A KEX2-RELATED ENDOPEPTIDASE ACTIVITY PRESENT IN RAT-LIVER SPECIFICALLY PROCESSES THE INSULIN PRORECEPTOR

The insulin proreceptor is cleaved by limited proteolysis posttranslationally at an Arg-Lys-Arg-Arg site to generate its mature alpha- and beta-subunit form. An S-35-labelled insulin proreceptor substrate preparation and a 15-mer peptide substrate that mimics the amino acid sequence around and including the insulin proreceptor processing site (IRP-peptide) has revealed an endopeptidase activity that catalyses insulin proreceptor cleavage in a rat liver subcellular fraction. Under optimal conditions, normal S-35-labelled insulin proreceptor substrate processing by this fraction was quantitative. This fraction was not able to process an S-35-labelled insulin proreceptor variant substrate (where the Arg(-1) of the tetrabasic cleavage site had been replaced by Ala(-1)), similarly to previous in vivo observations, suggesting that this endopeptidase activity has physiological relevance. Biochemical characterization of the insulin proreceptor/IRP-peptide processing revealed this rat liver endopeptidase activity to have a broad pH range (> 70% maximal activity between pH 5.5 and 10.0) and a pH optimum of pH 8-10. It was Ca2+-dependent activity, maximally active between 0.5 and 5 mM Ca2+ and half-maximally activated between 50 and 90 mu M Ca2+. Endoproteolytic activity was not inhibited by group-specific inhibitors of serine-, cysteinyl or aspartyl proteinases or by 1,10-phenanthroline; however, EDTA and 1,2-cyclohexanediaminetetraacetic acid did inhibit the activity, but this was accounted for by Ca2+ chelation. The IRP-peptide substrate assay enabled measurement of an apparent K-m of 22 mu M and a V-max of 18.6 pmol/min for this endopeptidase activity. These biochemical characteristics suggest the insulin proreceptor processing endopeptidase activity to be a legitimate member of the Kex2-related proprotein convertase family. Immunoblotting detected furin and PACE4 proteins (both members of this family) to be present in the rat liver subcellular fraction containing insulin proreceptor processing activity. Since the biochemical characteristics of the insulin proreceptor processing endopeptidase activity mostly resembled those of furin activity, it is likely that insulin proreceptor proteolytic maturation can be catalysed by furin in the liver.