HETEROGENEITY IN THE GLYCOSYLATION PATTERN OF HUMAN PANCREATIC RIBONUCLEASE

Different molecular forms of ribonuclease were isolated from fresh hum an pancreas obtained from healthy transplant donors. The purification procedure consists of the preparation of an acetone powder, followed b y (NH4)(2)SO4 precipitation and two chromatography steps (cationic exc hange and reversed-phase). Protein bands in gel electrophoresis with R NAase activity were monitored using a negative-staining zymogram techn ique. Several glycosylated enzyme forms with apparent molecular masses ranging from 14 to 40 kDa were separated. Peptides containing the thr ee Asn-Xaa-Thr/Ser acceptor sites for glycosylation were isolated and analysed. The site with Asn-34 was almost completely glycosylated, whi le the sites with Asn-76 and Asn-88 had carbohydrate in about half and a minor part of the molecules, respectively. The carbohydrate composi tions of the glycopeptides are different from those of the same gene p roduct isolated from human urine. C-Terminal threonine was present in part of the molecules, indicating partial degradation by carboxypeptid ase.