Heterogeneity in the glycosylation pattern of human pancreatic ribonuclease.

Different molecular forms of ribonuclease were isolated from fresh human pancreas obtained from healthy transplant donors. The purification procedure consists of the preparation of an acetone powder, followed by (NH4)2SO4 precipitation and two chromatography steps (cationic exchange and reversed-phase). Protein bands in gel electrophoresis with RNAase activity were monitored using a negative-staining zymogram technique. Several glycosylated enzyme forms with apparent molecular masses ranging from 14 to 40 kDa were separated. Peptides containing the three Asn-Xaa-Thr/Ser acceptor sites for glycosylation were isolated and analysed. The site with Asn-34 was almost completely glycosylated, while the sites with Asn-76 and Asn-88 had carbohydrate in about half and a minor part of the molecules, respectively. The carbohydrate compositions of the glycopeptides are different from those of the same gene product isolated from human urine. C-Terminal threonine was present in part of the molecules, indicating partial degradation by carboxypeptidase.