PURIFICATION AND CHARACTERIZATION OF RIBONUCLEASE FROM RABBIT RETICULOCYTES.

Abstract

A method is described for the purification of ribonmuclease (EC 2.7.7.16) from rabbit reticulocytes by heart treatment at pH 2.5 followed by chromatography on a carboxymethylcellulose column. The enzymei was purified 5000-fold, and was free from both acid and alkaline phosphatases (EC 3.I.3.2 and EC 3.I.3.I, respectively), non-specific phosphodiesterase (EC 3.I.4.I) and deoxyribonuclease (EC 3.I.4.5). The enzyme had a pH optimum at 5.8. It was heat and acid stable and could be stored at —20° for I year without loss of activity. The products of hydrolysis of yeast ribonucleic acid by the enzyme were found to contain both 2′- and 3′-purine and pyrimide nucleotides,t he ratio of ratio of adenylic to cytidylic acid being approximately I:I. An incomplete digest of yeast ribonucleic acid by the enzyme was separated on a diethylaminoethyl-cellulose column and both mono- and oligonucleotide fractions were identified. Nucleoside 2′- and 3′-cyclic phosphates were hydrolyzed to their corresponding non-cyclic mononucleotides by the enzyme. A possible physiological role of the enzyme is discussed on the basis of the following experiments: (I) degradation of rabbit reticulocyte ribosome by the enzyme; (2) chronological difference in its activity in rabbit reticulocytes; and (3) intracellular localization of the enzyme.