Antoinette E. Greco

PURIFICATION AND CHARACTERIZATION OF RIBONUCLEASE OF CALF SPLEEN

Several intracellular ribonuclease (RNase) activities have been de~cribedl-~ since i t was established6 that the RNase of spleen and thymus differed from the crystalline pancreatic RNase. The procedures used have provided RNase preparations that exhibit characteristic substrate specificities and that differ in their pH optima on ribonucleic acid (RNA), their ability to attack purine linkages, their magnesium ion requirements, and their heat stability. The spleen phosphodiesterase activities described by Heppel and Hilmoe3 and Maver and Greco4 have many characteristics in common, although the methods used differ markedly. Both preparations degrade RNA, “core”, and the benzyl esters of ribonucleotides to yield exclusively the nucleoside-3’-phosphates, and the 2 enzyme preparations hydrolyze the cyclic purine and pyrimidine nucleotides to form nucleoside-2’-phosphates, but the &laver and Greco preparation also forms some nucleoside-3’-phosphate. Both are more sensitive to heating than the pancreatic RNase, and their activity on RNA is inhibited by magnesium ions a t their pH optimum of 5.6 to 5.8. With a very different method of purification, Kaplan and Heppe15 have prepared a spleen RNase that closely resembles the pancreatic RNase in its catalytic properties and heat stability. RNA is not completely hydrolyzed, and only pyrimidine mononucleotides are formed. This spleen RNase differs from the pancreatic RNase only in its more acid PH optimum on RNA and in its behavior on the cation exchange resin XE-64. These investigators found that approximately 70 per cent of the RNase activity of the homogenate was destroyed in an initial heating step in their purification procedure, which involved 10 min. heating a t 60” C. a t fH 3.5. Most of the activity of the Rlaver and Greco preparations would have been destroyed by this treatment. The present work describes the RNase activities of fractions obtained when spleen nuclease preparations were subjected to chromatographic analysis on cellulose anion and cation exchangers. This report is an extension of preliminary experiments.’