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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)

Imbalances in the intracellular nucleotide precursor pools in mammalian cells can result in the induction of mutations during the DNA replication process. By using a shuttle vector system developed in our laboratory, we have analyzed the sequence specificity of mutations induced in mouse A9 cells by exposure of the cells to a high concentration of thymidine. The target for mutagenesis in these studies was the bacterial gpt gene stably integrated into the chromosomal DNA of the mouse cells. Previous studies in this laboratory had generated a large panel of xanthine guanine phosphoribosyl-transferase (EC 2.4.2.22)-negative mutant lines that possess single-base mutations within the gpt coding sequence. This study utilized four xanthine guanine phosphoribosyltransferase-negative mutant lines to assess the frequency of mutation induced by thymidine at guanine residues in four sequence contexts: the 5' and 3' guanine residues of a GG doublet, the middle guanine residue of a GGG triplet, and the 3' guanine residue of a GGGG quartet. The results of this study demonstrate that treatment of cultured cells with a high concentration of thymidine can result in G.C----A.T transition mutations that occur preferentially at the 3' guanine residue of a run of two or more adjacent guanines. Guanine residues flanked on their 3' side by other guanine residues are severalfold less mutable by thymidine than are guanine residues flanked on their 3' side by a different base. This study demonstrates a sequence-specific mode for thymidine-induced mutations and suggests implications for mutagenesis in vivo.
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PMID:Thymidine-induced mutations in mammalian cells: sequence specificity and implications for mutagenesis in vivo. 155 89

The enzymes that catalyse the salvage of purines in Entamoeba histolytica trophozoites have been surveyed. Adenine deaminase (EC 3.5.4.2), adenosine deaminase (EC 3.5.4.4), guanine deaminase (EC 3.5.4.3), adenine phosphoribosyltransferase (PRTase) (EC 2.4.2.7), xanthine PRTase (EC 2.4.2.22) and hypoxanthine PRTase (EC 2.4.2.8) were all detected in cell homogenates but only at low activities, whereas AMP deaminase (EC 3.5.4.6) and guanine PRTase (EC 2.4.2.8) were not found. Phosphorylases (EC 2.4.2.1) active in both anabolic and catabolic directions were present and all nucleosides tested were phosphorylated by kinases (EC 2.7.1.15, EC 2.7.1.20, EC 2.7.1.73). 3'-Nucleotidase (EC 3.1.3.6) and 5'-nucleotidase (EC 3.1.3.5) were found, the former being mainly particulate. Nucleotide interconversion enzymes (adenylosuccinate lyase, EC 4.3.2.2; adenylosuccinate synthetase, EC 6.3.4.4; IMP dehydrogenase, EC 1.2.1.14; GMP synthetase, EC 6.3.5.2 and GMP reductase, EC 1.6.6.8) were not detected. The results suggest that in E. histolytica the main route of nucleotide synthesis is from the individual bases through the actions of phosphorylases and kinases.
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PMID:Purine-metabolising enzymes in Entamoeba histolytica. 287 91

Extracts of Babesia divergens were examined for the enzymes which catalyse purine salvage. Adenosine deaminase (EC 3.5.4.4), guanine deaminase (EC 3.5.4.3), inosine phosphorylase (EC 2.4.2.1), purine phosphoribosyltransferases (EC 2.4.2.7, EC 2.4.2.8, EC 2.4.2.22) and nucleoside kinases (EC 2.7.1.15, EC 2.7.1.20, EC 2.7.1.73) were all detected at relatively high activities, whereas nucleotide interconverting enzymes were not detected. Coformycin and 4-amino-5-imidazolecarboxamide were found to be potent inhibitors of adenosine deaminase and guanine deaminase, respectively. The results suggest that B. divergens is capable of synthesizing purine nucleotides via two routes, one involving purine phosphoribosyltransferases and the other employing nucleoside kinases.
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PMID:Purine-metabolizing enzymes in Babesia divergens. 303 31

Giardia lamblia, a flagellated parasitic protozoan and the causative agent of giardiasis, lacks de novo purine biosynthesis and exists on salvage of adenine and guanine by adenine phosphoribosyltransferase and guanine phosphoribosyltransferase. Guanine phosphoribosyltransferase from G. lamblia crude extracts has been purified to apparent homogeneity by Sephacryl S-200 gel filtration followed by C-8-GMP-agarose and 2',3'-GMP-agarose affinity chromatography, resulting in an overall recovery of 77% and a purification of 83,000-fold. The molecular weight of the native enzyme as estimated by gel filtration and isokinetic sucrose gradients was found to be 58,000-63,000, with a subunit molecular weight of approximately 29,000, as shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Mono P chromatofocusing chromatography gives rise to a major activity peak eluting from the column at a pH of 6.75 and two minor activity peaks at pH of 5.3 and 5.2. Hypoxanthine and xanthine can be recognized by the enzyme as substrates but at Km values 20 times higher than that observed with guanine. G. lamblia guanine phosphoribosyltransferase is immunologically distinct from human hypoxanthine-guanine phosphoribosyltransferase and Escherichia coli xanthine-guanine phosphoribosyltransferase, and G. lamblia DNA fragments are incapable of hybridizing with mouse neuroblastoma hypoxanthine-guanine phosphoribosyltransferase DNA or E. coli xanthine phosphoribosyltransferase DNA under relatively relaxed conditions. All evidence presented suggests that G. lamblia guanine phosphoribosyltransferase may be qualified as a potential target for antigiardiasis chemotherapy.
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PMID:Purification and characterization of guanine phosphoribosyltransferase from Giardia lamblia. 308 75

Bacillus subtilis mutants defective in purine metabolism have been isolated by selecting for resistance to purine analogs. Mutants resistant to 2-fluoroadenine were found to be defective in adenine phosphoribosyltransferase (apt) activity and slightly impaired in adenine uptake. By making use of apt mutants and mutants defective in adenosine phosphorylase activity, it was shown that adenine deamination is an essential step in the conversion of both adenine and adenosine to guanine nucleotides. Mutants resistant to 8-azaguanine, pbuG mutants, appeared to be defective in hypoxanthine and guanine transport and normal in hypoxanthine-guanine phosphoribosyltransferase activity. Purine auxotrophic pbuG mutants grew in a concentration-dependent way on hypoxanthine, while normal growth was observed on inosine as the purine source. Inosine was taken up by a different transport system and utilized after conversion to hypoxanthine. Two mutants resistant to 8-azaxanthine were isolated: one was defective in xanthine phosphoribosyltransferase (xpt) activity and xanthine transport, and another had reduced GMP synthetase activity. The results obtained with the various mutants provide evidence for the existence of specific purine base transport systems. The genetic lesions causing the mutant phenotypes, apt, pbuG, and xpt, have been located on the B. subtilis linkage map at 243, 55, and 198 degrees, respectively.
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PMID:Genetic and physiological characterization of Bacillus subtilis mutants resistant to purine analogs. 311 Jan 31

1. The activities of the purine phosphoribosyltransferases (EC 2.4.2.7 and 2.4.2.8) in purine-analogue-resistant mutants of Schizosaccharomyces pombe were checked. An 8-azathioxanthine-resistant mutant lacked hypoxanthine phosphoribosyltransferase, xanthine phosphoribosyltransferase and guanine phosphoribosyltransferase activities (EC 2.4.2.8) and appeared to carry a single mutation. Two 2,6-diaminopurine-resistant mutants retained these activities but lacked adenine phosphoribosyltransferase activity (EC 2.4.2.7). This evidence, together with data on purification and heat-inactivation patterns of phosphoribosyltransferase activities towards the various purines, strongly suggests that there are two phosphoribosyltransferase enzymes for purine bases in Schiz. pombe, one active with adenine, the other with hypoxanthine, xanthine and guanine. 2. Neither growth-medium supplements of purines nor mutations on genes involved in the pathway for new biosynthesis of purine have any influence on the amount of hypoxanthine-xanthine-guanine phosphoribosyltransferase produced by this organism.
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PMID:The substrate specificity of purine phosphoribosyltransferases in Schizosaccharomyces pombe. 512 76

Myeloma, hybridoma, and thymoma cell lines have been successfully transfected for the Escherichia coli xanthine-guanine phosphoribosyltransferase gene (gpt) by using the plasmid vector pSV2-gpt. The transformed cells synthesize the bacterial enzyme 5-phospho-alpha-D-ribose-1-diphosphate:xanthine phosphoribosyltransferase (XGPRT; EC 2.4.2.22) and have been maintained in selective medium for over 4 months. Lymphoid cell lines expressing a K immunoglobulin light chain were obtained by transfecting cells with pSV2-gpt containing a rearranged K light chain genomic segment from the S107 myeloma cell line. The S107 light chain is synthesized in gpt-transformed J558L myeloma cells and is identical to the light chain synthesized by the S107 myeloma cell line, as judged by immunoprecipitation and two-dimensional gel electrophoresis. Furthermore, this light chain is synthesized and secreted as part of an intact antibody molecule by transformed hybridoma cells that normally secrete an IgGl (gamma, K) antibody molecule. No light chain synthesis was detected in a similarly transformed rat myeloma or a mouse thymoma line.
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PMID:Immunoglobulin gene expression in transformed lymphoid cells. 640 77

Cultured monkey (TC7) and mouse (3T6) cells synthesize an Excherichia coli enzyme, xanthine-guanine phosphoribosyltransferase (XGPRT; 5-phospho-alpha-D-ribose-1-diphosphate:xanthine phosphoribosyltransferase, EC 2.4.2.22), after transfection with DNA vectors carrying the corresponding bacterial gene, Ecogpt. In contrast to mammalian cells, which do not efficiently use xanthine for purine nucleotide synthesis, cells that produce E. coli XGPRT can synthesize GMP from xanthine via XMP. After transfection with vector-Ecogpt DNAs, surviving cells producing XGPRT can be selectively grown with xanthine as the sole precursor for guanine nucleotide formation in a medium containing inhibitors (aminopterin and mycophenolic acid) that block de novo purine nucleotide synthesis. Cells transformed for Ecogpt arise with a frequency of 10(-4) to 10(-5); they appear to be genetically stable in as much as there is no discernible decrease in XGPRT formation or loss on their ability to grow in selective medium after propagation in nonselective medium. Although several of the vector-gpt DNAs can replicate in monkey and mouse cells, none of the transformants contain autonomously replicating vector-gpt DNA. Rather, the gpt transformants contain one to five copies of the transfecting DNA associated with, and most probably integrated into, cellular DNA sequences. In several transformants, vector-coded gene products for which there was no selection are also synthesized. This suggests that recombinant DNAs containing Ecogpt as a selective marker can be useful for cotransformation of nonselectable genes.
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PMID:Selection for animal cells that express the Escherichia coli gene coding for xanthine-guanine phosphoribosyltransferase. 701 22

We have cloned and expressed the full-length gene encoding the hypoxanthine-guanine-xanthine phosphoribosyltransferase (HGXPRTase) from the anaerobic protozoan parasite Tritrichomonas foetus. This enzyme is essential in nucleic acid metabolism of T. foetus because the parasite is unable to synthesize purine nucleotides de novo and relies on the HGXPRTase activities for its purine requirements. Initially, a cDNA clone encoding part of the HGXPRTase was isolated by complementation of an Escherichia coli mutant, SO609, with a cDNA library of T. foetus. Northern blot analysis identified a single mRNA band of approximately 700-800 bases. The full-length genomic clone was then isolated and identified to have an open reading frame of 549 bp encoding an 183-amino acid sequence with an estimated size of 21.1 kDa. The sequence is only 27.3% identical to that of the human HGPRTase. The T. foetus HGXPRTase gene was subsequently cloned into the pBAce vector for expression in E. coli. This construct yields completely soluble and enzymatically active recombinant T. foetus HGXPRTase, which constitutes approximately 20% of the total cellular protein of the transformed E. coli. It has the same molecular weight as the authentic native enzyme, and the N-terminal amino acid sequence of the recombinant enzyme is identical to that predicted from the open reading frame. The high expression of this apparently native T. foetus HGXPRTase will provide large quantities of purified protein, necessary for detailed kinetic and structural analysis of this enzyme for its potential value as a target for antitrichomonial chemotherapy. To our knowledge, this is also the first time a gene from T. foetus was cloned and expressed.
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PMID:Isolation, sequencing and expression of the gene encoding hypoxanthine-guanine-xanthine phosphoribosyltransferase of Tritrichomonas foetus. 800 20

Tritrichomonas foetus, an anaerobic, flagellated protozoan parasite, is incapable of de novo purine nucleotide synthesis, and depends primarily on the salvage of purine bases from the host. The hypoxanthine-guanine-xanthine phosphoribosyltransferase (HGXPRTase) from this organism has been purified to homogeneity by ammonium sulfate precipitation and Sephacryl-HR100 gel filtration, followed by anion exchange FPLC. Hypoxanthine, guanine and xanthine phosphoribosyltransferase activities co-eluted in all the purification steps, suggesting that they are associated with the same enzyme protein. The molecular mass of the native protein, as estimated by gel filtration, is 24 kDa. The molecular mass estimated from sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) is also 24 kDa. Non-denaturing polyacrylamide gel electrophoresis of the purified protein, followed by activity staining with either [14C]hypoxanthine, [14C]guanine or [14C]xanthine, also demonstrates that the enzyme is a monomer of 24 kDa. This monomeric structure is distinctive from all the other reported PRTases which are either dimers or tetramers. Furthermore, unlike the mammalian HGPRTase, which is heat stable, the T. foetus enzyme is heat labile. Kinetic studies with the purified T. foetus HGXPRTase showed that the apparent Kms for hypoxanthine, guanine and xanthine were 4.1 microM, 3.8 microM and 52.4 microM respectively. This recognition of xanthine as a substrate by the parasite enzyme with only about a 10-fold higher Km value than those for hypoxanthine and guanine distinguishes it from the mammalian HGPRTase, which cannot use xanthine as a substrate, as well as the HGXPRTases of Eimeria tenella and Plasmodium falciparum, which are dimers, with xanthine about 100-times less proficient as a substrate.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:The hypoxanthine-guanine-xanthine phosphoribosyltransferase from Tritrichomonas foetus has unique properties. 823 11


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