Nucleases of membrane-bound rat liver ribosomes

Abstract

Highly purified cytoplasmic membrane-bound ribosomes from rat liver, containing 63 % RNA and characterized by latent ribo- and deoxyribonuclease activity were isolated. On treatment of ribosomes with 500 mM LiCl, NaCl, and NH 4Cl solutions, which caused 15–20 % loss of the ribosomal protein, only a partial loss of the nuclease activity was observed. Crude, unpurified ribosomes revealed both 3′-ribonuclease and 5′-ribonuclease activities, their pH optima being 8.1 and 7.2, respectively. Apart from that, they possessed two kinds of deoxyribonuclease activity with pH optima of 8.1 and 5.3. As the purification process proceeded ribosomes lost their 5′-ribonuclease and acid deoxyribonuclease activities whereas the 3′-ribonuclease and alkali deoxyribonuclease specific activity slightly increased. Several lines of evidence clearly indicate that both ribonuclease and deoxyribonuclease activities are confined solely to small ribosomal subunits, the large ones being completely devoid of it. Latency can be ascribed only to the ribonuclease of intact ribosomes since the large subunit seems to protect the 18-S RNA molecule to a certain extent from attack by the enzyme residing in the small subunit as long as the structure of the whole ribosomal particle is preserved. On the other hand, nearly immediate degradation of the 18-S RNA molecule began in small separated subunits (in the medium optimal for the action of ribosomal ribonuclease) even while kept at 0°C. The experimental data presented in this paper do not support the alternative view that latency of ribosomal ribonuclease may be the result of an inactive dissociable complex formed by the enzyme and a ribosomal protein(s) with inhibitory properties. Enzyme preparations obtained by fractionation with (NH 4) 2SO 4 (0.4–0.8 satn) from purified ribosomes displayed both ribonuclease and deoxyribonuclease activity. Successive chromatography of these preparations on a DEAE-Sephadex A-50 column enabled us to separate the two activities. Both ribosomal enzymes seemed to be endonucleases, for even exhaustive enzyme hydrolysis of RNA and DNA yielded only relatively small mononucleotide fractions (10 and 4–5 %, respectively). The ribosomal ribonuclease appears to differ from pancreatic and alkali ribonuclease (a) in pH optimum, (b) in its action both on pyrimidine 2′:3′-cyclic phosphate and on poly(A) and poly(U) and (c) in the base composition of the “core” and mononucleotide fractions of the enzyme RNA hydrolyzate. Analysis of these fractions indicated that the phosphodiester bonds formed by adenine (AX) were the most resistant to the action of the ribosomal ribonuclease. On the other hand, the bonds most accessible to the enzyme proved to be pyrimidylpyrimidine and guanidylpyrimidine. As to the specificity of ribosomal deoxyribonuclease, the most readily hydrolyzed internucleotide bonds in the DNA molecule appear to be those formed by the thymidyl residues (XT). The enzyme displayed a slight preference for native DNA as the substrate.