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)

We have developed a sensitive radioimmunoassay capable of detecting and quantitating 20 ng of hypoxanthine phosphoribosyltransferase (EC 2.4.2.8; IMP:pyrophosphate phosphoribosyltransferase) protein. For this assay, hypoxanthine phosphoribosyltransferase from human erythrocytes was iodinated with 125I under mild conditions using hydrogen peroxide and lactoperoxidase attached to Sepharose-4B. Antisera prepared against homogeneous human hypoxanthine phosphoribosyltransferase precipitates the iodinated enzyme as effectively as the unlabeled enzyme. The radioimmunoassay has been used to look for hypoxanthine phosphoribosyltransferase crossreacting material in hemolysates from sixteen different patients with a marked genetic deficiency of this enzyme characteristic of the Lesch-Nyhan syndrome. Fifteen hemolysates contained no detectable (less than 1% of normal) crossreacting material. One hemolysate contained a normal amount of crossreacting material. Hypoxanthine phosphoribosyltransferase from this patient (E.S.) has been shown to be a Km mutant enzyme.
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PMID:Radioimmune determination of hypoxanthine phosphoribosyltransferase crossreacting material in erythrocytes of Lesch-Nyhan patients. 106 95

We have previously reported that lead(II) is weakly mutagenic to Chinese hamster V79 cells. A transgenic cell line G12 containing a single copy of the E. coli gpt gene was developed in this laboratory from Chinese hamster V79 cells. The gpt locus in the G12 cells is more mutable by radiation and oxidative agents compared with the endogenous hprt locus of wild-type V79 cells. We have investigated the mutagenicity of two lead compounds at the gpt locus in G12 cells. Only at a toxic dose is lead acetate significantly mutagenic to G12 cells. Lead nitrate is not significantly mutagenic at any dose. Although both compounds are water-soluble, lead acetate, but not lead nitrate, forms a fine white insoluble precipitate upon addition to growth medium. A nick translation assay on cells treated with lead compounds and then permeabilized indicated that lead nitrate and, to a greater extent, lead acetate causes the appearance of nicks in chromosomal DNA. Lead ions in the presence of hydrogen peroxide, but not alone, introduced nicks into supercoiled plasmid DNA in vitro, suggesting that lead ions can partake in a Fenton reaction and thereby damage DNA. At lower nonmutagenic concentrations, lead acetate enhances the mutagenicity of MNNG and ultraviolet light. DNA damage by ultraviolet light is not enhanced by lead ions in vitro. Our data support the concept that non-toxic concentrations of lead(II) can inhibit DNA repair. Thus, at biologically relevant doses, lead(II) could act as a comutagen and possibly a cocarcinogen, but is not likely to act as an initiating genotoxic carcinogen.
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PMID:Mutagenesis and comutagenesis by lead compounds. 128 17

To test the hypothesis that reactive species in the oxygen cascade are responsible for spontaneous mutation, we examined the spectra of oxygen and hydrogen peroxide-induced mutations at the hprt locus in a human B-lymphoblastoid cell line. We compared these spectra with the spontaneous mutational spectrum. Large gene alterations were studied by Southern analysis of individual TGR clones. A combination of high fidelity polymerase chain reaction, denaturing gradient gel electrophoresis and direct DNA sequencing were used to detect and identify point mutations in exon 3 of hprt. With regard to spontaneous mutations, a previous study showed that 39% of the spontaneous TGR clones had large gene alterations. In the present study, the analysis of spontaneous point mutations within exon 3 revealed two hotspots. A one base-pair deletion (-A) at base-pair 256 or 257 and a two base-pair deletion (-GG) at base-pair 237 and 238, were detected in triplicate cultures. Each of the hotspots comprised about 1% of the TGR mutants. The analysis of individual oxygen-induced TGR clones (48 h, 910 microM-O2) showed 43% had large gene alterations similar to the spontaneous TGR clones. However, none of the spontaneous point mutation hotspots was found among triplicate oxygen-treated cultures. Two point mutations in common with H2O2-treated cultures were found in one of the three oxygen-treated cultures. Hydrogen peroxide-induced mutations (1 h, 20 microM) also differed from spontaneous mutations. Only 24% of the hydrogen peroxide-induced TGR clones had large gene alterations. The analysis of point mutations showed three hotspots within exon 3 of hprt. An AT to TA transversion at base-pair 259 had an average frequency of 3% of all TGR mutants (present in all of 3 H2O2-treated cultures). Two GC to CG transversions at base-pairs 243 and 202 were present at a frequency of 0.6% and 0.4%, respectively. A five base-pair deletion (base-pair 274 to 278) was present at an average frequency of 0.3%. The latter three mutations were detected in two of three H2O2-treated cultures. Thus, the point mutation spectra of both oxygen and hydrogen peroxide were significantly different from the spontaneous spectrum. The oxygen and hydrogen peroxide-induced spectra shared some features, suggesting that oxygen and hydrogen peroxide share some but not all pathways for induction of mutations within the DNA sequence studied here.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Mutational spectra in human B-cells. Spontaneous, oxygen and hydrogen peroxide-induced mutations at the hprt gene. 146 15

We have studied the mutagenicity and toxicity of physical and chemical agents in the Chinese hamster ovary (CHO) cell line K1-BH4 and its transformant, AS52. The AS52 cells lack the normal X-linked mammalian hypoxanthine-guanine phosphoribosyltransferase (hprt) gene but instead contain a single autosomally integrated copy of the bacterial equivalent, the xanthine-guanine phosphoribosyltransferase (gpt) gene. We found that X-rays and neutrons appear to be equitoxic to both cell types; however, these physical agents are approximately 10 times more mutagenic to the gpt gene of AS52 cells than to the hprt gene of K1-BH4 cells. We reasoned that if reactive oxygens were to mediate the mutagenic effects of both radiomimetic chemicals and radiation, then reactive oxygen-producing chemicals, such as streptonigrin and bleomycin, and oxidizing agents such as potassium superoxide and hydrogen peroxide, would exhibit similar levels of toxicity but different frequencies of mutants when assayed with the two cell lines. Our experiments fulfill such predictions. We postulate that the apparent hypermutability of AS52 cells probably results from a higher recovery of multi-locus deletion mutants in AS52 cells than in K1-BH4 cells, rather than a higher yield of induced mutants. Preliminary studies, using Southern blot and the polymerase chain reaction to analyze the mutational spectrum of the mutants, support our hypothesis that reactive oxygens induce deletion mutations in mammalian cells.
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PMID:Molecular analysis of reactive oxygen-species-induced mammalian gene mutation. 197 50

Chromium(VI) resistant Chinese hamster ovary (CHO) cell lines were established in this study by exposing parental CHO-K1 cells to sequential increases in CrO3 concentration. The final concentration of CrO3 used for selection was 7 microM for Cr7 and 16 microM for Cr16 cells. Cr16-1 was a subclone derived from Cr16 cells. Next, these resistant cells were cultured in media without CrO3 for more than 6 months. The resistance of these cells to CrO3 was determined by colony-forming ability following a 24-h treatment. The LD50 of CrO3 for chromium(VI) resistant cells was at least 25-fold higher than that of the parental cells. The cellular growth rate, chromosome number, and the hprt mutation frequency of these chromium(VI) resistant cells were quite similar to their parental cells. The glutathione level, glutathione S-transferase, catalase activity, and metallothionine mRNA level in Cr7 and Cr16-1 cells were not significantly different from their parental cells. Furthermore, Cr16-1 cells were as sensitive as CHO-K1 cells to free-radical generating agents, including hydrogen peroxide, nickel chloride, and methanesulfonate methyl ester, and emetine, i.e., a protein synthesis inhibitor. The uptake of chromium(VI) and the remaining amount of this metal in these resistant and the parental cell lines were assayed by atomic absorption spectrophotometry. Experimental results indicated that a vastly smaller amount of CrO3 entered the resistant cell lines than their parental cells did. A comparison was made of the sulfate uptake abilities of CHO-K1 and chromium(VI) resistant cell lines. These results revealed that the uptake of sulfate anion was substantially reduced in Cr7 and Cr16-1 cells. Extracellular chloride reduced sulfate uptake in CHO-K1 but not in Cr16-1 cells. Therefore, the major causative for chromium(VI) resistance in these resistant cells could possibly be due to the defects in SO4(2-)/C1- transport system for uptake chromium(VI).
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PMID:Long-term exposure to chromium(VI) oxide leads to defects in sulfate transport system in Chinese hamster ovary cells. 761 50

A large number of hprt-mutants were obtained by treating human lymphoblast cells (TK6) with 5 microM K2Cr2O7 for 5 hr and selecting by growth in 6-thioguanine. A combination of high fidelity polymerase chain reaction (PCR) and denaturing gradient gel electrophoresis (DGGE) allowed us to measure mutant frequencies as a function of DNA sequence. Chromium(VI) induced four hotspots in a 104 bp domain of hprt exon 3. Substitutions at G:C base pairs were the predominant mutations. One of the chromium-induced hotspots was located at the same position as previously determined hydrogen peroxide and benzo(a)pyrene diol epoxide hotspots.
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PMID:Use of denaturing-gradient gel electrophoresis to study chromium-induced point mutations in human cells. 784 3

In the present study 34 agents, related to carcinogenesis in different ways, were investigated with respect to their recombinogenic activity in mammalian cells. The induction of intrachromosomal recombination was studied using the spontaneous mutant clone SP5 derived from V79 Chinese hamster cells, which exhibits a duplication of exon 2 and its flanking regions in the hprt gene, which was found to be inserted between the two EcoR1 sites of intron 1. Earlier studies on the removal of this insertion fragment in the SP5 clone indicated that such loss involved intrachromosomal recombination and was detectable by using a reversion mutation assay. The categories of agents investigated here included monofunctional alkylating agents, polyaromatic hydrocarbons giving rise to bulky adducts, chlorinated compounds giving small cyclic adducts, intercalating agents, DNA cross-linkers, UV and ionizing radiation, inhibitors of DNA synthesis and topoisomerases, DNA bases and base analogues, radical formers and tumour promotors. Statistically significant enhancements in the frequency of reversion in SP5 cells were observed after treatment with aflatoxin B1, 9-aminoacridine, benzo[a]-pyrene-7,8-dihydrodiol, benzo[a]pyrene-7,8-diol-9,10-epoxide, camptothecin, dimethylbenzanthracene, dimethyl-nitrosamine, ethidium bromide, ethylmethanesulfonate, N-ethyl-N'-nitrosourea, fluorodeoxyuridine, ICR 191, N-methyl-N'-nitrosoguanidine, mitomycin C and UV irradiation. Only slight inducing effects were indicated in the case of methylmethanesulfonate, N-methyl-N'-nitrosourea and gamma irradiation, although not statistically significant. Negative results were found after treatment with 3-amino-benzamide, 5-azacytidine, bleomycin, 5-bromodeoxyuridine, 1,2-dichloroethane, ethylene oxide, etoposide, formaldehyde, hydrogen peroxide, methotrexate, propylene oxide, quercitin, sodium azide, 12-O-tetradecanoylphorbol-13-acetate, thymidine and a complex mixture consisting of a cigarette smoke condensate. Our results on chemically or physically induced recombinogenic effects in the endogenous SP5 hprt gene are in agreement with data obtained in non-endogenous gene systems based on transgenic cell lines containing integrates of tandem mutated tk or hygromycin resistance genes, but not completely consistent with findings on integrates based on the neo genes. This suggests that many factors influence the recombination process, including a difference in the mechanisms underlying inter- and intrachromosomal recombination. Consequently, the endogenous SP5/V79 system is suggested to be more representative than integrated systems for investigating induction of recombination, as well as for mechanistic studies of recombination at the molecular level, e.g. intrachromosomal recombination.
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PMID:Studies on intrachromosomal recombination in SP5/V79 Chinese hamster cells upon exposure to different agents related to carcinogenesis. 795 71

The crystal structure of HGPRTase with bound GMP has been determined and refined to 2.5 A resolution. The enzyme has a core alpha/beta structure resembling the nucleotide-binding fold of dehydrogenases, and a second lobe composed of residues from the amino and carboxy termini. The GMP molecule binds in an anti conformation in a solvent-exposed cleft of the enzyme. Lys-165, which forms a hydrogen bond to O6 of GMP, appears to be critical for determining the specificity for guanine and hypoxanthine over adenine. The location of active site residues also provides evidence for a possible mechanism for general base-assisted HGPRTase catalysis. A rationalization of the effects on stability and activity of naturally occurring single amino acid mutations of HGPRTase is presented, including a discussion of several mutations at the active site that lead to Lesch-Nyhan syndrome.
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PMID:The crystal structure of human hypoxanthine-guanine phosphoribosyltransferase with bound GMP. 804 44

Mammalian cells in culture have been used to study the genetic effects of physical and chemical agents. We have used Chinese hamster ovary (CHO) cells, clone K1-BH4, to quantify mutations at the X-linked, large (35 kb) hypoxanthine-guanine phosphoribosyltransferase (hprt) locus (the CHO/HPRT assay) induced by environmental agents. By transfecting an hprt-deletion mutant CHO cell line with the plasmid vector pSV2gpt, we isolated a transformant, AS52. AS52 cells carry a single functional copy of an autosomal, small (456 bp) xanthine-guanine phosphoribosyltransferase (gpt) gene (the bacterial equivalent of the mammalian hprt gene; AS52/GPT assay). We found that ionizing radiations such as X-rays and neutrons and oxidative genotoxic chemicals such as Adriamycin, bleomycin, hydrogen peroxide, and potassium superoxide are much more mutagenic to the gpt gene in AS52 cells than to the hprt locus in K1-BH4 cells. The hypermutability of the gpt gene probably results from a higher recovery of multilocus deletion mutants in AS52 cells than in K1-BH4 cells, rather than a higher yield of induced mutants. These results demonstrate that the use of the hprt locus alone could lead to an underestimate of the genetic risk of these agents. Analyses of the mutation spectrum using a polymerase chain reaction-based deletion screening and DNA sequencing procedure showed that a high proportion of HPRT- and GPT- mutants induced by X-rays carry deletion mutations. Thus, both the mutant frequency and mutation spectrum need to be considered in assessing the genetic risk of ionizing radiation and oxidative genotoxic chemicals.
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PMID:Quantitative and molecular analyses of genetic risk: a study with ionizing radiation. 814 20

Phosphoribosyltransferases (PRTases) are enzymes involved in the synthesis of purine, pyrimidine, and pyridine nucleotides. They utilize alpha-D-5-phosphoribosyl-1-pyrophosphate (PRPP) and a nitrogenous base to form a beta-N-riboside monophosphate and pyrophosphate (PPi), and their functional significance in nucleotide homeostasis is evidenced by the devastating effects of inherited diseases associated with the decreased activity and/or stability of these enzymes. The 2.6-A structure of the Salmonella typhimurium orotate phosphoribosyltransferase (OPRTase) complexed with its product orotidine monophosphate (OMP) provides the first detailed image of a member of this group of enzymes. The OPRTase three-dimensional structure was solved using multiple isomorphous replacement methods and reveals two major features: a core five-stranded alpha/beta twisted sheet and an N-terminal region that partially covers the C-terminal portion of the core. PRTases show a very high degree of base specificity. In OPRTase, this is determined by steric constraints and the position of hydrogen bond donors/acceptors of a solvent-inaccessible crevice where the orotate ring of bound OMP resides. Crystalline OPRTase is a dimer, with catalytically important residues from each subunit available to the neighboring subunit, suggesting that oligomerization is necessary for its activity. On the basis of the presence of a common PRPP binding motif among PRTases and the similar chemistry these enzymes perform, we propose that the alpha/beta core found in OPRTase will represent a common feature for PRTases. This generality is demonstrated by construction of a model of the human hypoxanthine-guanine phosphoribosyltransferase (HGPRTase) from secondary structure predictions for HGPRTase and the three-dimensional structure of OPRTase.
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PMID:Crystal structure of orotate phosphoribosyltransferase. 831 45


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