Guanosine anabolism for biosynthesis of nucleic acids in Novikoff ascites rat tumor cells in culture.

phosphate intermediates, judging from lack of contribution to adenine, uracil, and cytosine nucleosides in RNA and DNA. The group of enzymatic activities that catalyze the conversion of guanine and guanosine to guanosine triphosphate (namely, guanosine kinase, purine nucleoside phosphorylase, purine nucleoside monophosphate kinase, nu cleoside diphosphate kinase, and purine nucleoside triphosphate phosphatase) have been prepared from an extract of Novikoff ascites cells in a single procedure by the use of diethylaminoethyl cellulose. Gel permeation chromatography on Sephadex G-150 was used to resolve these enzymes sufficiently to permit determination of their individual activities, substrate specificities, molecular weight, and other characteristics. New rapid assays were developed for purine nucleoside kinase and phosphorylase, utilizing la beled nucleosides with chromatography on diethylamino ethyl paper. These techniques were designed to be useful for the measurement of the individual enzymes of the guanosine salvage pathway in studies of nucleotide metabolism and therapeutic effects. Practical methods for culture in suspension of Novikoff ascites tumor cells and for determination of the rates of incorporation of guanosine (and other nucleic acid precur sors) into RNA and DNA are described. RNA and DNA are extracted with hot 2.5 M potassium acetate and sepa rated by use of alkali in a form convenient for resolution of individual nucleotides and bases by electrophoresis and chromatography.