Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:2.4.2.8 (hypoxanthine-guanine phosphoribosyltransferase)
 2,527 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

In this study we examined the metabolism of hypoxanthine in fibroblast growth factor (FGF)-stimulated porcine aortic endothelial cells (PAEC). Our previous report indicated that hypoxanthine in fetal bovine serum (FBS) was an essential component for both basal and FGF-dependent growth of PAEC (Hayashi et al., Exp Cell Res 185: 217-228, 1989). Besides hypoxanthine, the addition of various purine bases and purine nucleosides, but not xanthine, xanthosine or any pyrimidine metabolites, restored the limited growth of PAEC cultured in medium containing 10% dialyzed FBS in the presence or absence of FGF. The metabolism of [14C]hypoxanthine was compared in PAEC treated with and without FGF. Treatment of PAEC with FGF for 24 hr enhanced the radioactivity incorporation from [14C]hypoxanthine into both the acid-soluble and -insoluble fractions approximately 2-fold. Upon chromatographic analyses of hypoxanthine metabolites in the acid-soluble nucleotide fraction, it was found that in control PAEC hypoxanthine was largely metabolized to IMP, adenine nucleotides and uric acid, whereas in FGF-treated cells it was converted to ATP, ADP, GTP, xanthine and uric acid. The radioactivity of IMP was lowered in FGF-stimulated cells. The addition of FGF to PAEC increased phosphoribosyl pyrophosphate (PRPP) synthetase activity by approximately 8-fold and the PRPP content by approximately 2-fold, but it did not increase hypoxanthine-guanine phosphoribosyltransferase (HGPRT) activity or hypoxanthine transport. On the other hand, methotrexate, an inhibitor of de novo synthesis of purine, did not affect the growth of PAEC. Analyses of the rate of [14C]formate incorporation into total purine compounds showed that PAEC had a low capacity to synthesize purines de novo, which was not stimulated by FGF. These data indicate that FGF stimulates the synthesis of PRPP necessary for the salvage synthesis of purine nucleotides in conjunction with purine bases, e.g. hypoxanthine.
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PMID:Fibroblast growth factor-dependent metabolism of hypoxanthine via the salvage pathway for purine synthesis in porcine aortic endothelial cells. 768 70

The effects of the differentiation-inducing agents sodium butyrate (NaOBt), dimethylsulfoxide (DMSO) and mycophenolic acid (MA), on purine nucleotide metabolism, was studied in an ovarian carcinoma cell line (GZL-8). Exposure to these agents inhibited cell proliferation, but did not affect cell viability. Three hours following exposure, NaOBt and DMSO moderately decelerated purine synthesis de novo, but MA accelerated it three-fold, this being associated with a two-fold increase in the excretion of hypoxanthine and xanthine into the incubation medium. NaOBt and DMSO did not affect the cellular nucleotide content, but MA caused a 73% decrease in GTP content and about a 50% increase in the cellular content of UTP. The following alterations in cellular enzyme activity were observed 72 h following exposure: NaOBt decreased the activity of hypoxanthine-guanine phosphoribosyltransferase and increased the activity of IMP and of AMP 5'-nucleotidases, DMSO increased the activity of IMP 5'-nucleotidase, and MA increased the activity of the two nucleotidases. The results suggest that, in the carcinoma cell line studied, the differentiation process induced by NaOBt and DMSO may be associated with a general shift in the direction of purine metabolism from anabolism to catabolism, whereas that induced by MA is associated with a specific decrease in the production of GTP.
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PMID:Effects of differentiation-inducing agents on purine nucleotide metabolism in an ovarian cancer cell line. 779 96

Tiazofurin and ribavirin are clinically used inhibitors of IMP dehydrogenase (DH), binding to the NAD and IMP sites, respectively, of the target enzyme. In patients with chronic granulocytic leukemia in blast crisis, daily tiazofurin infusions decreased the high IMP DH activity in blast cells and resulted in 77% response (G. Weber. In: R. A. Harkness et al., Purine and Pyrimidine Metabolism in Man, Vol. VII, Part B, pp. 287-292, 1991). However, patients relapsed in a few weeks with emergence of high IMP DH activity (G. Tricot et al., Int. J. Cell Cloning, 8: 161-170, 1990). The present study showed that the tiazofurin-induced depression of IMP DH activity in rat bone marrow can be maintained by ribavirin injection. Tiazofurin (150 mg/kg, i.p., once a day for 2 days) decreased IMP DH activity to 10% and ribavirin (250 mg/kg, i.p., once a day for the subsequent 3 days) maintained the enzymic activity at 20 to 30% of control values. In control rats where no ribavirin was given, IMP DH activity of the tiazofurin-treated rats rapidly returned to the range of untreated animals. The decrease of IMP DH activity (t1/2 = 2.6 h) sharply preceded that of the bone marrow cellularity (t1/2 = 17.4 h). In addition to the target enzyme, IMP DH, tiazofurin also decreased activities of the guanylate metabolic enzymes, guanine phosphoribosyltransferase and GMP reductase, and the pyrimidine salvage enzymes, deoxycytidine and thymidine kinases with t1/2 of 2.6, 4.7, 6.0, 3.4, and 6.5 h, respectively. In cycloheximide-treated rats, where much of protein biosynthesis was blocked, the t1/2(8) of these five enzymes in bone marrow were shorter, 1.6, 4.3, 3.0, 0.6, and 0.8 h, respectively. Thus, the impact of tiazofurin in the bone marrow entails a decrease in the activity of the target enzyme, IMP DH, and also of other enzymes in purine and pyrimidine biosynthesis as a result of the enzyme half-lives shortened by this drug. These novel observations should assist in achieving better protection and recovery of bone marrow during and after chemotherapy.
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PMID:Sequential impact of tiazofurin and ribavirin on the enzymic program of the bone marrow. 790 99

(1) The currently used clinical anti-metabolites are targeted against-key enzymes of de novo purine and pyrimidine biosynthesis. However, the activities of salvage enzymes in each of the biosynthetic segments are markedly higher than those of the rate-limiting enzymes of de novo biosynthesis. Enzyme-pattern-targeted chemotherapy has been suggested to overcome the circumvention activity of salvage. Combination of inhibition of de novo and salvage pathways does provide a synergistic impact. Examples that enzyme-pattern-targeted drug treatment yields synergism include the following: tiazofurin (against IMP DH) and allopurinol (by raising serum hypoxanthine levels it inhibits GPRT); methotrexate or 5-FU lead to inhibition of the dTMP synthase reaction and AZT (a competitive inhibitor of thymidine kinase) or dipyridamole (a nucleoside transport inhibitor); acivicin, an inhibitor and inactivator of glutamine-utilizing enzymes in the de novo pathways of purine and pyrimidine biosynthesis, and dipyridamole. (2) Administration of MTX, 5-FU, tiazofurin or acivicin causes inhibition and/or inactivation of target enzymes. That these drugs are effective in spite of the presence of highly active salvage enzymes is now accounted for, at least in part, by new observations showing that these drugs markedly reduce (but do not eliminate) the activities (amounts) of CdR and TdR kinases, dTMP synthase and GPRT. This action is attributed to the rapid decay rate of these enzymes. (3) Studies on the bone marrow enzymic programs indicate that there is a window of opportunity for strengthening therapy and for the protection of bone marrow by administering salvage metabolites when the salvage enzymes are still present in high enough activities, i.e., 2-6 hr after administration of the blockers of de novo enzyme activities. (4) These results are a strong argument for discovering and utilizing inhibitors of purine and pyrimidine salvage enzymes to achieve more successful enzyme-pattern-targeted chemotherapy and to avoid development of resistant clones of cancer cells. (5) These approaches provide greater explanatory coherence than the previous accounts because recognition of (a) the importance of salvage and (b) rapid decay of key and salvage enzymes reveals a paradigm shift. The problem-solving process in chemotherapy should now be not only data-driven but also explanation-driven.
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PMID:Targeted and non-targeted actions of anti-cancer drugs. 794 86

Nucleoside phosphotransferase acting on inosine and deoxyinosine has been partially purified from cultured Chinese hamster lung fibroblasts (V79). The activity is associated with a cytosolic 5'-nucleotidase acting on IMP and deoxyIMP. The transfer of the phosphate group from IMP to inosine catalyzed by this enzyme was activated by ATP and 2,3-bisphosphoglycerate. Inosine, deoxyinosine, guanosine, deoxyguanosine, and the nucleoside analogs 2',3'-dideoxyinosine and 8-azaguanosine are substrates, while adenosine and deoxyadenosine are not. IMP, deoxyIMP, GMP, and deoxyGMP are the best phosphate donors. The cytosolic 5'-nucleotidase/phosphotransferase substrate, 8-azaguanosine, was found to be very toxic for cultured fibroblasts (LD50 = 0.32 microM). Mutants resistant to either 8-azaguanosine and the correspondent base 8-azaguanine were isolated and characterized. Our results indicated that the 8-azaguanosine-resistant cells were lacking both cytosolic 5'-nucleotidase and hypoxanthine-guanine phosphoribosyltransferase, while 8-azaguanine resistant cells were lacking only the latter enzyme. Despite this observation, both mutants displayed 8-azaguanosine resistance, thus indicating that cytosolic 5'-nucleotidase is not essential for the activation of this nucleoside analog.
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PMID:Cytosolic 5'-nucleotidase/nucleoside phosphotransferase: a nucleoside analog activating enzyme? 815 32

The purpose of this paper was to clarify critical aspects of the behavior of signal transduction activity in normal and cancer cells. 1. Signal transduction activity in the conversion of phosphatidylinositol through PI and PIP kinases and PLC to IP3 is regulated at multiple sites. In liver, hepatomas and human carcinomas PIP kinase is the rate limiting enzyme and PLC activity is present in great excess. 2. The steady-state signal transduction activity as measured by the three enzyme activities and IP3 concentration was markedly up-regulated in rat hepatomas of different growth rates. The steady-state specific activities of the three signal transduction enzymes were elevated in ovarian carcinomas as compared to normal ovary. Increased enzyme activities were also observed in human breast carcinoma cells as compared to normal human breast parenchymal cells. In breast, ovarian and rat hepatoma cells as they go through lag, log and plateau phases, IP3 concentration in the early lag phase increased 4.5- to 20-fold and PI and PIP kinase activities peaked in mid-log phase. These events returned to baseline levels in the plateau phase. PLC activity did not change. 3. The bone marrow PI and PIP kinase activities in 3-day starvation were decreased to 13% and IP3 concentration was reduced to 24%; at 1-day refeeding they returned to normal. PLC activity changed little. These alterations are in line with the rapid t1/2 degradation rates (12 min) of PI and PIP kinases observed in studies with cycloheximide. By contrast, PLC has a long half-life. 4. The molecular action of tiazofurin entails inhibition of IMP DH activity, decrease in GTP and IP3 concentrations, reduction of ras and myc oncogene expression, and signal transduction enzyme activities. These events are followed by induced differentiation and apoptosis. There are also decreases in enzyme activities which have rapid turnover, including TdR kinase, dTMP synthase, and GPRT. In vitro studies indicated that these events are abrogated by addition of guanine which restores GTP concentrations. Therefore, most or all these events were brought about by the reduced GTP concentration in the tiazofurin target cells. 5. Quercetin and genistein are able to inhibit PI and PIP kinase activities and reduce IP3 concentration in vivo and in tissue culture systems. These flavonoids are also inhibitors of cell proliferation and clonogenic ability in rat hepatoma 3924A and in human OVCAR-5 and MDA-MB-435 cells. Quercetin down-regulated the expression of c-myc and Ki-ras oncogenes and led to induced differentiation and apoptosis in K562 cells. Genistein reduced IP3 concentration in vivo and in the tissue culture system. Genistein is antiproliferative and has cytototoxicity in human carcinoma cells. All three drugs, tiazofurin, quercetin and genistein, act, in part at least, through depression of cellular IP3 concentration although the mechanisms may not be identical. 6. Quercetin and genistein, which attack different targets and different phases of the cell cycle, proved to be synergistic in OVCAR-5 cells. The impact of tiazofurin, genistein and quercetin is of interest because the drugs crucially inhibit the display of the neoplastic program of cells and lead to induced differentiation and apoptosis.
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PMID:Regulation of the signal transduction program by drugs. 938 80

Tritrichomonas foetus, an anaerobic flagellated protozoan, causes urogenital trichomoniasis in cattle. Hypoxanthine-guanine-xanthine phosphoribosyl transferase (HGXPRTase), an essential enzyme in T. foetus required for salvaging exogenous purine bases, has been regarded as a promising target for anti-tritrichomonial chemotherapy. The steady-state kinetic analyses of synthesis and pyrophosphorolysis of IMP, GMP, and XMP and product inhibition studies have been used to elucidate the reaction mechanisms. Double-reciprocal plots of initial velocities versus the varying concentrations of one substrate at a fixed concentration of the other show intersecting lines indicating a sequential mechanism for both the forward and the reverse reactions. In terms of the kcat/Km ratios, hypoxanthine is the most effective substrate whereas guanine and xanthine are converted equally well into their corresponding nucleotides. The minimum kinetic model from the data in product inhibition studies is an ordered bi-bi mechanism, where the substrates bind to the enzyme (first PRPP followed by the purine bases), and the products released (first PPi followed by purine nucleotide) in a defined order. The Kms for PPi in the T. foetus HGXPRTase-catalyzed reactions are unusually high, close to the millimolar range. Since the crystal structure of this enzyme [Somoza et al. (1996) Biochemistry 35, 7032-7040] suggests potential binding between the threonine-47 in a conserved cis-peptide loop and PPi whereas human HGPRTase has lysine-68 [Eads et al. (1994) Cell 78, 325-334] at the corresponding position, we prepared a T47K enzyme mutant and found in the T47K-catalyzed reaction a 4-10-fold decrease of Km for PPi. The lack of ionic interactions between Thr-47 and PPi and an increased distance between the loop and the active site as compared to the human HGPRTase are thus proposed to be responsible for the high Km for PPi in the T. foetus HGXPRTase-catalyzed reaction.
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PMID:Steady-state kinetics of the hypoxanthine-guanine-xanthine phosphoribosyltransferase from Tritrichomonas foetus: the role of threonine-47. 952 25

Lesch-Nyhan syndrome is a hereditary disorder of purine metabolism causing overproduction of uric acid and neurological problems including spasticity, choreoathetosis, mental retardation, and compulsive self-mutilation. The syndrome is caused by a defect in the enzyme hypoxanthine-guanine phosphoribosyltransferase (HPRT), which converts guanine and hypoxanthine to the nucleotides GMP and IMP. There is evidence that the neurological problems are due to an adverse effect of the HPRT deficiency on the survival and/or development of dopaminergic neurons, specifically. Here we report that HPRT-deficient PC12 mutants that have a normal or near normal dopamine content (55-97% of that of wild-type cells) fail to undergo neuronal differentiation induced by nerve growth factor (NGF) when the de novo pathway of purine synthesis is partially inhibited. However, nerve growth factor-induced differentiation is near normal under these conditions in PC12 HPRT-deficient mutants containing much lower dopamine levels (<8% of that of wild type cells), indicating a neurotoxic effect of the endogenous dopamine in the mutants. The degree of inhibition of the de novo pathway of purine synthesis was the same in both classes of HPRT-deficient mutants. Expression of BCl-2 in a PC12 mutant that has a normal dopamine content allowed partial NGF-induced differentiation suggesting that the apoptotic pathway might be involved in the failure of differentiation when the de novo pathway of purine synthesis is partially inhibited.
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PMID:Impaired differentiation of HPRT-deficient dopaminergic neurons: a possible mechanism underlying neuronal dysfunction in Lesch-Nyhan syndrome. 967 Sep 94

The hypoxanthine-guanine-xanthine phosphoribosyltransferase (HGXPRTase) from Tritrichomonas foetus has been proven to be a target for potential anti-tritrichomonial chemotherapy. Using a structure-based approach, the base-binding region of the active site of this enzyme, which confers unique purine base specificity, was characterized using site-directed mutagenesis. Determining the roles of different active-site residues in purine specificity would form the basis for designing specific inhibitors toward the parasitic enzyme. A D163N mutant converts the HGXPRTase into a HGPRTase, which no longer recognizes xanthine as a substrate, whereas specificities toward guanine and hypoxanthine are unaffected. Apparently, the side-chain carboxyl of Asp163 forms a hydrogen bond through a water molecule with the C2-carbonyl of xanthine, which constitutes the critical force enabling the enzyme to recognize xanthine as a substrate. Mutations of Arg155, which orients and stacks the neighboring Tyr156 onto the bound purine base by forming a salt bridge between itself and Glu11, result in drastic increases in the Kms for GMP and XMP (but not IMP). This change leads to increased kcats for the forward reactions with guanine and xanthine as substrates without affecting the conversion of hypoxanthine to IMP. Thus, the apparent dislocation of Tyr156, resulted from mutations of Arg155, bring little effect on the hydrophobic interactions between Tyr156 and the purine ring. But the forces involved in recognizing the exocyclic C2-substituents of the purine ring, which involve the Tyr156 hydroxyl, Ile157 backbone carbonyl, and Asp163 side-chain carboxyl, may be weakened by the shifted conformation of the peptide backbone resulted from loss of the Glu11-Arg155 salt bridge. The conserved Lys134 was proven to be the primary determinant in conferring the specificity of the enzyme toward 6-oxopurines. By substituting the lysine residue for a serine, which can potentially hydrogen bond to either an amino or an oxo-group, we have successfully augmented the purine specificity of the enzyme. The K134S mutant recognizes adenine in addition to hypoxanthine, guanine, and xanthine as its substrates. Adenine and hypoxanthine are equivalent substrates for the mutant enzyme with similar Kms of 34.6 and 38.0 microM, respectively. The catalysis of an adenine phosphoribosyltransferase reaction by this mutant enzyme was further demonstrated by the competitive inhibition of AMP with an estimated Kis of 25.4 microM against alpha-D-5-phosphoribosyl-pyrophosphate (PRPP) in converting hypoxanthine to IMP. We have thus succeeded in using site-directed mutagenesis to convert T. foetusHGXPRTase into either a HGPRTase or a genuine AHGXPRTase.
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PMID:Altering the purine specificity of hypoxanthine-guanine-xanthine phosphoribosyltransferase from Tritrichomonas foetus by structure-based point mutations in the enzyme protein. 984 28

Inosine 5 -monophosphate dehydrogenase (IMPDH) is a rate-limiting enzyme for the synthesis of GTP and dGTP. Two isoforms of IMPDH have been identified. IMPDH Type I is ubiquitous and predominantly present in normal cells, whereas IMPDH Type II is predominant in malignant cells. IMPDH plays an important role in the expression of cellular genes, such as p53, c-myc and Ki-ras. IMPDH activity is transformation and progression linked in cancer cells. IMPDH inhibitors, tiazofurin, selenazofurin, and benzamide riboside share similar mechanism of action and are metabolized to their respective NAD analogues to exert antitumor activity. Tiazofurin exhibits clinical responses in patients with acute myeloid leukemia and chronic myeloid leukemia in blast crisis. These responses relate to the level of the NAD analogue formed in the leukemic cells. Resistance to tiazofurin and related IMPDH inhibitors relate mainly to a decrease in NMN adenylyltransferase activity. IMPDH inhbitors induce apoptosis. IMPDH inhitors are valuable probes for examining biochemical functions of GTP as they selectively reduce guanylate concentration. Incomplete depletion of cellular GTP level seems to down-regulate G-protein function, thereby inhibit cell growth or induce apoptosis. Inosine 5'-monophosphate dehydrogenase (IMPDH, EC 1.1.1.205) catalyzes the dehydrogenation of IMP to XMP utilizing NAD as the proton acceptor. Studies have demonstrated that IMPDH is a rate-limiting step in the de novo synthesis of guanylates, including GTP and dGTP. The importance of IMPDH is central because dGTP is required for the DNA synthesis and GTP plays a major role not only for the cellular activity but also for cellular regulation. Two isoforms of IMPDH have been demonstrated. IMPDH Type I is ubiquitous and predominately present in normal cells, whereas the IMPDH Type II enzyme is predominant in malignant cells. Although guanylates could be salvaged from guanine by the enzyme hypoxanthine-guanine phosphoribosyltransferase (EC 2.4.2.8), the level of circulating guanine is low in dividing cells and this route is probably insufficient to satisfy the needs of guanylates in the cells.
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PMID:Consequences of IMP dehydrogenase inhibition, and its relationship to cancer and apoptosis. 1039 Jun 1


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