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

Two major protease activities were present in out 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. (C) 2003 Elsevier Inc. All rights reserved.