The phosphorylation of dadenosine and dguanosine is a critical step in the accumulation of toxic levels of intracellular deoxynucleoside triphosphates. In addition, these reactions are essential for the activation of antiviral and anticancer nucleoside analogs. This proposal will focus upon the enzymes which phosphorylate dadenosine and dguanosine. We will test the hypothesis that the regulation of dadenosine and dguanosine phosphorylation is mainly dependent upon the concentration of dadenosine and dguanosine. The approach to this problem will include: a) Purification and characterization of dadenosine and dguanosine phosphorylating activities from human placental cytoplasm, and b) performance of careful kinetic studies of the purified activities. The methods will include radiochemical assays of nucleoside kinase enzymes, affinity chromatography, ion exchange chromatography, other enzyme fractionation techniques, initial velocity studies and product inhibition studies. This approach will lead to an understanding of the regulation of the enzymes which phosphorylate dadenosine and dguanosine. It is hoped that new insights will be obtained into the metabolic basis for the accumulation of toxic deoxynucleotides in human cells and the development of noval antiviral and anticancer nucleoside analogs will be facilitated.
