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:2.7.7.7 (DNA polymerase)
17,007 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The metabolism and mode of action of the anti-herpes compound buciclovir [R)-9-(3,4-dihydroxybutyl)-guanine, BCV) has been studied in herpes simplex virus-infected and uninfected Vero cells. In uninfected cells, a low and constant concentration of intracellular BCV was found, while in herpes simplex virus-infected cells, an increasing concentration of BCV phosphates was found due to metabolic trapping. The major phosphorylation product was BCV triphosphate (BCVTP) which was 92% of the total amount of BCV phosphates. BCV phosphates were accumulated to the same extent in cells infected with either a herpes simplex virus type 1 or a herpes simplex virus type 2 strain while thymidine kinase-deficient mutants of herpes simplex virus type 1 were 10 times less efficient in accumulating BCV phosphates. In uninfected Vero cells, the concentration of the phosphorylated forms of BCV was less than 1% of that found in herpes simplex virus-infected cells. The BCVTP formed in herpes simplex virus-infected cells was highly stable, as 80% of the amount of BCVTP was still present even 17 h after removal of extracellular BCV. BCV was a good substrate for herpes simplex virus type 1- and type 2-induced thymidine kinases but not for the cellular cytosol or mitochondrial thymidine kinases. BCV monophosphate could be phosphorylated by cellular guanylate kinase to BCV diphosphate. BCVTP was a selective and competitive inhibitor to deoxyguanosine triphosphate of the purified herpes simplex virus type 1- and type 2-induced DNA polymerases. BCVTP could neither act as an alternative substrate in the herpes simplex virus type 2 or cellular DNA polymerase reactions, nor could [3H]BCV monophosphate be detected in DNA formed by herpes simplex virus type 2 DNA polymerase, or be detected in nucleic acids extracted from herpes simplex virus type 1-infected cells. These data indicate that BCVTP may inhibit the herpes simplex virus-induced DNA polymerase without being incorporated into DNA.
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PMID:Metabolism and mode of action of (R)-9-(3,4-dihydroxybutyl)guanine in herpes simplex virus-infected vero cells. 241 21

Mouse macrophages grown from spleen cells were found to be very sensitive to the interferon (IFN) activity against herpes simplex virus type 1 (HSV-1). Therefore we have used these cells to investigate the level at which IFN blocks the replication of HSV-1. IFN treatment resulted in a strong inhibition of the induction of HSV DNA polymerase and other beta proteins. RNA hybridization experiments revealed that the amount of mRNA for the beta protein thymidine kinase was strongly reduced in IFN treated HSV-1 infected cells. Analysis of the effect of IFN on expression of the alpha genes indicated a strong inhibition of alpha protein synthesis. In contrast the synthesis of mRNA of the alpha protein ICP 4 was only moderately inhibited. The results indicate that IFN primarily acts on the translation of HSV alpha proteins.
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PMID:Synthesis of herpes simplex virus proteins and nucleic acids in interferon-treated macrophages. 242 81

The thymidine analog 3'-azido-3'-deoxythymidine (BW A509U, azidothymidine) can inhibit human immunodeficiency virus (HIV) replication effectively in the 50-500 nM range [Mitsuya, H., Weinhold, K. J., Furman, P. A., St. Clair, M. H., Nusinoff-Lehrman, S., Gallo, R. C., Bolognesi, D., Barry, D. W. & Broder, S. (1985) Proc. Natl. Acad. Sci. USA 82, 7096-7100]. In contrast, inhibition of the growth of uninfected human fibroblasts and lymphocytes has been observed only at concentrations above 1 mM. The nature of this selectivity was investigated. Azidothymidine anabolism to the 5'-mono-, di-, and -triphosphate derivatives was similar in uninfected and HIV-infected cells. The level of azidothymidine monophosphate was high, whereas the levels of the di- and triphosphate were low (less than or equal to 5 microM and less than or equal to 2 microM, respectively). Cytosolic thymidine kinase (EC 2.7.1.21) was responsible for phosphorylation of azidothymidine to its monophosphate. Purified thymidine kinase catalyzed the phosphorylations of thymidine and azidothymidine with apparent Km values of 2.9 microM and 3.0 microM. The maximal rate of phosphorylation with azidothymidine was equal to 60% of the rate with thymidine. Phosphorylation of azidothymidine monophosphate to the diphosphate also appeared to be catalyzed by a host-cell enzyme, thymidylate kinase (EC 2.7.4.9). The apparent Km value for azidothymidine monophosphate was 2-fold greater than the value for dTMP (8.6 microM vs. 4.1 microM), but the maximal phosphorylation rate was only 0.3% of the dTMP rate. These kinetic constants were consistent with the anabolism results and indicated that azidothymidine monophosphate is an alternative-substrate inhibitor of thymidylate kinase. This conclusion was reflected in the observation that cells incubated with azidothymidine had reduced intracellular levels of dTTP. IC50 (concentration of inhibitor that inhibits enzyme activity 50%) values were determined for azidothymidine triphosphate with HIV reverse transcriptase and with immortalized human lymphocyte (H9 cell) DNA polymerase alpha. Azidothymidine triphosphate competed about 100-fold better for the HIV reverse transcriptase than for the cellular DNA polymerase alpha. The results reported here suggest that azidothymidine is nonselectively phosphorylated but that the triphosphate derivative efficiently and selectively binds to the HIV reverse transcriptase. Incorporation of azidothymidylate into a growing DNA strand should terminate DNA elongation and thus inhibit DNA synthesis.
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PMID:Phosphorylation of 3'-azido-3'-deoxythymidine and selective interaction of the 5'-triphosphate with human immunodeficiency virus reverse transcriptase. 243 Feb 86

The mutagenic potential of the cytidine analog, 5-azacytidine (Aza Cyd), was tested at the thymidine kinase (TK) gene locus of L5178Y mouse lymphoma cells. 3-h exposure to as little as 20 ng/ml Aza Cyd yielded a substantial increase in TK-deficient L5178Y cells as measured by drug-induced resistance to trifluorothymidine (TFTres) 48 h later. This mutagenic effect was diminished up to 75% when Aza Cyd was tested in the presence of either enzymatically active or heat-denatured 9000 X g supernatant prepared from rat liver homogenate. The mutagenicity of Aza Cyd was also decreased in the presence of 1-5 X 10(-3) M thymidine and eliminated in the presence of greater than 1 X 10(-5) M cytidine. Two L5178Y TK-deficient cell lines had no selective survival advantage compared to TK-competent L5178Y cell stock when plated in soft-agar medium that contained Aza Cyd. Four other specific inhibitors of scheduled DNA synthesis in mammalian cells, deoxyadenosine, aphidicolin, 1-beta-D-arabinofuranosylcytosine, and hydroxyurea were also L5178Y/TK mutagens. These data along with other published results suggest that chemicals known to disrupt nucleotide biosynthesis, alter deoxyribonucleotide pools, or directly inhibit DNA polymerase can cause stable, heritable increases in TFT resistance through mechanisms dependent upon altered replicative DNA synthesis, yet not necessarily dependent upon DNA incorporation or the binding of these mutagenic agents to nuclear DNA.
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PMID:The mutagenicity of 5-azacytidine and other inhibitors of replicative DNA synthesis in the L5178Y mouse lymphoma cell. 243 24

Confirmatory evidence for the existence of a multienzyme complex of DNA precursor pathways in mammalian cells was obtained. Using neutral sucrose gradient centrifugation of cell lysates we found that at least five enzymes involved in DNA precursor metabolism in uninfected. S-phase BHK-cell fibroblasts cosediment at a common rate, indicative of a multienzyme complex. The enzymes include DNA polymerase thymidine kinase, ribonucleotide reductase, dihydrofolate reductase, and NDP-kinase. This complex was partially, but not completely, disrupted when lysates from GO-phase cells were centrifuged. Using lysates from cells infected with herpes simplex virus (HSV) type I some of the virus-induced ribonucleotide reductase and a minor proportion of the HSV-thymidine kinase cosedimented rapidly. The virus-induced DNA polymerase sedimented independently near the middle of the gradient, in contrast to the behaviour of the host polymerase. The enzyme associations observed were disrupted by NaCl or by inclusion of ethylenediamine tetraacetic acid during the cell lysis procedure, instead of the usual EGTA. These results indicate the importance of ionic forces in maintaining the enzyme complexes. The bulk of the DNA and the RNA present in the lysates did not sediment at the same rate as the complexes, showing that the enzymes were not simply adhering nonspecifically to these polyanions. Newly synthesised radiolabeled DNA (15 min pulse with [3H]thymidine) was not preferentially associated with the enzymes, but some functional DNA was evident in the enzyme complex fraction from the uninfected S-phase cells. DNA polymerase activity in this fraction did not require, nor was it stimulated by, exogenous "activated" DNA. Added DNA primer-template was required, however, for maximal activity of the polymerase in gradient fractions derived from GO-phase cells and from HSV-infected cells. No evidence for channeling of ribonucleotide precursors into DNA of permeabilized cells (uninfected or HSV-infected) was detected. Most rCDP was incorporated into RNA. In the uninfected, S-phase cells about 10 pmol/10(6) cells/90 min of rCDP residues was incorporated into DNA compared with 120 pmol/10(6) cells/90 min when radiolabeled dCTP was used. Nonradioactive dCTP present in equimolar concentration in the incubation with labeled rCDP did not, however, diminish the incorporation of label from the ribonucleotide. In permeabilized HSV-infected cells incorporation of radiolabel from rCDP into DNA was barely detectable.
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PMID:Search for multienzyme complexes of DNA precursor pathways in uninfected mammalian cells and in cells infected with herpes simplex virus type I. 244 4

Little is known about the cellular mechanisms responsible for the trophic effects of cholecystokinin (CCK) and secretin on the rat pancreas, and controversy exists with regard to the interaction between these two peptides. In the present study attempts were made to elucidate the time course of events leading to pancreatic growth and to clarify the interaction between the peptides when given as continuous, long-term intravenous infusions to rats. A cholecystokinin-like peptide (CCK-LP) and secretin were given as a continuous intravenous infusion to conscious and unrestrained animals with free access to food and water for 0.5, 1, 2, 4, 6, 8, 12, 24, 48, and 96 h. The pancreas was quickly removed and analyzed for variables indicating synthesis and accumulation of DNA, RNA, and polyamines. CCK-LP increased the activity of RNA polymerase already after 1 h, whereas an increase in the activity of ornithine decarboxylase (ODC) and the level of putrescine was seen at 4 h. Spermidine was increased after 12 h. The activities of DNA polymerase and thymidine kinase were increased at 12 and 24 h, respectively, whereas the total contents of DNA and RNA were first increased at 48 h. Secretin alone showed a marked but short-lived effect on polyamine synthesis and a weak effect on the variables indicating protein synthesis and growth. When the two peptides were given together, a large but transient potentiation of ODC activity was observed, whereas no interaction was seen on polyamines, RNA synthesis, or pancreatic growth. The present study confirms the trophic effects of CCK and secretin on the rat pancreas but fails to confirm an interaction between the two peptides on growth. Both peptides stimulate polyamine synthesis, and ODC appears to be an early and sensitive indication of their trophic effect. The initiation of RNA synthesis appears to be independent of the ODC activity.
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PMID:Short- and long-term effects of secretin and a cholecystokinin-like peptide on pancreatic growth and synthesis of RNA and polyamines. 247 84

The role played by the polyamines in mediating the pancreatic growth and secretory responses to hormonal stimulation is uncertain. The effect of an inhibitor of ornithine decarboxylase (ODC), alpha-difluoromethylornithine (DFMO), on rat pancreatic protein secretion and synthesis and on growth in response to hormonal stimulation was therefore studied. Anesthetized rats were given an intravenous injection of DFMO (50, 100, or 150 mg/kg), followed by a 7-h continuous infusion (15, 25, or 35 mg/kg/h, respectively). After a basal 1-h period an intravenous infusion of 2.5 micrograms/kg/h of the cholecystokinin-like peptide Thr28Nle31CCK25-33 (CCK-LP) was added and continued for 6 h. The control rats received CCK-LP only. The ODC activity in the pancreas was markedly reduced by DFMO, but DFMO did not affect pancreatic juice volume or protein output. In another series conscious rats were given a continuous intravenous infusion of 2.5 micrograms/kg/h of CCK-LP for 8, 24, and 48 h or 5.0 micrograms/kg/h of secretin for 8 and 48 h, with or without DFMO (100 mg/kg as an injection initially and thereafter 25 mg/kg/h). The ODC activity and putrescine concentration in the pancreas were significantly reduced by DFMO at 8 and 24 h but not at 48 h. DFMO also significantly reduced the activities of RNA polymerase, DNA polymerase, and thymidine kinase at 24 h, but not at 48 h. The present study thus indicates that polyamines play a role in the initiation of the growth response to hormonal stimulation but does not support a similar dependence for early pancreatic protein synthetic and secretory responses.
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PMID:Inhibition of polyamine synthesis by alpha-difluoromethylornithine and its effects on pancreatic secretion and growth in the rat. 247 85

Acute and long-term changes of ornithine decarboxylase and polyamines during pancreatic adaptation in response to cholecystokinin administration (1 microgram kg-1 body wt every 8 h) were studied in rats. alpha-difluoromethylornithine, an irreversible and specific inhibitor of ornithine decarboxylase, was applied simultaneously to elucidate the essential role of polyamines in pancreatic growth. In the cholecystokinin-treated animals ornithine decarboxylase activity was increased after 2 h, reached a maximum after 8 h (444.6 pmol 14CO2 h-1 mg-1 DNA, about 65-fold greater than controls, P less than 0.001) followed by a significant increase of putrescine after 6 h and spermidine after 24 h while spermine remained unchanged. The trophic parameters increased in the following time sequence: thymidine kinase (12 h), DNA polymerase (24 h), pancreatic weight (2 days), protein (2 days) and DNA (5 days). alpha-difluoromethylornithine significantly delayed the increase in ornithine decarboxylase, putrescine and spermidine as well as all trophic parameters. Increases in ornithine decarboxylase, polyamines and all trophic parameters were completely inhibited by simultaneous application of the CCK receptor antagonist L-364,718. These data indicate an important role for ornithine decarboxylase and polyamines in cholecystokinin-induced pancreatic growth in rats.
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PMID:Ornithine decarboxylase and polyamines in cholecystokinin-induced pancreatic growth in rats: effects of alpha-difluoromethylornithine and the CCK receptor antagonist L-364,718. 247 58

3'-Azido-2',3'-dideoxyuridine-5'-triphosphate was found to be a potent and highly selective inhibitor of human immunodeficiency virus type 1 and simian immunodeficiency virus reverse transcriptases. The affinity of 3'-azido-2',3'-dideoxyuridine-5'-triphosphate for the reverse transcriptases was similar to that observed for 3'-azido-3'-deoxythymidine-5'-triphosphate. Both compounds were competitive inhibitors with respect to the normal substrate dTTP and served at least 100 times better as substrates than did dTTP. In contrast, cellular DNA polymerase alpha showed an about 60-times-higher preference for dTTP as substrate than for either inhibitor. The phosphorylation of thymidine in human peripheral blood mononuclear cell extracts was inhibited in a competitive manner by both 3'-azido-2',3'-dideoxyuridine and 3'-azido-3'-deoxythymidine, with apparent inhibition constants of 290 and 3.4 microM, respectively. The Michaelis-Menten constant, Km, for thymidine was 7.0 microM. 3'-Azido-2',3'-dideoxyuridine and 3'-azido-3'-deoxythymidine both served as substrates, with apparent Km values of 67 and 1.4 microM, respectively. The maximal rates of phosphorylation with 3'-azido-2',3'-dideoxyuridine and 3'-azido-3'-deoxythymidine were 40 and 30%, respectively, of the rate with thymidine. The different affinities of 3'-azido-2',3'-dideoxyuridine and 3'-azido-3'-deoxythymidine for the thymidine kinase and the Km values observed with these compounds as substrates may explain the difference in effects on human immunodeficiency virus type 1 replication in infected peripheral blood mononuclear cells observed when equimolar concentrations of the two compounds are compared.
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PMID:Phosphorylation of 3'-azido-2',3'-dideoxyuridine and preferential inhibition of human and simian immunodeficiency virus reverse transcriptases by its 5'-triphosphate. 248 79

The dose dependence of a cholecystokinin-like peptide (CCK-LP) on the trophic response in the rat pancreas was studied. Graded doses of Thr28Nle31CCK25-33 (0.02, 0.1, 0.5, 2.5, and 12.5 micrograms/kg/h) or saline were given as a continuous intravenous infusion to conscious and fed rats for 8 and 48 h. Secretin (5.0 micrograms/h) was given alone or combined with the three highest doses of CCK-LP for 48 h. CCK-LP showed a dose-dependent stimulating effect on pancreatic growth and synthesis of RNA and polyamines. The threshold dose ranged from 0.02 to 0.5 micrograms/kg/h and was lowest for stimulation of ornithine decarboxylase (ODC). The maximal effects on protein, RNA, and DNA contents were achieved with 2.5 micrograms/kg/h. These same variables markedly decreased with 12.5 micrograms/kg/h, whereas marked further increases were found for the activities of RNA polymerase, DNA polymerase, and thymidine kinase. This same dose of CCK-LP caused after 8 h of treatment a marked and transient increase in pancreatic weight, activity of ODC, and concentration of putrescine. When secretin was added to 0.5 and 2.5 micrograms/kg/h of CCK-LP, no additional effect (except for ODC) was found. When secretin was added to the highest dose of CCK-LP, the decreased contents of protein and RNA were significantly increased, and the markedly increased activities of RNA- and DNA-synthesizing enzymes were significantly decreased. The present study shows a clear dose-response relationship for the trophic effect of CCK-LP on the rat pancreas and indicates that the growth effect of a supramaximal dose includes components of regeneration secondary to damage.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:The effects of graded doses of a cholecystokinin-like peptide with and without secretin on pancreatic growth and synthesis of RNA and polyamines in rats. 248 Jun 34


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