Development of an Improved Assay for Purine Nucleoside Kinase Activity in Cell Extracts and Detection of Inosine Kinase Activity in Brevibacterium acetylicum ATCC 953, Related Species, and Corynebacterium flaccumfaciens ATCC 6887

Abstract

  An improved assay was developed to detect direct purine nucleoside phosphorylating activity in cell-free extracts. Direct inosine phosphorylating activity was detected in 2 of 70 species tested. Both activities, which depended on magnesium ion and ATP, phosphorylated a hydroxyl group at the 5′ position of inosine. The new assay was shown to be useful for screening of direct purine nucleoside phosphorylating activity and have the potential to detect inosine kinase in the presence of a background of nucleoside phosphorylase and purine phosphoribosyltransferase activities. Previously, the latter two activities made it difficult to correctly detect direct phosphorylation of inosine by inosine kinase.