Deoxycytidine Kinase: II. PURIFICATION AND GENERAL PROPERTIES OF THE CALF THYMUS ENZYME

Abstract

Abstract Deoxycytidine kinase has been purified 500-fold from calf thymus. It is concluded that the enzyme will catalyze the phosphorylation of deoxycytidine, 1-β-d-arabinofuranosyl cytosine, deoxyguanosine, and deoxyadenosine since the protein catalyzing these activities showed coincident purification. Also, this protein could not be further fractionated by isoelectric focusing, gel filtration, or sucrose density gradient centrifugation, and the nucleosides mutually inhibited each other's phosphorylation. However, the deoxycytidine-phosphorylating capacity of the preparation is considerably more stable than all other nucleoside-phosphorylating activities. The sedimentation constant, partial specific volume, and Stokes radius of the enzyme were estimated by zonal centrifugation, isodensity centrifugation, and gel filtration, respectively. These parameters were then used to calculate the molecular weight (56,000) and frictional ratio (1.36) of deoxycytidine kinase. The enzyme is very sensitive to sulfhydryl group inhibitors and is unstable in the absence of thiol; activity can be restored by incubating the enzyme with thiol compounds. Enzyme activity is fairly constant over the pH range 6 to 10, but there are marked effects of pH on nucleotide inhibition and heat stability. UDP, dTDP, dCMP, dCDP, and dCTP are all inhibitors of deoxycytidine kinase, but these inhibitions are reversed by UTP, dUTP, and dTTP.