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 B lymphoblast lines severely deficient in hypoxanthine-guanine phosphoribosyltransferase (HGPRT) were selected for resistance to 6-thioguanine from cloned normal and phosphoribosylpyrophosphate (PP-Rib-P) synthetase-superactive cell lines and were compared with their respective parental cell lines with regard to growth and PP-Rib-P and purine nucleotide metabolism. During blockade of purine synthesis de novo with 6-methylthioinosine or aminopterin, inhibition of growth of all HGPRT-deficient cell lines was refractory to addition of Ade at concentrations which restored substantial growth to parental cell lines. Ade-resistant inhibition of growth of parental lines by 6-methylthioinosine, however, occurred during Ado deaminase inhibition. Insufficient generation of IMP (and ultimately guanylates) to support growth of lymphoblasts lacking HGPRT activity and blocked in purine synthesis de novo best explained these findings, implying that a major route of interconversion of AMP to IMP involves the reaction sequence: AMP----Ado----Ino----Hyp----IMP. PP-Rib-P generation and purine nucleoside triphosphate pools were unchanged by introduction of HGPRT deficiency into normal lymphoblast lines, in agreement with the view that accelerated purine synthesis de novo in this deficiency results from increased availability of PP-Rib-P for the pathway. Cell lines with dual enzyme defects did not differ from PP-Rib-P synthetase-superactive parental lines in rates of PP-Rib-P and purine synthesis despite 5-6-fold increases in PP-Rib-P concentrations, excretion of nearly 50% of newly synthesized purines, and diminished GTP concentrations. Fixed rates of purine synthesis de novo in PP-Rib-P synthetase-superactive cells appeared to reflect saturation of the rate-limiting amidophosphoribosyltransferase reaction for PP-Rib-P. In combination with accelerated purine excretion, increased channeling of newly formed purines into adenylates, and impaired conversion of AMP to IMP, fixed rates of purine synthesis de novo may condition cell lines with defects in HGPRT and PP-Rib-P synthetase to depletion of GTP with consequent growth retardation.
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PMID:Regulation of purine nucleotide synthesis in human B lymphoblasts with both hypoxanthine-guanine phosphoribosyltransferase deficiency and phosphoribosylpyrophosphate synthetase superactivity. 131 6

Adenine phosphoribosyltransferase (APRTase) and hypoxanthine-guanine phosphoribosyltransferase (HGPRTase) have been purified from Artemia cysts and nauplii to apparent homogeneity, as determined by SDS-PAGE. The purification includes affinity chromatography on AMP-Sepharose, which binds both enzymes, and they are eluted at different 5-phospho-alpha-D-ribosyl diphosphate (PP-Rib-P) concentrations. The purified enzymes from Artemia cysts were similar to nauplii enzymes with respect to Mr in denaturing gel electrophoresis and gel filtration, pH and cation dependence and kinetic constants for substrates and inhibitors. By Sephadex G-100 filtration, the native Mr of the adenine and hypoxanthine-guanine enzymes was estimated to be Mr 28,000 and 66,000, respectively. Analysis by SDS-PAGE revealed that the APRTase was a dimer of Mr 15,000 sub-units and the HGPRTase, a tetramer of four identical Mr 19,000 sub-units. The pH profile of the HGPRTase shows two apparent buffer-independent pH optima, at 7.0 and 9.5, while the APRTase has just one, at about pH 8-9. The purine phosphoribosyltransferase activity with adenine was highest, about tenfold the HGPRTase activity with hypoxanthine and fivefold that with guanine. Both enzymes exhibited similar requirements for divalent cations, either Mg2+, Mn2+ or Zn2+, while Ca2+ is highly inhibitory. The Km values of APRTase for adenine and PP-Rib-P are 2 and 30 microM, respectively, and the Km values of HGPRTase for hypoxanthine, guanine and PP-Rib-P are less than 1, less than 1 and 15 microM, respectively. Plots of the reciprocal enzyme activities versus reciprocal concentrations of one substrate at several fixed levels of the second one yield a pattern of inhibition by guanine and hypoxanthine. Product-inhibition studies indicated that AMP is a competitive inhibitor with respect to PP-Rib-P in the APRTase reaction, while the HGPRTase shows a mixed inhibition by GMP.
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PMID:Artemia purine phosphoribosyltransferases. Purification and characterization. 185 Sep 82

A novel method for measuring AMP-deaminase activity in human erythrocytes is presented, based on the determination of the reaction product, IMP, using high performance liquid chromatography. IMP formation was found to be proportional both to the incubation time and the amount of haemolysate over a wide range. The minimal detectable AMP-deaminase activity was more than 1000 times lower than the mean activity found in healthy controls (1083 nmol/h/mg Hb). No marked difference of activity was found in the patients with the following inherited purine disorders: familial juvenile gouty nephropathy and deficiencies of adenosine deaminase, hypoxanthine-guanine phosphoribosyltransferase or adenine phosphoribosyltransferase. The activity in the erythrocytes of patients with chronic renal failure was also similar to controls. The existence of subjects with low erythrocyte AMP-deaminase activity in the population has been confirmed.
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PMID:A high performance liquid chromatographic assay for AMP-deaminase activity in the erythrocytes of healthy subjects and patients with inherited purine disorders. 191 25

The proliferative effect of insulin on de novo purine synthesis and on the expression of various enzymes of purine metabolism were studied in primary cultured rat hepatocytes. Insulin greater than 1.5 x 10(-8) M increased DNA and de novo purine synthesis to 260-390 and 270-420%, respectively, 24 and 8 h after the administration. Insulin at 1.5 x 10(-7) M increased the specific activity of amidophosphoribosyltransferase (ATase) to 154-180%, hypoxanthine-guanine phosphoribosyltransferase to 129%, and adenine phosphoribosyltransferase (APRT) to 205%, in contrast to unchanged xanthine dehydrogenase at 80%. Enzyme induction was supported by the results of kinetic analysis and the inhibition of the insulin-induced increase in enzyme activities by protein synthesis inhibitors. Insulin increased ATP to 127% and decreased AMP, ADP, 5'-guanylic acid (GMP), and guanosine 5'-diphosphate (GDP), respectively, to 73, 69, 73, and 69%. Insulin increased adenylate energy charge from 0.83 to 0.90 without changing total feedback inhibitory potential on ATase. No obvious increase of 5-phosphoribosyl-1-pyrophosphate supply was suggested, although its apparent availability for purine ribonucleotide synthesis was increased to 208-245%, reflecting mainly induced APRT activity to 205%. It is concluded that hepatocyte proliferation by insulin, as evidenced by purine metabolism, is mediated by the selective gene activation of anabolic enzymes and increased ATP as the basis to activate multiple metabolic pathways without remarkable changes of substrate availability or feedback inhibition.
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PMID:Increased de novo purine synthesis by insulin through selective enzyme induction in primary cultured rat hepatocytes. 218 59

The activities (Vmax) of several enzymes of purine nucleotide metabolism were assayed in premature and mature primary rat neuronal cultures and in whole rat brains. In the neuronal cultures, representing 90% pure neurons, maturation (up to 14 days in culture) resulted in an increase in the activities of guanine deaminase (guanase), purine-nucleoside phosphorylase (PNP), IMP 5'-nucleotidase, adenine phosphoribosyltransferase (APRT), and AMP deaminase, but in no change in the activities of hypoxanthine-guanine phosphoribosyltransferase (HGPRT), adenosine deaminase, adenosine kinase, and AMP 5'-nucleotidase. In whole brains in vivo, maturation (from 18 days of gestation to 14 days post partum) was associated with an increase in the activities of guanase, PNP, IMP 5'-nucleotidase, AMP deaminase, and HGPRT, a decrease in the activities of adenosine deaminase and IMP dehydrogenase, and no change in the activities of APRT, AMP 5'-nucleotidase, and adenosine kinase. The profound changes in purine metabolism, which occur with maturation of the neuronal cells in primary cultures in vitro and in whole brains in vivo, create an advantage for AMP degradation by deamination, rather than by dephosphorylation, and for guanine degradation to xanthine over its reutilization for synthesis of GMP. The physiological meaning of the maturational increase in these two ammonia-producing enzymes in the brain is not yet clear. The striking similarity in the alterations of enzyme activities in the two systems indicates that the primary culture system may serve as an appropriate model for the study of purine metabolism in brain.
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PMID:Developmental changes in the activity of enzymes of purine metabolism in rat neuronal cells in culture and in whole brain. 232 47

The Authors present a procedure for the determination of adenine phosphoribosyltransferase (APRT) and hypoxanthine phosphoribosyltransferase (HPRT) in lymphocytes which exhibits high sensitivity and requires low quantities of lymphocytes. 5 normal subjects and 4 patients affected by chronic lymphocytic leukemia (CLL) were considered. Human lymphocytes were prepared and treated as previously reported. To the incubation mixtures buffered with 50 mM TRIS-HCl pH 7.4 either 14C-adenine or 14C-hypoxanthine was added: after deproteinization and neutralization we followed the formation of either 14C-adenylic acid (AMP) or 14C-inosinic acid (IMP) by HPLC. A Supelcosil C18 5 microns (250 X 4.5 mm) column was used: IMP was eluted with 20 mM KH2PO4 pH 5.5 while AMP with a linear gradient to 40% B in 20 min., where A was 20 mM KH2PO4 pH 5.5 and B methanol/water 60:40. Evaluation of AMP and IMP formed was carried out by determination of the radioactivity of the collected peaks. The values of APRT in leukemic patients were enhanced when referred to the proteins and those of HGPRT decreased: the Authors propose to complete the study evaluating the intracellular content of adenine and hypoxanthine.
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PMID:[Behavior of the enzymes of the salvage pathway of purine bases in leukemia lymphocytes]. 239 7

Human DNA was used to transform adenosine kinase (AK)-deficient BHK cells followed by selection of AK+ cells in medium containing alanosine, adenosine, and uridine (AAU medium). Twenty AAUr isolates were analyzed, and none of them contained AK activity. Several purine salvage enzymes were, however, found to be affected in these cells. The levels of hypoxanthine-guanine phosphoribosyltransferase and adenylosuccinate synthetase activities were elevated, while the adenylosuccinase activity was reduced. AAU-resistance may be explained by elevated activity of adenylosuccinate synthetase to overcome the alanosine block; thus AAUr cells were able to convert exogenous adenosine----inosine----hypoxanthine----IMP----AMPS----AMP. Moreover, these AAUr cells required exogenous purines for growth. HPLC analyses of endogenous nucleotide pools of AAUr cells showed that the levels of adenine nucleotides have diminished to less than 10% of the parental levels. These results suggest that the AAU-resistant mutation, which elicits pleiotropic phenotypes in BHK cells, affects an important component in the regulation of adenine nucleotide synthesis. By including erthyro-9-(2-hydroxy-3-nonyl)adenine in the AAU medium (renamed as AAUE medium) to block deamination of adenosine, AK+ BHK cells were isolated.
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PMID:Imbalance of purine nucleotides in alanosine-resistant baby hamster kidney cells. 253 26

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

In LLC-PK1 cells, a cyclic AMP (cAMP)-elevating peptide hormone, calcitonin, induces urokinase-type plasminogen activator (uPA) gene transcription without concomitant protein synthesis. To understand the molecular mechanism of the uPA gene regulation by cAMP, we developed a system which allows us to obtain mutant cells with modified regulatory proteins. A uPA-gpt hybrid gene was constructed, in which the regulatory region of the uPA gene was linked to a bacterial xanthine-guanine phosphoribosyltransferase gene (gpt), and it was transfected into LLC-PK1 cells. A stably transformed cell line, which expressed gpt only in the presence of calcitonin, was obtained, and then these cells were treated with a chemical mutagen, ethyl methanesulfonate. Cells were screened for constitutive gpt expression and, as mutations in regulatory proteins should affect the two genes at the same time, cells were further screened for an increased basal uPA mRNA level. Several such clones were obtained and none of them had modified cAMP-dependent protein kinase activity, suggesting that mutations were in the post-protein kinase step in the pathway of hormone action. Five clones were fused with the parent LLC-PK1 cells, and all of the fusion cells showed reduced basal uPA mRNA levels, indicating that they were recessive mutants. One clone was analyzed further for sensitivity to calcitonin in the induction of uPA mRNA, and it showed a significantly different dose-response pattern compared with parent cells. These results suggest that the uPA gene is regulated, at least partly, by a negatively regulating factor and that the action of cAMP is linked to this factor.
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PMID:A new genetic approach for studying hormonal regulation of urokinase-type plasminogen activator gene expression in LLC-PK1 cells. 283 Apr 99

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


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