SOLUBLE AND MEMBRANE-BOUND FORMS OF TRYPSIN-LIKE ENZYMES IN MUSCA-DOMESTICA LARVAL MIDGUTS

An enzyme hydrolyzing benzoyl-DL-arginine p-nitroanilide (BAPA) was purified from acid extracts of M. domestica larval midguts by using an S-Sepharose column and a soybean trypsin inhibitor (SBTI) agarose column. The yield, purification factor and final specific activity were, respectively: 67%, 154-fold, 20 U/mg of protein. The purified enzyme sediments as a molecule with M(r) 33,000 and displays two protein bands after SDS-polyacrylamide gel electrophoresis with M(r) 33,000 and M(r) 35,000. The enzyme has a pH optimum around pH 8.5, K(m) values toward N-benzoyl-DL-arginine p-nitroanilide of 0.121 mM and is inhibited by tosyl-L-lysine chloromethylketone, benzamidine, SBTI, and phenylmethyl-sulfonylfluoride. The enzyme is not affected by inhibitors of cysteine-proteinases or metallo-proteinases supporting the assertion it is a trypsin. Microvilli isolated from M. domestica posterior midguts have a trypsin-like enzyme that is solubilized by Triton X-100. The soluble purified trypsin, the microvillar, and the microvillar Triton X-100-solubilized trypsin display the same properties, except in relation to heating stability and to the enzyme-SBTI dissociation constant. Since these differences may be related to physical strains imposed on the enzyme in the membrane or in detergent micelles, the data suggest that most of the polypeptide chains of both the soluble and the membrane-bound trypsin are identical. These results support a postulated model for trypsin secretion by M. domestica larvae, according to which soluble trypsin contained in secretory vesicles is derived from a trypsin form that is associated with the vesicle membranes.