PROTEASE ACTIVITIES IN THE LARVAL MIDGUT OF HELIOTHIS-VIRESCENS - EVIDENCE FOR TRYPSIN AND CHYMOTRYPSIN-LIKE ENZYMES

Protease activities in the midgut contents of larvae of the tobacco bu dworm, Heliothis virescens, were investigated, Two major activities we re present: (i) a trypsin-like enzyme, hydrolysing the synthetic subst rate 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 containin g more than one amino acid, but not N-benzoyl-tyrosine p-nitroanilide and was strongly inhibited by chymostatin, The latter activity was con sidered to represent a hitherto ill-characterised family of insect chy motrypsins, which differ: from their mammalian counterparts in activit y 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), p-nitroanilide) were also detected, The two major enzyme a ctivities were shown to account for almost all the protease activity o f gut contents towards protein substrates, The proteases showed differ ent sensitivities to inhibition by plant protein protease inhibitors, which were effective in protecting exogenous proteins from digestion b y gut extracts.