Preparation and properties of sea-urchin ribonuclease

Abstract

1. 1. A method is described for the purification of a ribonuclease (polyribonucleotide 2-oligonucleotide transferase (cyclizing), EC 2.7.7.16) from mature, unfertilized eggs of the sea urchin Psammechinus miliaris. The enzyme was purified 19 000-fold with a yield of 48% with the aid of chloroform treatment, (NH 4) 2SO 4 fractionation, and by chromatography on Sephadex G-100, on SE-Sephadex C-50 and on CM-Sephadex C-50. 2. 2. The enzyme had a pH-optimum at 5.3–5.5. 3. 3. The enzyme was heat labile, loosing about 50% of its activity in 3 min at 60° and pH 5.3, 70% of its activity in 3 min at 60° and pH 2.1, and 80% of its activity in 3 min at 40° and pH 9.0. 4. 4. The enzyme hydrolyzed yeast RNA completely to 2′,3′-cyclic nucleotides, and finally hydrolyzed the purine cyclic nucleotides slowly to their corresponding 3′-isomers. 5. 5. The enzyme hydrolyzed guanosine 2′-3′-cyclic phosphate about three times faster than adenosine 2′,3′-cyclic phosphate. 6. 6. The purified enzyme was found to be free of deoxyribonuclease and non-specific phosphodiesterase activity. 7. 7. A molecular weight of about 37 000 was estimated for the enzyme by comparative gel filtration experiments.