat brain enzyme and of 25000-27000 for the subunit of human and rat enzyme. Guanylate kinase does not interfere with the purification of hypoxanthine phosphoribosyltransferase on GMP agarose and moreover is itself partially purified by this chromatography.
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PMID:Facilitated purification of hypoxanthine phosphoribosyltransferase. 99 64

The molecular weights of the subunits of the enzyme hypoxanthine-guanine phosphoribosyltransferase (EC 2.4.2.8) from human erythrocytes were determined with a simple novel method, including electrophoresis in sodium dodecyl sulphate gels, gel slicing, elution of protein from the gel slices and enzyme reactivation in the presence of the substrate 5-phosphorylribose-1-pyrophosphate. As molecular weight standards glutaraldehyde-polymerized polypeptides of human haemoglobin were used. The experiments clearly showed the existence of molecular weight differences in human erythrocyte hypoxanthine-quanine phosphoribosyltransferase.
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PMID:Determination of the subunit molecular weight of hypoxanthine-guanine phosphoribosyltransferase from human erythrocytes by recovery of enzyme activity from sodium dodecyl sulphate gels. 120 54

Arsenic, a potent carcinogen, fails to induce gene mutations in mammalian cells. However, posttreatment of ultraviolet light (UV) irradiated cells with sodium arsenite synergistically enhances the mutation frequency on the hypoxanthine (guanine) phosphoribosyltransferase locus. To investigate the molecular mechanism of the comutagenic effects of sodium arsenite, we characterized the alterations of nucleotide sequences in 30 UV-induced and 39 sodium arsenite enhanced hprt mutants from Chinese hamster ovary K1 cells by direct sequencing of mRNA-PCR amplified cDNA. The majority of sequence alterations derived from UV irradiation (80%) and from sodium arsenite posttreatment (70%) were single base substitutions. UV irradiation induced all types of base substitutions. Among them, 57% were transversions. The frequency of transversions increased to 70% in sodium arsenite enhanced mutants. While base substitutions observed in UV-induced mutants were evenly distributed along with the whole coding region, exons 3 and 8 were most frequently mutated in sodium arsenite enhanced mutants. Sodium arsenite posttreatment did not alter the strand bias for mutation induction, i.e., 73% and 78%, of the mutations were located on the non-transcribed strand in UV-induced and sodium arsenite enhanced mutants, respectively. In contrast to UV-induced mutations, bases at the 5' position of TT and the 3' position of CT sequences were the most frequent mutation sites observed in sodium arsenite enhanced mutants. We hypothesize that sodium arsenite may interfere with the process of mutation fixation of TT and CT dimers during DNA replication.
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PMID:Posttreatment with sodium arsenite alters the mutational spectrum induced by ultraviolet light irradiation in Chinese hamster ovary cells. 139 6

2-Amino-N6-hydroxyadenine (AHA) is a remarkably efficient and specific inducer of point mutations in Neurospora, with few or no larger scale events being detected (de Serres et al., 1985). In the present studies, AHA is shown to be a potent point mutagen at the tk +/-, hprt+ and Na+/K+ ATPase loci in L5178Y/tk (+/-)-3.7.2C mouse lymphoma cells. Both large and small colony tk-/- mutants were analyzed at the molecular level and a preliminary assessment was made of small colony mutant karyotypes (230 bands/haploid metaphase cell; large colony mutants typically have normal karyotypes and were not analyzed). AHA induced greatly delayed (7-9 cell doublings) cytotoxicity, suggestive of a mutational mechanism (e.g., base-pair substitution) requiring DNA replication prior to its phenotypic expression. Approximately one-third of the tk -/- mutants formed small colonies, a phenotype which is typically associated with alterations to chromosome 11b, the site of the functional tkb allele in the parental cells. However, banded karyotypes have provided convincing evidence for alterations chromosome 11b in only 2 of the 7 small colony mutants analyzed. Southern blot analysis showed that 78% (21/27) of these small colony mutants have retained the Nco-1 6.3-kb band, which is diagnostic of the tkb allele. This makes AHA unique among the mutagens examined so far in inducing small colony mutants without inducing large losses of tkb DNA. Although a dose-dependent increase in the proportion of small colony mutants was noted, no significant dose-dependent differences were seen at the molecular level in the relatively few mutants analyzed. The majority of AHA-induced tk -/- mutants formed large colonies. Southern blot analysis showed that 86% (25/29) of these had retained the Nco-1 6.3-kb band which is diagnostic of the tkb allele. It is concluded that AHA induces primarily micromutations (less than 100 base pairs), probably through a base-pair substitution mechanism, at the tk, hprt and Na+/K+ ATPase loci in this system, with some larger scale damage (kilobases of DNA at the molecular level; chromosome 11b damage at the cytogenetic level) also occurring.
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PMID:Mutagenicity of 2-amino-N6-hydroxyadenine (AHA) at three loci in L5178Y/tk+/- mouse lymphoma cells: molecular and preliminary cytogenetic characterizations of AHA-induced tk-/- mutants. 165 47

The induction of mutants at the hypoxanthine-guanine phosphoribosyltransferase and Na+/K+ ATPase loci by photoaddition of two bifunctional psoralens was compared in normal and in Fanconi's anemia lymphoblasts from the genetic complementation group A. For the two loci, the frequency of mutants was significantly lower in Fanconi's anemia than in normal cells. This is true whether the data are expressed as a function of dose or as a function of survival level. It is suggested that the chromosomal instability characteristic of Fanconi's anemia is responsible for the cancer proneness rather than the mutability at the gene level.
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PMID:Mutagenic response of Fanconi's anemia cells from a defined complementation group after treatment with photoactivated bifunctional psoralens. 215 78

When 7,12-dimethylbenz[a]anthracene (DMBA) and aflatoxin B1 (AFB1) were activated by hepatocytes from Fischer 344 rats fed a diet containing 2% butylated hydroxyanisole (BHA), frequencies of mutation to 6-thioguanine resistance (TGR) at the HGPRTase gene locus and to ouabain resistance (OuR) at the Na+,K(+)-ATPase gene locus in V79 cells were 30-70% less than those obtained with hepatocytes from untreated controls. A difference in the mutation frequency did not occur when dimethylnitrosamine (DMN) was activated by BHA induced- rather than control-hepatocytes. Analysis of hepatocytes from rats fed 2% BHA showed a small (1.5-fold), but significant, increase in glutathione levels over that in the controls but no change in activity of cytochrome P450. Cytosolic glutathione S-transferase (GST) activity was increased 2-3-fold in hepatocytes from rats fed the 2% BHA diet. These results suggest that mutagenic response to DMBA and AFB1 is reduced, at least in part, because of BHA-induction of hepatocyte GST activity; while activation of DMN can occur by pathway(s) unaffected by BHA-induction of these liver enzymes. In contrast to mutation frequencies, significant differences between BHA- and control-activation in the production of sister-chromatid exchange (SCE) and micronucleus formation (MN) were not detected with any of the genotoxins. It was concluded that the mechanism(s) by which SCE and MN occur are likely unrelated to the capacity of BHA to induced activity of hepatic enzymes, e.g. the GSH S-transferases, that directly or indirectly affect mutation end-points.
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PMID:Comparative genotoxicity of 3 procarcinogens in V79 cells as related to glutathione S-transferase activity of hepatocytes from untreated rats and those fed 2% butylated hydroxyanisole. 216 83


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