[42] UMP-CMP kinase from Tetrahymena pyriformis

Publisher Summary This chapter describes the purification procedure of uridine monophosphate–cytidine monophosphate (UMP-CMP) kinase enzyme from Tetrahyrnena pyriformis organism. As purified from Tetrahymena pyriformis , this kinase phosphorylates both UMP and CMP, and the ratio of these two activities remains constant during the purification. The purified enzyme also retains appreciable kinase activity for deoxycytidine monophosphate (dCMP). The present enzyme preparation was purified for the phosphorylation of UMP and, as a corollary, of CMP. In contrast to this substrate specificity, CMP–dCMP and UMP kinase activities can be separated in Escherichia coli extracts, and in Salmonella typhimurium , UMP and CMP kinases do not appear to be encoded by the same structural gene. The assay measures the conversion of radioactive UMP to labeled uridine diphosphate (UDP) with chromatographic isolation of the products formed. It is specific enough for use in either crude or purified fractions of the enzyme. The fractions from Sephadex are essentially free of UDP kinase, adenosine triphosphatase, and uridine diphosphatase; all of these activities are present in the original lysate.