Isolation and Characterization of Proinsulin C-Peptide from Bovine Pancreas

Abstract Although the enzymes that catalyze the transformation of proinsulin to insulin have not yet been identified, studies of intermediate forms of proinsulin isolated from bovine pancreas indicate the existence of a mechanism in which the interchain connecting peptide is cleaved with elimination of a pair of basic residues from each end to yield insulin and the intact remainder of the connecting peptide segment. This acidic peptide, designated the C-peptide, has been isolated from fresh bovine pancreas and purified by means of a procedure involving acid-ethanol extraction, gel filtration, carboxymethylcellulose chromatography, paper electrophoresis, and partition chromatography. The peptide was found to occur on an equimolar basis with insulin, as expected from the stoichiometry of the cleavage process. The yield of both peptides was approximately 10 µmoles per kg of pancreas. Analysis of the amino acid sequence of the peptide and its chymotryptic fragments demonstrated its structural identity with the authentic proinsulin C-peptide prepared by tryptic digestion of the bovine proinsulin intermediate fraction. These findings are in accord with the hypothesis that the cleavage of proinsulin occurs within newly formed secretory granules and the products are retained therein until they are discharged together by means of fusion of the granule membrane with the cell membrane in the process of emiocytosis.