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:2.7.1.21 (thymidine kinase)
7,561 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Although most mammalian cell lines can utilize either nicotinic acid or nicotinamide for the biosynthesis of nicotinamide adenine dinucleotide (NAD), thymidine kinase-deficient, mouse 3T3-4F cells are unable to utilize nicotinic acid. When 3T3-4E cells were fused with human D98/AH2 cells, autoradiography showed that the resultant heterokaryons synthesized NAD from nicotinic acid at rates comparable to the human parental cell. The rate of nicotinic acid utilization in heterokaryons remained unchanged over the four-day period of study following cell fusion. In contrast to the results observed with heterokaryons, nicotinic acid utilization was markedly reduced in hybrid cells. Of 100 hybrid clones examined at four or five days following cell fusion, 60 utilized nicotinic acid at rates less than one tenth that of the parental human cell. Similar results were observed in hybrid clones at nine or ten days following fusion. Uniformly high rates of NAD biosynthesis were observed in hybrid clones with nicotinamide as the precursor. This excludes the possibility that the reduction in nicotinic acid utilization in hybrid cells is due to a general metabolic dysfunction. The biochemical mechanism by which nicotinic acid utilization is markedly reduced has not been determined with certainty, however, several observations suggest genetic suppression.
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PMID:Autoradiographic studies of nicotinic acid utilization in human-mouse heterokaryons and inhibition of utilization in newly-formed hybrid cells. 17 18

Poly(ADP-ribose) polymerase activity in nuclei isolated from differentiating cardiac muscle of the rat has been characterized and its activity measured during development. Optimum enzyme activity is observed at pH 8.5. Poly(ADP-ribose) polymerase is inhibited by ATP, thymidine, nicotinamide, theophylline, 3-isobutyl-1-methylxanthine and caffeine and stimulated by actinomycin D. The activity measured under optimal assay conditions increases during differentiation of cardiac muscle and is inversely related to the rate of DNA synthesis and to the activities of DNA polymerase alpha and thymidine kinase. When DNA synthesis and the activity of DNA polymerase alpha are inhibited in cardiac muscle of the 1-day-old neonatal rat by dibutyryl cyclic AMP or isoproterenol, the specific activity of poly(ADP-ribose) polymerase measured in isolated nuclei is increased. The concentration of NAD+ in cardiac muscle increases during postnatal development. In the adult compared with the 1-day-old neonatal rat the concentration of NAD+ relative to fresh tissue weight, DNA or protein increased 1.7-fold, 5.2-fold or 1.4-fold respectively. The concentration of NAD+ in cardiac muscle of the 1-day-old neonatal rat can be increased by approx. 20% by dibutyryl cyclic AMP. These data suggest that NAD+ and poly(ADP-ribose) polymerase may be involved with the repression of DNA synthesis and cell proliferation in differentiating cardiac muscle.
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PMID:Poly(adenosine diphosphate ribose) polymerase activity and nicotinamide adenine dinucleotide in differentiating cardiac muscle. 18 Sep 77

The bioreductive alkylating agent, 2,3-bis(chloromethyl)-1,4-napthoquinone (CMNQ), has been shown to inhibit the growth of Sarcoma 180 ascites cells in vivo. Evidence for the reductive activation of this agent via the mitochondrial respiratory chain was provided by CMNQ-induced oxidation of reduced nicotinamide adenine dinucleotide; the interaction was shown to be on the substrate side of the site of rotenone inhibition. Consistent with the concept that reduction of CMNQ to a hydroquinone results in the generation of an alkylating species (i.e., a quinone methide) was the finding that radioactivity from [14C]CMNQ present in Sarcoma 180 ascites cells was associated with DNA, RNA, and protein for a period of up to 72 hr after exposure to tumor-bearing animals to this agent. Inhibition of the incorporation of [3H]thymidine, [3H]uridine, and [14C]leucine into DNA, RNA, and protein, respectively, of Sarcoma 180 ascites cells was produced by this agent, with DNA biosynthesis being the most susceptible. The inhibitory effect of CMNQ on the formation of DNA was, at least in part, the result of a prevention of the conversion of thymidine to its nucleotide forms. This action was due to (a) a drug-induced decrease in intracellular levels of adenosine 5'-triphosphate, presumably resulting from uncoupling of oxidative phosphorylation by CMNQ; and (b) a partial loss of thymidine kinase activity in Sarcoma 180 cells, which did not appear to be due to direct inhibition of the enzyme by the drug. Although the primary event produced by CMNQ at the mitochondrial level appeared to be release of respiratory control, other effects of mitochondrial metabolism occurred. These included inhibition of reduced nicotinamide adenine dinucleotide and succinoxidase activities, as previously demonstrated, and mitochondrial swelling, which suggested interaction of CMNQ with the inner mitochondrial membrane. These findings indicate a variety of biochemical lesions are associated with the antineoplastic activity of CMNQ and demonstrate a relationship between the effects of this drug on mitochondrial respiratory control and DNA biosynthesis.
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PMID:Mode of action of the bioreductive alkylating agent, 2,3-bis(chloromethyl)-1,4-naphthoquinone. 18 23

The suitability of non-replicating thymidine kinase deficient herpes simplex virus type 1 expressing bacterial beta-galactosidase (tk-lacZ HSV-1) as a transfer vehicle into sympathetic preganglionic neurons in vivo was assessed. Many sympathoadrenal preganglionic neurons (451 +/- 105) with normal morphology were identified using beta-galactosidase histochemistry two days after inoculation of tk-lacZ HSV-1 into the adrenal gland of hamsters. Beta-galactosidase activity co-localized with nicotinamide adenine dinucleotide phosphate-diaphorase-positive sympathetic preganglionic neurons in the nucleus intermediolateralus, pars principalis. The maximal number of beta-galactosidase expressing neurons was found two days post-inoculation but this number dropped dramatically after this time. An inflammatory infiltrate was abundant around infected neurons and in the white matter at five days and infected neurons appeared morphologically abnormal. At 26 days, the infiltrate was still present but no infected sympathoadrenal preganglionic neurons were detected. Approximately 25% fewer nicotinamide adenine dinucleotide phosphate-diaphorase-positive neurons in the nucleus intermediolateralis, pars principalis were counted ipsilaterally than contralaterally in animals infected for 14, 21 or 26 days with tk-lacZ HSV-1, compared to the 3% difference in animals mock-infected for 26 days. Approximately 33% of the estimated number of sympathoadrenal preganglionic neurons infected with tk-lacZ HSV-1 at five days were apoptotic or necrotic. About 60% of neurons infected with tk-lacZ HSV-1 at two days no longer expressed nicotinamide adenine dinucleotide phosphate-diaphorase at 14-26 days. In conclusion, the non-replicating thymidine kinase deficient HSV-1 was efficiently retrogradely transported from the adrenal gland to infect sympathoadrenal preganglionic neurons. These gene transfer experiments using tk-lacZ HSV-1 suggest that foreign gene expression in sympathetic preganglionic neurons in vivo may be maximal two days after inoculation when beta-galactosidase was expressed in the greatest number of sympathetic preganglionic neurons. After two days, fewer neurons expressed beta-galactosidase and the presence of tk-lacZ HSV-1 appeared to be altering protein expression in sympathetic preganglionic neurons and/or leading to the demise of the infected neuron.
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PMID:Gene transfer into sympathetic preganglionic neurons in vivo using a non-replicating thymidine kinase-deficient herpes simplex virus type 1. 927 1

The ThyA gene that encodes for thymidylate synthase (TS) is absent in the genomes of a large number of bacteria, including several human pathogens. Many of these bacteria also lack the genes for dihydrofolate reductase (DHFR) and thymidine kinase and are totally dependent on an alternative enzyme for thymidylate synthesis. Thy1 encodes flavin-dependent TS (FDTS, previously denoted as TSCP) and shares no sequence homology with classical TS genes. Mechanistic studies of a FDTS from Thermotoga maritima (TM0449) are presented here. Several isotopic labeling experiments reveal details of the catalyzed reaction, and a chemical mechanism that is consistent with the experimental data is proposed. The reaction proceeds via a ping-pong mechanism where nicotinamide binding and release precedes the oxidative half-reaction. The enzyme is primarily pro-R specific with regard to the nicotinamide (NADPH), the oxidation of which is the rate-limiting step of the whole catalytic cascade. An enzyme-bound flavin is reduced with an isotope effect of 25 (consistent with H-tunneling) and exchanges protons with the solvent prior to the reduction of an intermediate methylene. A quantitative assay was developed, and the kinetic parameters were measured. A significant NADPH substrate inhibition and large K(M) rationalized the slow activity reported for this enzyme in the past. These and other findings are compared with classical TS (ThyA) catalysis in terms of kinetic and molecular mechanisms. The differences between the FDTS proposed mechanism and that of the classical TS are striking and invoke the notion that mechanism-based drugs will selectively inhibit FDTS and will not have much effect on human (and other eukaryotes) TS. Since TS activity is essential to DNA replication, the unique mechanism of FDTS makes it an attractive target for antibiotic drug development.
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PMID:Mechanistic studies of a flavin-dependent thymidylate synthase. 1530 27

The gene coding for arylformamidase (Afmid, also known as kynurenine formamidase) was inactivated in mice through the removal of a shared bidirectional promoter region regulating expression of the Afmid and thymidine kinase (Tk) genes. Afmid/Tk -deficient mice are known to develop sclerosis of glomeruli and to have an abnormal immune system. Afmid-catalyzed hydrolysis of N-formyl-kynurenine is a key step in tryptophan metabolism and biosynthesis of kynurenine-derived products including kynurenic acid, quinolinic acid, nicotinamide, NAD, and NADP. A disruption of these pathways is implicated in neurotoxicity and immunotoxicity. In wild-type (WT) mice, Afmid-specific activity (as measured by formyl-kynurenine hydrolysis) was 2-fold higher in the liver than in the kidney. Formyl-kynurenine hydrolysis was reduced by approximately 50% in mice heterozygous (HZ) for Afmid/Tk and almost completely eliminated in Afmid/Tk knockout (KO) mice. However, there was 13% residual formyl-kynurenine hydrolysis in the kidney of KO mice, suggesting the existence of a formamidase other than Afmid. Liver and kidney levels of nicotinamide plus NAD/NADP remained the same in WT, HZ and KO mice. Plasma concentrations of formyl-kynurenine, kynurenine, and kynurenic acid were elevated in KO mice (but not HZ mice) relative to WT mice, further suggesting that there must be enzymes other than Afmid (possibly in the kidney) capable of metabolizing formyl-kynurenine into kynurenine. Gradual kidney deterioration and subsequent failure in KO mice is consistent with high levels of tissue-specific Afmid expression in the kidney of WT but not KO mice. On this basis, the most significant function of the kynurenine pathway and Afmid in mice may be in eliminating toxic metabolites and to a lesser extent in providing intermediates for other processes.
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PMID:Effect of arylformamidase (kynurenine formamidase) gene inactivation in mice on enzymatic activity, kynurenine pathway metabolites and phenotype. 1586 19

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