Isolation and specificity of the intracellular ribonuclease from Tetrahymena pyriformis

Abstract

Ribonuclease was isolated from Tetrahymena pyriformis and found to be heterogeneous, consisting of three enzymatically active components (I, II, III). Following acid extraction, the components were separated by elution from SE-Sephadex buffered in 0.1 M sodium acetate (pH 4.5) and using a linear NaCl gradient. Rechromatography of these components in the same system resulted in a purification of 400-, 215-, and 140-fold for I, II, and III, respectively. These three components did not hydrolyze DNA, p-nitrophenyl phosphate or bis- p-nitrophenyl phosphate in the presence or absence of EDTA. All three components exhibited maximum activity at pH 5.0. Both EDTA and sodium citrate at 0.0088 M brought about a stimulation of ribonuclease activity. Urea, at 4.0 M, increased the activity of each component approx. 30 %, while at 8.0 M the components were differentially inhibited, i.e., 14 %, 51 % and greater than 99 % for I, II, and III, respectively. The three components degrade synthetic polyribonucleotides with different initial velocities. Polyadenylic acid (poly (A)) and polyinosinic acid (poly (I)) are hydrolyzed readily in the following order: Component III > II ⪢ I. Polycytidylic acid (poly(C)) however, is hydrolyzed most rapidly by Component I, to a lesser degree by Component II and not at all by Component III, unless urea (4.0 M) is added to the reaction mixture. All three components degrade polyuridylic acid (poly(U)) at the same initial rate, but quantitative differences in mononucleotides are found at equilibrium. Each polymer is hydrolyzed to 3′-nucleotides via a 2′,3′-cyclic intermediate.