Biochemical characterization of equine herpesvirus type 3-induced deoxythymidine kinase purified from lytically infected horse embryo dermal fibroblasts.
Infection of horse KyED cells with equine herpesvirus type 3 (EHV-3) resulted in a sevenfold increase in cytosol deoxythymidine kinase (dTK) activity. The EHV-3 dTK was purified from KyED cytosol dTK by affinity chromatography on deoxythymidine-Sepharose and characterized with respect to its electrophoretic mobility, molecular weight, substrate specificity, phosphate donor specificity, and immunological specificity. The purified EHV-3 dTK migrated in polyacrylamide gels with an Rf of 0.30 and sedimented in glycerol gradients with an S value of 5.13, corresponding to a molecular weight of 83,000. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis yielded a single band with a molecular weight of 38,000 to 40,000. Antiserum prepared against the EHV-3 dTK induced in KyED cells neutralized the EHV-3-induced enzyme activity but not the dTK purified from uninfected cells. EHV-3 dTK was less sensitive to feedback inhibition to dTTP and had a lower Ki for the antiviral compound 1-beta-D-arabinofuranyosylthymine and a lower Km for the substrate deoxythymidine. These results indicate that infection of cells with EHV-3 results in the induction of a new virus-coded dTK activity which meets the criteria of Jensen for an evolutionary primitive enzyme.
D000818:Animals; D002413:Cations, Divalent / Q000494:pharmacology; D002460:Cell Line; D004790:Enzyme Induction; D005347:Fibroblasts; D006564:Herpesviridae / Q000201:enzymology* / Q000254:growth & development; D006736:Horses; D007700:Kinetics; D008970:Molecular Weight; D009711:Nucleotides / Q000378:metabolism; D013379:Substrate Specificity; D013937:Thymidine Kinase / Q000302:isolation & purification / Q000378:metabolism*
D002413:Cations, Divalent; D009711:Nucleotides; D013937:Thymidine Kinase