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)

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

Inactivation of hypoxanthine phosphoribosyltransferase caused by periodate-oxidized GMP is irreversible, even under the conditions of polyacrylamide gel electrophoresis and during affinity chromatography on GMP-Sepharose. Partial binding of the inhibitor to the enzyme protein can be demonstrated on dodecyl sulfate gel electrophoresis: The substrate, phosphoribosyl diphosphate in the presence of Mg2, and the product GMP protect the enzyme against inactivation. Periodate-oxidized GMP, AMP and oxidized purine nucleosides do not influence ribosephosphate pyrophosphokinase, 5'-nucleotidase, purine-nucleoside phosphorylase and guanylate kinase. A variety of other purine nucleosides and nucleotides, tested in their periodateoxidized form, do not lead to a compound comparable or superior to oxidized GMP in its effect on hypoxanthine phosphoribosyltransferase. In an erythrocyte system it is clearly demonstrated that oxidized GMP cannot act across an intact cell membrane.
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PMID:Irreversible inhibition of hypoxanthine phosphoribosyltransferase. Further studies on the specificity of periodate-oxidized GMP. 20 May 44

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

Human hypoxanthine/guanine phosphoribosyltransferase (EC 2.4.2.8) was purified from red blood cells by the following two methods. Method A includes (a) elimination of hemoglobin by DEAE-cellulose, (b) DEAE-Sephadex chromatography, (c) specific elution of the enzyme from CM-Sephadex by pyrophosphate and (d) Sephadex G-100 gel filtration. Method B includes (a) elimination of hemoglobin by DEAE-cellulose, (b) acid treatment at pH 4.5, (c) ammonium sulfate fractionation, (d) DEAE-Sephadex chromatography, (e) heat treatment at 85 degrees C and (f) Sephadex G-100 gel filtration. Homogeneous enzyme preparation was obtained by the two methods with 8000-9000-fold purification. The sedimentation coefficient (S20,w) was 5.5--5.6 S, and the molecular weight of the enzyme was estimated at about 85000 by the sedimentation equilibrium method. The subunit molecular weight of the untreated protein and S-carboxymethylmaleyl protein was estimated as 41000--45000 by the sedimentation equilibrium method in the presence of guanidine hydrochloride. However, the subunit size estimated by the sodium dodecylsulfate gel electrophoresis was only 26000. Amino acid composition of the enzyme was determined. Glucosamine, sialic acid and hexose were not detected in the enzyme.
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PMID:Purification and characterization of human hypoxanthine/guanine phosphoribosyltransferase. 88 Sep 43

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 adenine phosphoribosyltransferase (APRTase) and hypoxanthine-guanine phosphoribosyltransferase (HGPRTase) activities from promastigotes of Leishmania donovani have been purified to homogeneity using ammonium sulfate precipitation, DEAE-cellulose exclusion, and either AMP-agarose (APRTase) or GTP-agarose (HGPRTase) affinity chromatography. The specific activities of the affinity-purified APRTase and HGPRTase fractions were 326-fold and 1341-fold greater than those in the 40-80% ammonium sulfate precipitate, respectively. The purified APRTase migrated as a single band on sodium dodecyl sulfate (SDS) polyacrylamide gels with a size of 29 kDa, while HGPRTase was also determined to be homogeneous by SDS gel electrophoresis with a size of 24 kDa. In addition, a mutant cell line, APPB2, partially deficient in APRTase activity, still contained quantities of purifiable APRTase protein, while a clonal secondary derivative of the APPB2 cell line that is completely deficient in APRTase activity, APPB2-640A3, failed to express purifiable APRTase protein. The homogeneous enzymes possessed apparent Km values for their nucleobase substrates between 2.0 and 5.0 microM, and both enzymes were inhibited by their immediate or ultimate reaction endproducts, APRTase by AMP and PPi and HGPRTase by GMP, GTP, and PPi. The generation of homogeneous preparations of APRTase and HGPRTase protein will serve as a prerequisite for the generation of immunological and molecular biological probes to analyze the leishmanial phosphoribosyltransferases.
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PMID:Purification and characterization of the adenine phosphoribosyltransferase and hypoxanthine-guanine phosphoribosyltransferase activities from Leishmania donovani. 270 89

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

A facile and rapid purification procedure, based upon the heat denaturation of extraneous proteins and GMP-Sepharose affinity chromatography, has been used to purify hypoxanthine phosphoribosyltransferase from human brain. A homogeneous enzyme preparation, as judged by sodium dodecyl sulfate and gradient polyacrylamide gel electrophoresis, was obtained. The subunit molecular weight of the enzyme was estimated as 24,000 by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The native molecular weight, determined by gradient gel electrophoresis, was approximately 100,000. These results suggest human brain hypoxanthine phosphoribosyltransferase is a tetramer, consistent with recent results reported for the human erythrocyte enzyme. At least three charge variant forms of the human brain enzyme were distinguished by nondenaturing polyacrylamide gel electrophoresis, electrofocusing, and chromatofocusing. Acidic pI values of approximately 5.7, 5.5, and 5.0 were estimated for the three major species.
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PMID:Hypoxanthine phosphoribosyltransferase from human brain: purification and partial characterization. 609 78

Somatic cell hybrid clones were derived from the fusion of hypoxanthine phosphoribosyltransferase (HPRT; EC 2.4.2.8)-deficient mouse cells and two different human fibroblast strains, each carrying an X chromosome-autosome translocation. One of these had an X/11 translocation [46,X,t(X;11)(p21;q13)] and the other had an X/19 translocation [46,X,t(X;19)(q22;q13)]. The structurally normal human X chromosome is the late-replicating (genetically inactive) chromosome in these two cell strains; the rearranged X chromosome is early replicating (genetically active). One primary hybrid clone carrying both the translocated X chromosome and the structurally normal X chromosome was isolated in hypoxanthine/aminopterin/thymidine medium from each of these two cell fusion experiments. These clones were then selected in medium containing 8-azaguanine to achieve the loss of the active human HPRT locus. Five subclones from the cell hybrid with the X/11 translocation failed to express two known human X-chromosome markers [glucose-6-phosphate dehydrogenase (G6PD; EC 1.1.1.49) and phosphoglycerate kinase (PGK; EC 2.7.2.3)] but did express human microsomal steroid sulfatase (STS; sterol-sulfate sulfohydrolase, EC 3.1.6.2). Three of these were cytogenetically analyzed and found to contain a structurally normal human X chromosome but not the X/11 translocation. Two subclones were isolated in 8-azaguanine from the hybrid with the X/19 translocation. Cytogenetic analysis of these two clones showed the presence of a structurally normal human X chromosome; the X/19 translocation was not present. They did not express human G6PD, PGK, or HPRT but did express human STS. These results indicate that human STS is expressed from a locus on the inactive human X chromosome and support our earlier finding that the STS locus escapes X-inactivation in man.
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PMID:Expression of an X-linked gene from an inactive human X chromosome in mouse-human hybrid cells: further evidence for the noninactivation of the steroid sulfatase locus in man. 693 82

Hypoxanthine-guanine phosphoribosyltransferase (EC 2.4.2.8) from beef brain has been purified 3100-fold to apparent homogeneity using a purification procedure based on GMP-Sepharose affinity chromatography. The native enzyme has a molecular weight of 84,000 as determined by gel filtration studies. A subunit molecular weight of 26,000 was obtained by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, suggesting that the enzyme is a trimer. Two forms of the enzyme have been separated by nondenaturing polyacrylamide gel electrophoresis and isoelectric focusing. Basic pI values of 7.85 and 8.10 were obtained for the two forms. These values are much higher than have been observed with any other purified phosphoribosyltransferase. The amino acid composition of the enzyme is 18 Lys, 6 His, 9 Arg, 1 Trp, 6 Cys, 28 Asx, 12 Thr, 16 Ser, 19 Glx, 10 Pro, 23 Gly, 16 Ala, 17 Val, 5 Met, 11 Ile, 19 Leu, 9 Tyr, and 8 Phe. An unusual basic amino acid, yet to be identified, was also present. The enzyme exhibits Km values of 0.42 microM for guanine, 0.99 microM for hypoxanthine, 18.6 microM for P-Rib-PP in the presence of guanine, and 2.9 microM for P-Rib-PP in the presence of hypoxanthine.
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PMID:Studies of an unusually basic hypoxanthine-guanine phosphoribosyltransferase. 735 77


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