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)

The bacterial alkaline phosphatase (phoA) promoter and signal peptide have been used previously to control recombinant expression and secretion of eukaryotic proteins in Escherichia coli. Other reports have shown that this expression system can generate relatively modest levels of active hypoxanthine/guanine phosphoribosyltransferase (HPRT; hypoxanthine phosphoribosyltransferase; IMP:pyrophosphate phosphoribosyltransferase, EC 2.4.2.8), which carries part of the signal peptide but remains in the cytosol of the bacteria. Herein, the phoA promoter without its associated signal peptide is used to regulate expression of the HPRT of Schistosoma mansoni and the ornithine decarboxylase (ODC; L-ornithine carboxy-lyase, EC 4.1.1.17) of Trypanosoma brucei, two enzymes that have been identified as potential targets for antiparasitic chemotherapy. The levels of recombinant expression range from 20% to 60% of the total bacterial protein, and the majority of both recombinant enzymes was soluble. The specific activity for the recombinant trypanosomal ODC was one-third to two-thirds that of the authentic native enzyme and yields were predicted to be 15-30 mg of active enzyme per liter of bacterial culture. The specific activity for the recombinant schistosomal HPRT was equivalent to that for the native enzyme purified from schistosomes and up to 10 mg of enzymatically active HPRT has been purified from a 0.5-liter culture of treated bacteria. These results represent a break-through in recombinant expression of HPRT and ODC.
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PMID:High level expression in Escherichia coli of soluble, enzymatically active schistosomal hypoxanthine/guanine phosphoribosyltransferase and trypanosomal ornithine decarboxylase. 200 85

The enzymatic pattern of five enzymes involved in the purine salvage pathway, namely purine nucleoside phosphorylase (EC 2.4.2.1), adenosine deaminase (EC 3.5.4.4), 5'-nucleotidase (EC 3.1.3.5), alkaline phosphatase (EC 3.1.3.1), and hypoxanthine-guanine phosphoribosyltransferase (EC 2.4.2.8) has been evaluated both in human intestinal and breast carcinomas and compared to that of normal tissues. A higher level of hypoxanthine-guanine phosphoribosyltransferase was associated with tumor tissues. This metabolic alteration should lead to an elevated synthesis of nucleotides in cancer cells, might confer selective growth advantages to neoplastic tissues, and account, at least in part, for the difficulties encountered in the chemotherapy of human tumors, by using compounds affecting only the purine de novo biosynthesis.
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PMID:Purine salvage enzyme activities in normal and neoplastic human tissues. 212 39

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.
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PMID:The hypoxanthine-guanine phosphoribosyltransferase of Schistosoma mansoni. Further characterization and gene expression in Escherichia coli. 219 39

Mercaptopurine (MP) is a purine antimetabolite widely used for remission maintenance in the therapy of acute lymphoblastic leukemia. In order to study the biochemical parameters affecting MP activity, leukemic cells were obtained from ten patients with acute lymphoblastic leukemia at the time of diagnosis and from the same patients at the time of their initial marrow relapse. Hypoxanthine phosphoribosyltransferase (HPRT), the enzyme that converts MP to its active, nucleotide metabolite, thioinosine monophosphate; alkaline phosphatase, the primary catabolic enzyme of thioinosine monophosphate; and 5-phosphoribosyl-1-pyrophosphate (PRPP), the cellular ribose-phosphate donor essential for MP activation, were all measured within the patients' leukemic cells. There was marked interpatient variability in the three biochemical parameters studied with a greater than 10-fold range in alkaline phosphatase activity and an approximately 100-fold range in HPRT activity and PRPP levels. Four patients developed changes in biochemical parameters that influence MP activity at the time of relapse. In three of the four patients, alterations in more than one of these three biochemical parameters were noted. Three of four patients had a greater than 50% decrease in intracellular HPRT activity, four of four had a greater than 50% decrease in intracellular PRPP, and two of four had a greater than 9-fold increase in intracellular alkaline phosphatase activity at relapse. Two of four patients demonstrated changes in all three parameters at relapse in the directions that could have resulted in decreased MP sensitivity (i.e., decreased HPRT, decreased PRPP, and increased alkaline phosphatase). There was no correlation between pretreatment values of HPRT, PRPP, and alkaline phosphatase and remission duration. These results indicate that: (a) there is marked variation in HPRT, PRPP, and alkaline phosphatase in patients with acute lymphoblastic leukemia and b) following MP-containing maintenance chemotherapy, some patients develop biochemical changes that may result in decreased sensitivity to MP.
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PMID:Biochemical parameters of mercaptopurine activity in patients with acute lymphoblastic leukemia. 241 5

Using density gradient centrifugation, human trophoblastic cells were enriched from mixed cell populations of enzymatically dispersed first- and third-trimester placentae. Over 95 per cent of the cells recovered were of epithelial (i.e., trophoblastic) origin, as evidenced by their cytokeratin intermediate filament positivity and vimentin negativity, examined using indirect immunofluorescence, and also by their high content of human chorionic gonadotrophin. The activities of key enzymes involved in purine degradation and re-utilization (5'-nucleotidase; AMP-deaminase; hypoxanthine phosphoribosyltransferase (HPRT); xanthine dehydrogenase/oxidase) as well as the total activity of alkaline phosphatase were measured in the trophoblastic cells. A six-fold increase in the trophoblastic alkaline phosphatase activity was noted between the first and third trimester. A 40 per cent decrease was noted in the activity of 5'-nucleotidase, which, on the basis of kinetic properties, appears to have a dominant role in the dephosphorylation of placental nucleoside-5'-monophosphates. The trophoblastic activities of AMP-deaminase, HPRT, and xanthine dehydrogenase/oxidase did not change as a function of the gestational age. In view of the relative activities of the latter two enzymes, hypoxanthine formed in the trophoblast appears more likely to be re-utilized than degraded to uric acid.
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PMID:Activities of key enzymes of purine degradation and re-utilization in human trophoblastic cells. 283 9

A screening method using high-performance liquid chromatography (HPLC) for the simultaneous detection of deficiencies of adenine phosphoribosyltransferase (APRT) and hypoxanthine phosphoribosyltransferase (HPRT) activities in human erythrocytes is described. Both enzyme reactions of APRT and HPRT in lysates treated with a charcoal-dextran were simultaneously carried out in the same reaction tube and the enzyme activities were determined by measuring the increases in absorbance at 260 nm of adenosine and inosine converted from adenosine-5'-monophosphate and inosine-5'-monophosphate with alkaline phosphatase. Adenosine and inosine were separated from adenine and hypoxanthine by a reversed-phase column. The method could detect 1% of normal APRT activity and 0.3% of normal HPRT activity. The within-run coefficients of variation for APRT and HPRT activities were 3.2 and 3.4%, respectively.
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PMID:Screening for adenine and hypoxanthine phosphoribosyltransferase deficiencies in human erythrocytes by high-performance liquid chromatography. 343 62

3-Deazaguanosine containing a 14C label in the ribose moiety was prepared using [U-14C]inosine as the [14C] ribose donor and commercial purine-nucleoside phosphorylase (EC 2.4.2.1) both to degrade the inosine, in the presence of phosphate, and to synthesize [14C-ribosyl]3-deazaguanosine in reduced phosphate and an excess of 3-deazaguanine. Purification was by high-pressure liquid chromatography (HPLC). [14C-ribosyl]3-Deazaguanosine was metabolized by Chinese hamster ovary cells to two metabolites, one major and one minor, eluting in the triphosphate region after HPLC analysis, and appeared to be incorporated into perchloric acid-insoluble material. Cell line TGR-3, deficient in hypoxanthine-guanine phosphoribosyltransferase (EC 2.4.2.8) and resistant to 3-deazaguanine, also formed both metabolites. Line TGR-1/DGRR-9, deficient in hypoxanthine-guanine phosphoribosyltransferase and resistant to both 3-deazaguanine and 3-deazaguanosine, formed greatly reduced levels of the major metabolite. 3-Deazaguanosine 5'-triphosphate, prepared enzymically from authentic 3-deazaguanosine 5'-monophosphate, co-eluted with the major metabolite peak during HPLC analysis. Treatment of a metabolite-containing extract with bacterial alkaline phosphatase (EC 3.1.3.1) resulted in the formation of 3-deazaguanosine. These observations indicate that 3-deazaguanosine can be metabolized, in Chinese hamster ovary cells, to the triphosphate derivative in lieu of the action of hypoxanthine-guanine phosphoribosyltransferase.
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PMID:3-Deazaguanosine is metabolized to the triphosphate derivative in Chinese hamster cells deficient in hypoxanthine-guanine phosphoribosyltransferase. 370 Mar 97

Hypoxanthine phosphoribosyltransferase-deficient (HPRT-) mouse embryonic stem (ES) cells, HM-1 cells (genotype XY), were fused with adult female DD/c mouse spleen cells. As a result, a set of HAT-resistant clones was isolated. Four hybrid clones most similar in morphology and growth characteristics to the HM-1 cells were studied in detail with respect to their pluripotency. Of these, three clones contained 41-43 chromosomes, and one clone was nearly tetraploid. All the clones had the XXY set of sex chromosomes and expressed the HPRT of the somatic partner only. The hybrid clones shared features with the HM-1 cells, indicating that they retained their pluripotent properties: (1) embryonic ECMA-7 antigen, not TROMA-1 antigen, was present in most cells; (2) the hybrid cells showed high activity of endogenous alkaline phosphatase (AP); (3) all the hybrid clones were able to form complex embryoid bodies containing derivatives of all the embryonic germinal layers; (4) the hybrid cells contained synchronously replicating X chromosomes, indicating that they were in an active state; and (5) a set of chimeric animals was generated by injecting hybrid cells into BALB/c and C57BL/6J mouse blastocysts. Evidence for chimerism was provided by the spotted coat derived from 129/Ola mice and identification of 129/Ola glucose phosphate isomerase (GPI) in many organs. Thus the results obtained demonstrated that the hybrid cells retain their high pluripotency level despite the close contact of the "pluripotent" HM-1 genome with the "somatic" spleen cell genome during hybrid cell formation and the presence of the "somatic" X chromosome during many cell generations. The presence of HPRT of the somatic partner in many organs and tissues, including the testes in chimeric animals, shows that the "somatic" X chromosome segregates weakly, if at all, during development of the chimeras. There were no individuals with the 129/Ola genotype among the more than 50 offspring from chimeric mice. The lack of the 129/Ola genotype is explained by the imbalance of the sex chromosomes in the hybrid cells rendering the passage of hybrid cell descendants through meiosis in chimeras impossible. As a result, chimeras become unable to produce gametes of the hybrid cell genotype.
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PMID:In vitro and in vivo study of pluripotency in intraspecific hybrid cells obtained by fusion of murine embryonic stem cells with splenocytes. 959 May 28