Isolation and Characterization of Ovine Ribonuclease A, B, and C from Pancreatic Secretion

Chromatography over columns of CM-cellulose and Amberlite IRC-50 was used to fractionate the ribonucleases present in the pancreatic secretion of sheep. The chromatographic behavior of these enzymes was similar to that of the enzymes found in bovine pancreatic secretion, as was also their relative distribution. The principal component, ribonuclease A, accounted for ∼85% of the ribonuclease in the secretion. The remainder was present as several minor components, designated ribonucleases B, C, and D. Ribonucleases A, B, and C were isolated in a chromatographically homogeneous condition. Ribonuclease D, which may be a mixture of several components, was not studied further. Ribonucleases A, B, and C possessed the same amino acid composition, which differs by 8 residues from the composition of bovine ribonuclease A. Ovine ribonuclease B and C were found to be glycoproteins; ovine ribonuclease A was devoid of carbohydrate. Ribonuclease B contained ∼2 residues of N-acetylglucosamine and ∼6 of mannose. Ribonuclease C contained ∼5 residues of N-acetylglucosamine, ∼6 of mannose, ∼2 of galactose, ∼2 of fucose, and no sialic acid. Glycopeptides derived from residues 34 through 39 in the amino acid sequence of ovine ribonuclease (see Kobayashi, R., and Hirs, C.H.W. (1973) J. Biol. Chem. 248, 7833–7837) were isolated from tryptic hydrolysates of reduced, S-aminoethylated ribonuclease B and C. These glycopeptides have the NH2-terminal sequence Asn-Leu-Thr, a sequence homologous with the sequence in bovine ribonuclease B at which there is a site of carbohydrate-peptide attachment.