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

---

Query: EC:2.4.2.8 (hypoxanthine-guanine phosphoribosyltransferase)
2,527 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

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.
...
PMID:Tryptic peptide analysis of normal and mutant forms of hypoxanthine phosphoribosyltransferase from HeLa cells. 26 86

Hypoxanthine phosphoribosyltransferase (IMP:pryophosphate phosphoribosyltransferase, EC 2.4.2.8) from human erythrocytes has been purified 13 000-fold to apparent homogeneity. The native enzyme has a sedimentation coefficient of 5.9 S, determined by analytical ultracentrifugation, and a molecular weight of 81 000-83 000, determined by sedimentation equilibrium centrifugation. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicates a subunit molecular weight of 26 000, suggesting that the enzyme is a trimer. Isoelectric focusing resolves three peaks of enzyme activity at pH 5.6, 5.7 and 5.9. The amino acid composition of hypoxanthine phosphoribosyltrasferase is 17 Lys, 5 His, 12 Arg, 0 Trp, 31 Asx, 12 Thr, 14 Ser, 16 Glx, 14 Pro, 19 Gly, 12 Ala, 5 Cys, 18 Val, 5 Met, 11 Ile, 20 Leu, 10 Tyr, and 9 Phe. The enzyme appears to have a blocked N terminus.
...
PMID:Human hypoxanthine phosphoribosyltransferase. Purification and properties. 86 Dec 17

Due to the lack of de novo purine nucleotide biosynthesis, hypoxanthine-guanine phosphoribosyltransferase (HGPRTase) is an essential enzyme in the human parasite Schistosoma mansoni for supplying guanine nucleotides and has been proposed as a potential target for antiparasitic chemotherapy. While the enzyme can be purified from adult schistosome worms, yields are too low to allow extensive structural and kinetic studies. We therefore cloned and sequenced the cDNA and gene encoding the schistosomal enzyme but were unable to positively identify the amino-terminal sequence of the enzyme from the DNA sequence. Knowledge of the exact amino terminus was necessary before accurate expression of active enzyme could be attempted. Therefore, we purified the HGPRTase from crude extracts of the adult worms. The purified enzyme has a subunit molecular mass of 26 kDa and an amino-terminal sequence of Met-Ser-Ser-Asn-Met. This sequence matched one of the potential initiation sites predicted from the cDNA and gene sequence. We next expressed the correct size cDNA of the S. mansoni HGPRTase in Escherichia coli using a vector that is regulated by a bacterial alkaline phosphatase promoter and uses an E. coli signal peptide for secretion of expressed product into the periplasmic space. Using this expression system, some of the recombinant enzyme is secreted and found to have a correct amino terminus. That remaining in the cytoplasm has part of the signal peptide attached to the amino terminus. The recombinant schistosomal HGPRTase isolated from the periplasm of the transformed E. coli was purified and found to have kinetic and physical properties identical to those of the native enzyme.
...
PMID:The hypoxanthine-guanine phosphoribosyltransferase of Schistosoma mansoni. Further characterization and gene expression in Escherichia coli. 219 39

A plasmid, pRG1, has been constructed by incorporating the coding sequence of human hypoxanthine-guanine phosphoribosyltransferase (HPRT) into the expression vector pT7-7. Expression of human HPRT has been achieved in HPRT- Escherichia coli cells transformed with pRG1 and pGP1-2, as shown by: (1) exclusive labelling with [35S]methionine of a polypeptide with the same mobility as purified human HPRT on SDS-PAGE; and (2) measurement of HPRT activity after cell lysis. Although the majority of the recombinant HPRT was present in the particulate fraction after cell lysis and centrifugation, sufficient HPRT activity was present in the supernatant fraction to allow comparison with the HPRT purified from human erythrocytes and the activity in human haemolysates and lymphoblast lysates. Small differences in electrophoretic mobility on native gels were found between HPRT activity from these sources. The Km values of recombinant HPRT for the substrates 5-phospho-alpha-D-ribosyl-1-pyrophosphate and guanine were compared with those of lymphoblast and erythrocyte HPRT.
...
PMID:Expression of active human hypoxanthine-guanine phosphoribosyltransferase in Escherichia coli and characterisation of the recombinant enzyme. 222 82

The isoenzyme of hypoxanthine-guanine phosphoribosyltransferase (HPRT, E.C.2.4.2.8) functions in the metabolic salvage of purines. Partial HPRT deficiency is associated with gouty arthritis, while absence of activity results in Lesch-Nyhan (LN) syndrome. We characterized five unrelated patients with HPRT deficiency to understand the spectrum of molecular defects using Southern and Northern blot, polymerase chain amplification of HPRT mRNA and DNA sequencing, and oligonucleotide hybridization analysis of the HPRT gene. Southern blot analysis of DNA indicated that mutations leading to HPRT deficiency in our five patients were not the result of major chromosomal rearrangements or deletions. Sequencing analysis of the amplified DNA from three different patients with HPRT deficiency implied three unique molecular abnormalities: 1) one single-base substitution at codon 54 (from ATG to CTG) resulting in the replacement of methionine with leucine in an LN patient, 2) two single-base substitutions at codon 179 (from GTT to GGT) and at codon 180 (from GGA to AGA) resulting in the replacement of valine with glycine and glycine with arginine in a gouty patient, and 3) 51 nucleotide deletion between nucleotides 747 and 797 resulting in the formation of shorter sized HPRT mRNA and putative two amino-acid deleted HPRT protein in another gouty patient. These results are the direct molecular evidence of genetic heterogeneity in mutant HPRT.
...
PMID:Molecular analysis of hypoxanthine-guanine phosphoribosyltransferase mutations in five unrelated Japanese patients. 257 41

Human HPRT deficiency leads to two major forms of human disease. Partial enzyme deficiency results in gouty arthritis, while an almost complete deficiency leads to the Lesch-Nyhan disease. The latter is characterized by severe neurological dysfunction in addition to gouty arthritis, including retardation, choreoathetosis and aggressive and compulsive self-mutilation. The biochemical basis for the neurological symptoms is not understood. The human and mouse cDNA (RNA copy) genes have been isolated and sequenced. In addition, the amino acid sequence of the human protein has been directly determined. The human and mouse proteins differ at 7 amino acids out of the total, (including the N terminal methionine, which is processed off during maturation) of 218. There are 42 out of 654 nucleotide differences between the human and mouse genes in the amino acid coding region. The mouse genomic structure has been determined. It has 9 exons and 8 introns with a total size of approximately 36 kb. The human gene is very similar with identical intron-exon junction points and approximately the same total gene size. Both mouse and human presumed promotor region at the 5' end, lack a recognizable promotor in the form of a "TATAA" box and are very G-C rich, though not the same. This may be a feature of most "housekeeping" genes. HPRT gene point mutations in three gouty arthritis and one Lesch-Nyhan patient have been identified by peptide sequencing. Six gross gene rearrangements have been identified in Lesch-Nyhan HPRT genes. However it is likely that most mutations are point mutations or small deletions. So far all gene mutations identified are different from all others. The gene has been engineered into retrovirus vehicles which allows its efficient introduction into a wide variety of cells, including mouse marrow stem cells. This may allow treatment of Lesch-Nyhan patients as a model of gene therapy.
...
PMID:The role of the HPRT gene in human disease. 287 30

HPRT Ann Arbor is a variant of hypoxanthine (guanine) phosphoribosyl-transferase (HPRT: EC 2.4.2.8), which was identified in wo brothers with hyperuricemia and nephrolithiasis. In previous studies, this mutant enzyme was characterized by an increased Km for both substrates, a normal Vmax, a decreased intracellular concentration of enzyme protein, a normal subunit molecular weight and an acidic isoelectric point under native isoelectric focusing conditions. We have cloned a full-length cDNA for HPRT Ann Arbor and determined its complete nucleotide sequence. A single nucleotide change (T----G) at nucleotide position 396 has been identified. This transversion predicts an amino acid substitution from isoleucine (ATT) to methionine (ATG) in codon 132, which is located within the putative 5'-phosphoribosyl-1-pyrophosphate (PRPP)-binding site of HPRT.
...
PMID:Identification of a single nucleotide change in a mutant gene for hypoxanthine-guanine phosphoribosyltransferase (HPRT Ann Arbor). 289 20

The results of our previous studies suggested that differences in the primary structures of the hypoxanthine phosphoribosyltransferase (HPRT) A and B proteins (EC 2.4.2.8) of mice are associated with altered turnover of these proteins in reticulocytes. On the basis of nucleotide sequence comparisons of their corresponding cDNAs, we show here that the HPRT A and B proteins differ at two positions; there is an alanine/proline substitution at amino acid position 2 and a valine/alanine substitution at amino acid position 29 (HPRT A/B proteins, respectively; total protein length, 218 amino acids). On the basis of results obtained from sequencing of the N termini of the purified HPRT A and B proteins, we also show that these amino acid substitutions are associated with differences in processing of the proteins; HPRT B, which is encoded as N-terminal Met-Pro, has a free N-terminal proline residue; HPRT A, which is encoded as N-terminal Met-Ala, lacks a free N-terminal alpha-amino group and is presumed to be acetylated following removal of the N-terminal methionine (i.e. AcO-Ala). These observations are discussed in reference to the idea that the N terminus of a protein plays a role in determining the rate at which the protein is degraded in erythroid cells.
...
PMID:Altered turnover of allelic variants of hypoxanthine phosphoribosyltransferase is associated with N-terminal amino acid sequence variation. 337 61

We defined the amino acid sequence of adenine phosphoribosyltransferase isolated from human erythrocytes. Peptide fragments formed by cleavage at arginine, lysine, glutamic acid, and methionine were purified by high pressure liquid chromatography and sequenced by manual Edman degradation. The complete primary structure of human adenine phosphoribosyltransferase was established by sequence analysis of 19 peptide fragments. Presumed homology between the human and rodent enzymes was used to order fragments that had inadequate overlapping sequences. The enzyme has 179 residues with a calculated subunit molecular weight of 19,481. Mass spectrometry indicated that the NH2-terminal residue is acetylated. Human adenine phosphoribosyltransferase has sequence homology with xanthine-guanine phosphoribosyltransferase from Escherichia coli in 110-amino acid region encompassing the NH2-terminal section of the enzyme.
...
PMID:Human adenine phosphoribosyltransferase. Complete amino acid sequence of the erythrocyte enzyme. 353 Dec 9

The entire amino acid sequence of hypoxanthine-guanine phosphoribosyltransferase from human erythrocytes has been defined. Peptide fragments formed by cleavage at arginine, glutamic acid, and methionine residues were analyzed by Edman degradation or digestion with carboxypeptidase. The complete primary structure of human hypoxanthine-guanine phosphoribosyltransferase was established by sequence analysis of 17 peptide fragments, 15 of which were purified by reverse-phase high pressure liquid chromatography. The enzyme is 217 residues long with a molecular weight equal to 24,470. Mass spectroscopy indicated that the NH2-terminal alanine is acetylated.
...
PMID:Human hypoxanthine-guanine phosphoribosyltransferase. Complete amino acid sequence of the erythrocyte enzyme. 710 41


1 2 Next >>

---