Protease activity in gut of Daphnia magna: evidence for trypsin and chymotrypsin enzymes

Abstract

Two major protease activities were present in gut homogenates of the cladoceran crustacean Daphnia magna: (i) a trypsin activity that hydrolysed the synthetic substrate N-benzoyl- dl-arginine p-nitroanilide and was strongly inhibited by N-p-tosyl-lysine chloroketone (TLCK) and 4-(amidinophenyl)methanesulfonyl fluoride (APMSF) and not inhibited by chymostatin; and (ii) a chymotrypsin activity that hydrolysed synthetic chymotrypsin substrates containing more than one amino acid, did not hydrolyse N-benzoyl- l-tyrosine p-nitroanilide, and was strongly inhibited by chymostatin and not by TLCK and APMSF. Both activities had alkaline pH optima (pH 7–10), but were shown to be due to distinct types of proteases. These two enzyme activities accounted for 75–83% of the proteolytic activity of gut contents. Substrate SDS-polyacrylamide gel electrophoresis revealed nine different proteases ranging from 15 to 73 kDa.