Substrate-specific Analysis of Proteolytic Enzymes in the Larval Midgut of Calliphora vicina1

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Abstract
Data obtained from the hydrolysis of the trypsin-specific substrates, N-benzoyl-DL-arginine-p-nitroanilide and Nbenzoyl-L-arginine ethyl ester, by separate fore-midgut, mid-midgut, and hind-midgut homogenates at pH 7.0 confirm that trypsin-like or chymotrypsin-like enzymes are responsible for the basic proteolytic activity in each midgut segment of 3rd-stage larvae of Calliphora vicina (Robineau-Desvoidy). At least 2 such enzymes in each midgut segment are indicated by electrophoresis, and their mobilities are distinctly less than that of mammalian trypsin. Inhibition by soybean trypsin inhibitor of basic proteolytic activity of homogenates of each midgut segment confirms the presence of trypsin-like and chymotrypsinlike enzymes. Evidence for the presence of chymotrypsin is based on the hydrolysis of N-acetyl-L-phenylalanyl diiodotyrosine, which is specific for chymotryptic activity at pH 7.0. Hydrolysis of the pepsin-specific substrates, N-carbobenzoxy- a-glutamyl-L-tyrosine and N-acetyl-L-phenylalanyl diiodotyrosine, by fore-midgut and mid-midgut homogenates at pH 2.5 indicates that a pepsin-like enzyme is responsible for acidic proteolytic activity in these segments. Inhibition of acidic proteolytic activity by diphenyldiazomethane of fore-midgut and mid-midgut homogenates confirms the presence of a pepsin-like enzyme. Electrophoresis indicates that it is a single enzyme. Results of pH runs with aseptic larvae are similar to those with conventional larvae, indicating that the enzymes are autogenous.

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