Purification and characterization of Locusta migratoria chymotrypsin.
A chymotrypsin-like enzyme (CTLE) was isolated from the digestive tract of the African migratory locust Locusta migratoria migratorioides by ion-exchange chromatography on diethylaminoethyl (DEAE) cellulose followed by affinity chromatography on phenylbutylamine (PBA) Sepharose. The purity and homogeneity of CTLE have been shown by SDS-PAGE and on cellulose acetate strips. The enzyme has a molecular weight of 24,000, determined by SDS-PAGE and on a Sephadex G-75 calibrated column. It has an isoelectric point of 10.1 and contains 0-1 half cystine residues. Sequence analysis of the first 20 N-terminal amino acids has shown 25% homology with bovine chymotrypsin and 40% homology with Vespa crabo and Vespa orientalis chymotrypsins and with Hypoderma lineatum trypsin. The optimal pH for enzyme activity and stability was in the range of 8.5-9.0. The Km and kcat values, determined on substrates for proteolytic, esterolytic and amidolytic activity, similar to those for bovine chymotrypsin. CTLE was inactivated by PMSF and TPCK indicating the involvement of serine and histidine in its active site. The enzyme was fully inhibited by the proteinaceous, double-headed, chymotrypsin-trypsin inhibitors BBI from soybeans and CI from chickpeas, by chicken ovomucoid (COM) and turkey ovomucoid (TOM), as well as by the Kunitz soybean trypsin inhibitor (STI) which hardly inhibits bovine chymotrypsin. Inhibition studies of CTLE with amino acid and peptide-chloromethylketones point towards the existence of an extended binding site.
Sakal
E
E
Department of Biochemistry, Faculty of Agriculture, Hebrew University of Jerusalem, Rehovot, Isreal.
Applebaum
S W
SW
Birk
Y
Y
D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D002846:Chromatography, Affinity; D002848:Chromatography, DEAE-Cellulose; D002850:Chromatography, Gel; D002918:Chymotrypsin / Q000302:isolation & purification*; D004064:Digestive System / Q000201:enzymology; D004591:Electrophoresis, Polyacrylamide Gel; D006110:Grasshoppers / Q000201:enzymology*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D012689:Sequence Homology, Nucleic Acid; D013045:Species Specificity
D002918:Chymotrypsin