Soluble aminopeptidases from cytosol and luminal contents of Rhynchosciara americana midgut caeca : Properties and phenanthroline inhibition

Abstract

Rhynchosciara americana midgut caecal cells contain two major cytosolic aminopeptidases which are resolved by electrophoresis but which have the same M r value (115,700), as determined by gradient ultracentrifugation, and have pI values of 8.7 and 7.8. Electrophoretic migration of the two aminopeptidases in polyacrylamide gels of different concentration suggests they differ only in net charge. Thermal inactivation of both cytosolic aminopeptidases follow apparent first-order kinetics with identical half-lives. The two cytosolic aminopeptidases have the same K m values when either leucine-p- nitroanilide or arginine-p- nitroanilide are substrates. Midgut luminal fluid displays two major aminopeptidases resolved by electrophoresis which have the same properties as the two cytosolic aminopeptidases. The cytosolic aminopeptidases purified by electrophoresis have the same pH optimum of 8.0 and Tris ( K i 107 mM) and 1,10-phenanthroline ( K i 14 μM) both act as simple linear competitive inhibitors. The enzymes are true aminopeptidases with a broad specificity towards aminoacyl-β-naphthylamides and are more active on tetra and tripeptides than on dipeptides. The data support the assumption that the cytosolic aminopeptidases from caecal cells, which are similar to those in luminal fluid, are enzymes en route to their being secreted and that they differ only in net charge. Furthermore, the properties of the aminopeptidases are in accordance to their proposed role of oligopeptide digestion in the ectoperitrophic fluid.