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)

The kinetics of formation, the stability at 40 degrees C and the serological properties of thymidine kinase and deoxycytidine kinase activities induced by herpes simplex virus have been examined. The results are consistent with the hypothesis that both activities are carried on the same molecule-a deoxypyrimidine kinase. Mutants deficient in deoxypyrimidine kinase have been used to produce, by absorption of general antisera, deoxypyrimidine kinase-specific antisera. Using immunoprecipitation and SDS-polyacrylamide gel electrophoresis, only one size of polypeptide (mol. wt. 42400 plus or minus 200) has been found, constituting the type 2 enzyme. This is close to published values for the type i enzyme but co-electrophoresis demonstrated that the polypeptide of the type i enzyme was slightly bigger.
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PMID:Deoxypyrimidine kinases of herpes simplex viruses types 1 and 2: comparison of serological and structural properties. 16 87

To investigate the size of herpes simplex virus (HSV) thymidine kinase (TK) polypeptides, procedures have been devised to purify the enzyme from infected cells labeled with 35S-methionine by (i) affinity chromatography on Sepharose-5'-amino-5'-deoxythymidine; (ii) preparative isoelectric focusing or preparative PAGE; and (iii) glycerol gradient centrifugation. Portions of enzyme fractions, at each purification step, were also treated with an immunoadsorbent, Sepharose-anti-HSV-1 TK immunoglobulin (IgG). Labeled polypeptides eluted from the immunoadsorbent were analyzed by electrophoresis in SDS slab gels and autoradiography. The results demonstrate that the molecular weights of HSV TK polypeptides are about 40,000. TK-negative HSV-1 mutant B2006 failed to induce the 40 K dalton polypeptide.
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PMID:Detection of herpes simplex virus thymidine kinase polypeptides in cells labeled with 35S-methionine. 20 86

The HSV thymidine kinase genetic system has a number of advantages for the study of virus and cell genetics. Both forward and reverse mutants are readily selected. The enzyme can be assayed directly in the presence of the cell TK by the choice of the appropriate substrate, for example, 125IdC. The gene product, HSV TK, can be identified by SDS-polyacrylamide gel electrophoresis and mutants can be found with alterations in the electrophoretogram. The in vitro synthesis of HSV TK in response to HSV mRNA has been achieved and can be used to analyse specific mutant virus.
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PMID:The thymidine kinase gene of herpes simplex virus type 1: cell-free protein synthesis and substrate specificity studies. 22 38

We constructed a recombinant thymidine kinase-negative herpes simplex virus type 1 (HSV-1) that expressed the rotavirus major outer capsid glycoprotein, VP7. In the recombinant HSV-1, a promoter from the 5' noncoding region of the HSV-1 glycoprotein B locus regulated the expression of VP7 as a HSV-1 gamma 1 gene product. HSV-1-expressed VP7 resembled rotavirus-expressed VP7 in its SDS-PAGE mobility, high mannose-type glycosylation, disulfide bonding, perinuclear to cytoplasmic localization, intracellular retention, and reactivity with polyclonal antisera and nonneutralizing antibodies. Unlike rotavirus-expressed VP7, HSV-1-expressed VP7 lacked several neutralizing epitopes by immuno-histochemical staining and by ELISA. One neutralizing epitope identified on HSV-1-expressed VP7 by ELISA was masked by paraformaldehyde fixation of recombinant HSV-1- but not rotavirus-infected cells. Neutralizing epitopes were restored to HSV-1-expressed VP7 by coinfection of cells with the HSV-1 recombinant and a heterologous rotavirus that lack the neutralizing epitopes. The recovered neutralizing epitopes were detected on double-shelled rotavirus particles produced in the coinfected cells. This study indicates that the formation of several neutralizing epitopes on rotavirus VP7 requires interaction of VP7 with other rotavirus proteins. In addition, HSV-1 was a useful vector for studying the localization, processing, and antigenicity of an RNA virus glycoprotein.
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PMID:Neutralizing epitopes on herpes simplex virus-1-expressed rotavirus VP7 are dependent on coexpression of other rotavirus proteins. 137 Oct 25

Previous papers have reported that the syncytial mutant HSV-1(13)S11 carries three segregable syn mutations and exhibits its altered phenotype in four different cell lines, i.e. HEp-2, VERO, BHK and HEL both at 34 degrees C and 39 degrees C. Those studies have shown that one of three syncytial loci, designated Syn 5, is located in the Bam HI Q fragment spanning map units 0.296-0.317 of the prototype arrangement. Recombinants obtained from marker transfer experiments with donor BamHI Q fragment, have shown that locus Syn 5 is able to induce cell-to-cell fusion in VERO, BHK and HEL but not in HEp-2 cells. In this paper we have characterized the syn mutant HSV-1(13)S11 with regard to plaque morphology, synthesis of viral polypeptides and glycoproteins, thymidine kinase activity and physical map position of locus Syn 5 on the genome. Pertinent to the syn phenotype, earlier papers claimed that two different polypeptides, thymidine kinase (TK) and glycoprotein H (gH), whose genes map in BamHI Q, may be responsible for the fusion activity. Functional studies on the TK of the syn mutant HSV-1(13)S11 indicate that this polypeptide accumulates normally in infected cells and is a fully active enzyme. The other gene product, gH, has been studied with SDS-PAGE and in radioimmunoprecipitation (RIP) experiments using specific monoclonal antibodies. The results indicate that the amount of gH accumulation in the syn mutant-infected cells is greater than its parental strain. However, new marker transfer experiments described here located locus Syn 5 in 663 base pairs between SstI and EcoRI restriction endonuclease sites at the right end of the BamHI Q fragment, where TK gene overlaps in opposite orientation with UL 24 gene. Altogether these results indicate that the Syn 5 locus segregates from the gene specifying gH, to a region encompassing portions of the TK and UL 24 genes, and that the syn mutation does not affect the expression or activity of TK.
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PMID:Phenotypic and genotypic characterization of locus Syn 5 in herpes simplex virus 1. 164 2

We have cloned the v-src gene of ts339B77 RSV into a new Moloney-based retroviral expression vector (YN). In the resulting construct (ts339YNsrc), the ts339 gene is transcribed from the viral LTR, while a selectable resitance marker, the Tn5 neomycin phosphotransferase (neor) gene, is transcribed from an internal thymidine kinase (Tk) promoter. G418 resistance and focus formation were induced in NIH3T3 cells at comparable efficiencies within the permissive temperature range (33-37 degrees C). At 39 degrees, on the other hand, focus induction was reduced 15-fold with no corresponding decrease in expression of G418 resistance. In cells infected with ts339YNsrc, phosphoproteins were elevated and similar in pattern on SDS PAGE regardless of whether the cells were grown at the permissive or restrictive temperature. The ts339YNsrc virus will be useful for the study of effects of v-src expression, and may also be of help in identifying relevant substrates of the v-src product.
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PMID:A selectable temperature-sensitive v-src Moloney retrovirus. 248 57

It was revealed that thymidylate kinase was purified together with cytosolic thymidine kinase from human term placenta by p-aminophenyl thymidine-3'-phosphate-CH-Sepharose affinity column chromatography, which has been commonly used for purification of thymidine kinase. In addition, it was noted that mitochondrial thymidine kinase and nucleoside diphosphate kinase were concurrently eliminated. In the presence of ATP, cytosolic thymidine kinase and thymidylate kinase could be separated from each other by Ultrogel AcA 34 filtration, and their molecular weights were estimated to be 70,000 and 50,000, respectively. On SDS-polyacrylamide gel electrophoresis, thymidine kinase protein exhibited a band of 26,000, which was compatible with the molecular weight of the enzyme subunit calculated from its cDNA, while thymidylate kinase protein showed 24,000. Thymidylate kinase could utilize either ATP or dATP as an efficient phosphate donor, and showed substrate specificity for dTMP.
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PMID:Co-purification of thymidylate kinase and cytosolic thymidine kinase from human term placenta by affinity chromatography. 253 59

Dose-response experiments show that the presence of 300 microM cicloxolone sodium (CCX) or 500 microM carbenoxolone sodium (CBX) during the HSV replication cycle reduced the infectious virus yield by 10,000- to 100,000-fold: CCX is the more potent anti-herpes agent. HSV-2 replication was consistently more severely restricted by either drug than was that of HSV-1. The ED50 values obtained for either drug against HSV-1 or HSV-2 correlate well with data from dose-response curves. CCX, and to a lesser extent CBX, can be cytotoxic but the degree of cytotoxicity varies between cell lines and is also affected by the physiological state of the cells. Triterpenoid drugs exhibit some activity against virus particles in suspension but the effect is small and contributes little to the overall antiviral effect. The drugs appear to be active throughout the replication cycle. In contrast to all other anti-herpesvirus agents in clinical use the triterpenoid compounds do not appear to act directly to block virus DNA synthesis. HSV mutants resistant to ACG and PAA, or lacking a thymidine kinase gene, appear as sensitive as wild-type virus to CCX inhibition. HSV growth in the presence of the drugs resulted in a lower number of assembled virus particles but reduced to a much greater extent the infectious virus yield: thus the progeny virus quality is greatly diminished. Thermolability of progeny virus correlated well with this diminution of quality in increasing CCX concentration. SDS PAGE analysis of the structural proteins of virus particles made in cells treated with 300 microM CCX revealed numerous differences in the relative intensities of protein bands, which is in keeping with the changed quality of the drug-produced virus. SDS PAGE analysis of the polypeptides induced in drug treated infected cells revealed two effects; some polypeptides were synthesised in reduced amounts and the nuclear/cytoplasmic distribution of certain proteins was affected. Post-translational processing by glycosylation and sulphation of both cellular and HSV induced proteins was strongly inhibited by the triterpenoid drugs, while phosphorylation of only a few polypeptides appeared to be affected.
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PMID:The antiviral activity against herpes simplex virus of the triterpenoid compounds carbenoxolone sodium and cicloxolone sodium. 302 56

Two hundred and thirty virus-induced polypeptides have been detected in BHK cells infected with herpes simplex virus type 1 (HSV-1; strain 17) by means of two-dimensional gel electrophoresis. The polypeptides have been characterized by both relative mobility following isoelectric focusing and apparent mol. wt. in SDS-polyacrylamide gels. Some polypeptides, visualized as a single band on a one-dimensional SDS-polyacrylamide gel, were resolved into several spots. Three were identified in Vmw43, the band thought to contain thymidine kinase activity. Not all the observed polypeptides are unique species: some appear to be related and have altered mobilities as a consequence of post-translational modification events. Pulse-chase experiments and treatment of infected cells with neuraminidase suggested that glycoproteins gB, gC and gD contain sialic acid and that synthesis of gB and gD occurs by at least 15 and 10 discrete steps respectively.
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PMID:Two-dimensional gel analysis of HSV type 1-induced polypeptides and glycoprotein processing. 626 77

L(O)cl 3 and L(K)cl 1 cells, biochemically transformed by varicella-zoster virus (VZV), were labelled with L-[35S]methionine and [32P]orthophosphate. Cell extracts were immunoprecipitated with anti-VZV monkey serum and analysed by SDS-polyacrylamide gel electrophoresis. Four polypeptides of apparent mol. wt. 135000 (135K), 48K, 44K and 35K were detected in the L-[35S]methionine-labelled extracts, and, of these, the 35K band showed marked intensity. However, this band was not detected in extracts from cells infected with a VZV tk- strain (Kanno strain). Also, the 35K polypeptide showed very low intensity when immunoprecipitated from extracts of transformed cells grown in non-selective (NS) medium, i.e. cells that had a very low thymidine kinase (tk) activity. In the case of [32P]orthophosphate-labelled cells, polypeptides of apparent mol. wt. 180K, 81K, 48K, 44K and 37K were obtained. In both instances, the 44K polypeptide was not immunoprecipitated from L(K)cl 1 cell extracts. From our data it is postulated that the expression of the 35K polypeptide is correlated with the VZV-specific tk activity of the cells.
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PMID:Expression of varicella-zoster virus-related antigens in biochemically transformed cells. 630 13


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