The ribonucleases of bovine skeletal muscle

Abstract

Bovine skeletal muscle contains small amounts of at least six heat- and acid-stable RNA-degrading enzymes. Our results are the first evidence for multiple ribonucleases in skeletal muscle. Three of these have been highly purified, and each has been shown to be a pyrimidine-specific endoribonuclease by use of a rapid sequencing technique employing gel electrophoresis. However, synthetic co-polymers containing adenylate or guanylate residues in addition to pyrimidine residues are hydrolysed at higher rates than are the pyrimidine homopolymers. With 0.63 mM yeast RNA as substrate, all three enzymes (ribonucleases I, II and III) are optimally active in alkaline solution (pH 7.5-8.5) containing 0.05-0.15 M univalent salts, do not require bivalent cations, and have molecular weights of 13 000-20 000. The properties of muscle ribonuclease I are very similar to those of bovine pancreatic ribonuclease A. Muscle ribonucleases II and III have characteristics similar to those of ribonucleases found in various other bovine tissues. In common with all previously studied pyrimidine-specific endoribonucleases, the bovine muscle ribonucleases are inhibited by such purine homopolynucleotides as polyadenylate. Furthermore, polyamines, present in low concentrations, can reverse or regulate the amount of inhibition of enzyme activity.

Use of polynucleotide/polyacrylamide-gel electrophoresis as a sensitive technique for the detection and comparison of ribonuclease activities

“…These interactions would not lead to enzyme-catalysed degradation of substrate. In this connection, it is noteworthy that the rate of hydrolysis of the synthetic polymer by an enzyme does not serve as a guide to the migration of that enzyme in the presence of the polynucleotide (compare Table 1 with Table 3 of Davies et al, 1980). It is conceivable that the low concentrations of polynucleotides used cause changes in the average pore size of the gel, and that this change is therefore related to individual polynucleotides.…”

“…Synthetic polynucleotides were the products of Miles Laboratories, Elkhart, IN, U.S.A. RNAase A from bovine pancreas was supplied by Worthington Biochemical Corp., Freehold, NJ, U.S.A. Details of the purification of bovine muscle RNAases, as well as the procedure for measuring enzyme activity, are described in the preceding paper (Davies et al, 1980).…”

“…In the preceding paper (Davies et al, 1980) the properties of three polycationic RNAases from bovine muscle were described. In many respects these enzymes presented considerable difficulties in protein characterization because of their low tissue concentrations.…”