Trypsin secretion in Musca domestica larval midguts : A biochemical and immunocytochemical study

Abstract

Musca domestica larvae have trypsin in their midgut cells and contents, mainly at the posterior region. Both soluble and membrane-bound forms of trypsin are found. Membrane-bound trypsin is not affected by papain or phosphatidylinositol-specific phospholipase C, although it is solubilized with equal efficiency by detergents with high and low critical micellar concentrations. The solubilization increases at high pH values in the presence or absence of detergent. Once solubilized, membrane-bound trypsin behaves as a hydrophilic protein. M. domestica trypsin was purified to homogeneity and used to raise antibodies in a rabbit. A Western blot of M. domestica membrane-bound and soluble midgut proteins, after sodium dodecyl sulfate-polyacrylamide gel electrophoresis using trypsin antiserum, showed only bands co-migrating with trypsin. With this antiserum, trypsin was immunolocalized in the membranes of secretory and Golgi vesicles, surface of microvilli and midgut contents of M. domestica posterior midgut. The data suggest that trypsin is bound to the secretory vesicle membrane by a hydrophobic peptide anchor. Upon exocytosis, most trypsin is solubilized apparently because the neutral pH of the luminal contents causes a conformational change which hinders part of the trypsin anchor.