Protease activities in the larval midgut of Heliothis virescens: Evidence for trypsin and chymotrypsin-like enzymes

Abstract

Protease activities in the midgut contents of larvae of the tobacco budworm, Heliothis virescens, were investigated. Two major activities were present: (i) a trypsin-like enzyme, hydrolysing the synthetic substrate N-benzoyl-arginine p-nitroanilide (BApNA), strongly inhibited by N-p-tosyl-lysine chloroketone (TLCK) and not inhibited by chymostatin at concentrations where this inhibitor is chymotrypsin-specific; (ii) an enzyme which hydrolysed synthetic chymotrypsin substrates containing more than one amino acid, but not N-benzoyl-tyrosine p-nitroanilide and was strongly inhibited by chymostatin. The latter activity was considered to represent a hitherto ill-characterised family of insect chymotrypsins, which differ from their mammalian counterparts in activity towards synthetic substrates. Both activities had strongly alkaline pH optima, in the pH range 10–11, but were shown to be due to distinct proteases. Low levels of elastase-like activity (hydrolysing succinyl (alanine) 3 p-nitroanilide) were also detected. The two major enzyme activities were shown to account for almost all the protease activity of gut contents towards protein substrates. The proteases showed different sensitivities to inhibition by plant protein protease inhibitors, which were effective in protecting exogenous proteins from digestion by gut extracts.