Purification and characterization of trypsins from the digestive tract of Locusta migratoria.
Two trypsin-like enzymes were isolated from the digestive tract of the African migratory locust Locusta migratoria migratorioides. Primary purification was carried out on a DEAE-cellulose column, from which the two trypsins emerged in the anionic fraction. Further purification was achieved by affinity chromatography on a p-aminobenzamidine (PABA)-Sepharose column, which also separated the two trypsins (TLEAff.1. and TLEAff.2.), or by HPLC on an anion exchange column. The purity and homogeneity of the trypsins were demonstrated by electrophoresis of cellulose acetate strips and in polyacrylamide gels, with and without SDS. The molecular weights of TLEAff.1 and TLEAff.2, as determined by SDS-PAGE, were 17,000 and 24,000 respectively. The amino acid compositions of the locust trypsins were similar to those of trypsins from the digestive systems of other insects, which are characterized by the lack or low content of half cystines. The isoelectric points were 3.2 for TLEAff.1 and 3.5 fold for TLEAff.2. Since most of the locust trypsin comprised TLEAff.2, the latter served as the main object of this study. TLEAff.2 was unstable at low pH, differing in this respect from mammalian trypsins. The optimum activity was at pH 8.5-9.0. The Km and kcat, values were similar to those for bovine trypsin. Activation by substrate, a phenomenon in bovine trypsin, was also observed for TLEAff.2. The locust trypsin was full inhibited by the proteinaceous trypsin inhibitors Bowman-Birk (BBI) and Kunitz from soybeans, CI from chickpeas, chicken ovomucoid (COM), and turkey ovomucoid (TOM). It was inactivated by phenylmethylsulfonyl fluoride (PMSF) and tosyl-L-lysine chloromethyl ketone (TLCK), indicating the involvement of serine and histidine in the active site.
Sakal
E
E
Department of Biochemistry and Human Nutrition, Faculty of Agriculture, Hebrew University of Jerusalem, Rehovot, Israel.
Applebaum
S W
SW
Birk
Y
Y
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002417:Cattle; D002848:Chromatography, DEAE-Cellulose; D004064:Digestive System / Q000201:enzymology; D004591:Electrophoresis, Polyacrylamide Gel; D006110:Grasshoppers / Q000201:enzymology*; D007700:Kinetics; D008970:Molecular Weight; D014357:Trypsin / Q000302:isolation & purification*; D014361:Trypsin Inhibitors / Q000494:pharmacology
D000596:Amino Acids; D014361:Trypsin Inhibitors; D014357:Trypsin