The chromatographic separation and characterization of the acid and alkaline ribonucleases of bovine spleen and liver.

The occurrence of both acid and alkaline ribonuclease activities in liver and kidney was reported in 1954 by Roth (1) and by de Lamirande et al. (2). Since then, investigators have used various methods to purify the intracellular ribonuclease activities (3-8). In our studies, comparatively mild procedures were used for the nuclease preparations (5)) with the hope that the nuclease activities as they occur in the tissues could be detected and later separated chromatographically. Previously (9), the acid and alkaline ribonuclease activities of spleen nuclease preparations were separated from each other by chromatographic fractionation. The further purification and characterization of these intracellular ribonucleases has been impeded by the presence of at least two enzymes which fractionate very closely with the ribonuclease activities on diethylaminoethyl cellulose. These enzymes are an acid phosphatase, which readily dephosphorylates adenylic acid, and a phosphodiesterase without ribonuclease activity, which hydrolyzes cyclic adenylic acid to yield 2’-adenylic acid (10). In this report, a chromatographic procedure is described which removes these enzymes from the acid and alkaline ribonucleases. The digests of yeast ribonucleic acid by the ribonucleases were mapped by paper electrophoresis followed by descending paper chromatography. Both the acid and the alkaline ribonucleases hydrolyzed yeast ribonucleic acid to cyclic mononucleotides, which were further hydrolyzed to 3’nucleotides after exhaustive digestion. When digestion was not complete, distinct preferential hydrolysis occurred in that the acid ribonuclease digests contained little or no cytidylic acid, depending upon the extent of digestion, and the amounts of adenylic acid in the alkaline ribonuclease digests were low.