CHARACTERIZATION OF PROTEINASE CLASSES IN LANGOSTILLA ( PLEURONCODES PLANIPES ) AND CRAYFISH ( PACIFASTACUS ASTACUS ) EXTRACTS

Proteinases (EC 3.4.21‐24) in langostilla extract and crayfish hepatopancreas were classified using site specific effectors or chelators for either serine, cysteine, aspartic or metallo classes. Azocasein hydrolysis by langostilla and crayfish preparations was inhibited 50% and 40%, respectively, when assayed in the presence of serine proteinase inhibitor, phenylmethylsulfonyl fluoride. A similar degree of inhibition was observed with a trypsin inhibitor. However, no inhibition occurred with a chymotrypsin inhibitor. Less inhibition of azocasein hydrolysis, i.e., 20% and 14%, respectively, resulted with 1,10 phenanthroline. Inhibitors for cysteine and aspartic proteinases did not reduce the azocasein hydrolysis activities significantly. The amidase activity, with N‐benzoyl‐DL‐Arg‐p‐nitroanilide as substrate was more sensitive, in both extracts, to serine proteinase inhibitors than the azocasein hydrolase activity. Results showed the presence of serine proteinases, i.e., trypsin but not chymotrypsin, as the major component and some minor activity of metallo dependent proteinases in the decapods extracts. Zymograms obtained after SDS‐PAGE showed a dozen proteinases in each extract. Some of them were inhibited by a serine proteinase inhibitor and two to three were inhibited by 1,10 phenanthroline, supporting the results of the test tube proteinase activity assays. Furthermore, the reported technique for zymograms allowed direct comparison between extracts and provided information about their composition and the molecular weight of the targeted enzymes.