Characteristics of proteases from stomachless aquatic organisms

Proteases were partially purified from the pyloric ceca of mullet (Mugil cephalus) and hepatopancreas from Louisiana swamp crawfish (Procambarus clarkii) by chromatographic procedures.; Partially purified mullet proteases were effective in inactivating commercial orange pectinesterase at room temperature.; Four trypsin-like enzymes, purified from the hepatopancreas of crawfish, were designated as CP-I, CP-II, CP-III and CP-IV in order of elution following DEAE-Sepharose chromatography. These were inhibited by protease inhibitors such as phenyl methyl sulfonyl fluoride, soybean trypsin inhibitor, aprotinin, tosyl lysine chloromethyl ketone but not by the chymotrypsin inhibitor tosyl phenylalanine chloromethyl ketone. Therefore, these crawfish proteases could be considered as serine-type proteases and classified as trypsin-like. The apparent molecular weights of CP-I, CP-II, CP-III and CP-IV were determined to be 35.0, 41.2, 37.9 and 39.5 kDa, respectively, using sodium dodecyl sulfate polyacrylamide gel electrophoresis.; The proteases had optimal esterase activity at pH 8.0-8.5 and at a temperature between at 60-70{dollar}spcirc{dollar}C. Crawfish proteases were rich in acidic amino acids. Activation energies for hydrolysis of tosyl arginine methyl ester by crawfish proteases were 6.98-8.34 kcal/mole. Unlike other serine proteases, the activities of CP-I and CP-II were activated by mercury chloride (HgCl{dollar}sb2){dollar} while CP-III and IV were inhibited.; Immunological study showed that the four crawfish proteases are crossreactive and, therefore, they have shared structural components. Crawfish proteases were able to inactivate orange peel pectinesterase, tomato pectinesterase and pectate lyase c at room temperature.; Using pectate lyase c from Erwinia chrysanthemi as a model protein substrate of known sequence and three-dimensional structure, the peptide fragmentation patterns generated by each crawfish proteases was determined by Matrix Assisted Laser Desorption Time-of-flight mass spectrometry. Cleavage sites in native substrate were at lysine residue. The pectate lyase c cleavage hydrolyzed by crawfish proteases were similar to each other but clearly different from trypsin.