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Guanosine Anabolism for Biosynthesis of Nucleic Acids in Novikoff Ascites Rat Tumor Cells in Culture¹

The Department of Biochemistry, The University of Texas M.D. Anderson Hospital and Tumor Institute at Houston, Houston, Texas 77025

The utilization of labeled guanosine for the biosynthesis of RNA and DNA has been studied in cells cultured from the Novikoff ascites tumor of the rat. Guanosine contributed primarily to guanine moieties of RNA and DNA, whereas labeled adenosine contributed to both adenine and guanine moieties. The labeled ribose moiety of uniformly labeled guanosine-¹⁴C did not enter a pool of ribose 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, nucleoside 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 labeled nucleosides with chromatography on diethylaminoethyl 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 precursors) into RNA and DNA are described. RNA and DNA are extracted with hot 2.5 M potassium acetate and separated by use of alkali in a form convenient for resolution of individual nucleotides and bases by electrophoresis and chromatography.

¹ This work has been supported by NIH Research Grant CA-10407 to A. O. Development of the methods for cell culture and the "hot potassium acetate" procedures for extraction and separation of nucleic acids was supported by The American Cancer Society Research Grant P-146 to R. B. H. Additional support was provided by Grants G-460 and G-447 from the Robert A. Welch Foundation.

Received 12/20/72. Accepted 6/ 8/73.