Studies on salivary gland ribonucleases. III. Purification and properties of three ribonucleases from bovine parotid gland.

Three alkaline ribonucleases [EC 3.1.4.22] were purified 4,500- to 7,850-fold from bovine parotid gland by repeated CM-Sephadex C-25 chromatography and Sephadex G-50 gel filtration, with a total recovery of about 33%. They were designated as RNase BP1, BP2, and BP3, based on their order of elution from a CM-Sephadex C-25 column. The molecular weights of these enzymes were estimated by gel electrophoresis to be 18,500, 18,000, and 14,000, respectively. These enzymes are very similar to RNase A in that they are inhibited by heparin, show preferential hydrolysis of C5'-O-P linkages adjacent to a cytosine nucleotide rather than a uracil nucleotide, and in their antigenic properties. Spermine was found to stimulate the activities of these enzymes; the degree of stimulation was in the order of RNase BP3 greater than BP2 greater than BP1. The stimulation by spermine is mainly due to the increased cleavage of C5'-O-P linkages adjacent to cytosine nucleotides. From the results of amino acid analysis, glycosidase digestion and amino-terminal sequencing, it was suggested that the differences in molecular weights of RNase BP1, BP2, and BP3 are due to the differences in carbohydrate contents. In addition, it was suggested that RNase BP3 is identical with RNase A.