Minor aminopeptidases purified from the plasma membrane of midgut caeca cells of an insect ( Rhynchosciara americana) larva

Abstract

Rhynchosciara americana midgut caecal cells display in their plasma membranes, as judged by electrophoresis, a major (T 1) and three minor (T 2, T 3 and T 4) aminopeptidases which are solubilized by Triton X-100, and a major (P 1) and two minor (P 2 and P 3) aminopeptidases which are released by papain treatment. Previous work showed that T 1 corresponds to P 1. Aminopeptidases T 1 and T 3 have the same M r value as determined by density-gradient centrifugation and by electrophoresis, and display different pI values, as judged by isoelectric focusing and electrophoresis. Furthermore, T 1 and T 3 purified by electrophoresis display identical K m values for arginine p- nitroanilide and leucine p- nitroanilide and the same K i values for leucine hydroxamate, hydroxyl amine and isoamyl alcohol. The data suggest that T 1 and T 3 differ only in net charge, implying that T 2 and T 4 should be related to P 2 and P 3. P 2 and P 3 purified by electrophoresis display identical K i values for arginine hydroxamate, leucine hydroxamate, hydroxylamine and isoamyl alcohol and also identical substrate specificities. P 2 and P 3 show a broad specificity in relation to the N-terminal aminoacyl residue and hydrolyses tetrapeptides more efficiently than tripeptides and much more efficiently than dipeptides. The data suggest that T 2 and T 4 as well as P 2 and P 3 are respectively different aggregation states of a minor native membrane bound aminopeptidase solubilized by detergent and released by papain. The native aminopeptidase is probably a tetramer of identical (or similar) subunits, which, due to its substrate specificities, produces short oligopeptides (mainly tripeptides) which are the preferred substrates for the major membrane-bound aminopeptidase.