Vaccinia virus-induced ribonucleotide reductase can be distinguished from host cell activity.
Increased ribonucleotide reductase activity has been detected in vaccinia virus-infected BSC-40 cells. We have studied certain biochemical and kinetic properties of CDP reduction in extracts from infected and uninfected cells. ATP inhibited reductase activity in crude extracts by rapid and extensive substrate phosphorylation. Substitution of adenylylimido-diphosphate (AMP-PNP), a noncleavable analog that functions as positive activator for reductase, but inhibits phosphorylation and cleavage of substrate, allowed us to reliably measure reductase activity. In the presence of AMP-PNP, CDP reduction by extracts from infected or uninfected cells was linear with time for 60 min and with enzyme concentration, except at very low enzyme levels. Activities from both sources were optimally active at pH 8.1. Variation of AMP-PNP and Mg2+ concentrations revealed, however, that in the absence of exogenous Mg2+, AMP-PNP strongly stimulated virus-induced CDP reduction, but inhibited endogenous CDP reduction. In the presence of the activator, increasing Mg2+ concentrations progressively inhibited the induced activity, but stimulated the endogenous activity up to a 1:2 Mg2+/activator molar ratio. The vaccinia virus-induced activity was highly dependent on AMP-PNP and was not detectable over underlying cellular activity in its absence. Determination of substrate kinetics with respect to CDP revealed a threefold-lower Km for the virus-induced enzyme as compared with the cellular enzyme. These data suggest, but do not prove, that a novel ribonucleotide reductase is expressed on infection by vaccinia virus.
D000255:Adenosine Triphosphate / Q000494:pharmacology; D000266:Adenylyl Imidodiphosphate / Q000494:pharmacology; D000818:Animals; D002460:Cell Line; D002472:Cell Transformation, Viral; D002522:Chlorocebus aethiops; D003565:Cytidine Diphosphate / Q000378:metabolism; D004790:Enzyme Induction; D006863:Hydrogen-Ion Concentration; D007668:Kidney; D007700:Kinetics; D012264:Ribonucleotide Reductases / Q000096:biosynthesis* / Q000378:metabolism; D014616:Vaccinia virus / Q000201:enzymology*
D000266:Adenylyl Imidodiphosphate; D003565:Cytidine Diphosphate; D000255:Adenosine Triphosphate; D012264:Ribonucleotide Reductases