Limited trypsinolysis of porcine and equine colipases. Spectroscopic and kinetic studies

Abstract

Porcine and equine colipases have been submitted to mild tryptic digestion. Proteolysis occurs at the Arg 5Gly 6 bond with the loss of the N-terminal pentapeptide. Studies of native and trypsin-treated colipases by circular dichroism and laser chemically induced dynamic nuclear polarization indicate that proteolysis induces conformational changes in the region of the tyrosine cluster. Experiments in the presence of phospholipid provide further evidence showing that these residues are in or close to the region of the protein interacting with aggregated lipids. Kinetic studies of the reaction of bile salt-inhibited lipase with emulsified triolein in the absence and in the presence of lecithin show that tryptic hydrolysis of the protein cofactor increases its affinity for the enzyme in the presence of lipid substrate. In both cases, it was found that the apparent dissociation constant of the lipase-colipase complex is decreased by one order of magnitude. Our results confirm that the biological activity of the lipase cofactor is enhanced by specific tryptic cleavage in the amino terminal region of the polypeptide and support the suggestion by Borgström et al. (Borgström, B., Wieloch, T., Erlanson-Albertsson (1981) FEBS. Lett. 108, 407–410) that the secreted form of colipase is a precursor.