title: Identification and partial characterization of proteases in larval preparations of the cereal leaf beetle (Oulema melanopus, Chrysomelidae, Coleoptera).
abstract: We determined some biochemical properties of Oulema melanopus larval gut proteases. We found adult midgut enzyme preparations yielded results similar to whole-larval preparations, permitting studies of the very small whole-larval preparations. Protein preparations were analyzed using FITC-casein as a substrate. Acidic pH is optimal for proteolytic activity (range 3.0-4.0). Cysteine protease activity increased at acidic pH and in the presence of β-mercaptoethanol. Protease activities at all pH values were maximal at 45°C. Enzyme activity in larval preparations was inhibited by addition of Fe(2+) , Ca(2+) , Mg(2+) , Zn(2+) , and K(+) (10 mM). Fe(2+) and Zn(2+) significantly decreased enzyme activity at all pH values, Ca(2+) and Mg(2+) at pH 6.2 and Mg(2+) at pH 4.0. Inhibitors, including pepstatin A, showed the greatest inhibition at pH 4.0; phenylmethylsulfonyl fluoride, N-p-tosyl-l-phenylalanine chloromethyl ketone at pH 6.2; and phenylmethylsulfonyl fluoride, Nα -tosyl-l-lysine chloromethyl ketone hydrochloride, N-p-tosyl-l-phenylalanine chloromethyl ketone, trans-epoxysuccinyl-l-leucylamido-(4-guanidino) butane at pH of 7.6. Inhibition assays indicated that cysteine, aspartyl (cathepsin D), serine (trypsin, chymotrypsin-like) proteases and metalloproteases act in cereal leaf beetle digestion.
authors.affiliation: Department of Agrophages´ Forecasting Methods and Agricultural Economics, Institute of Plant Protection - National Research Institute, Poznań, Poland.
authors.forename: Beata
authors.initials: B
authors.lastname: Wielkopolan
authors.forename: Felicyta
authors.initials: F
authors.lastname: Walczak
authors.forename: Andrzej
authors.initials: A
authors.lastname: Podleśny
authors.forename: Robert
authors.initials: R
authors.lastname: Nawrot
authors.forename: Aleksandra
authors.initials: A
authors.lastname: Obrępalska-Stęplowska
mesh_terms: D000818:Animals; D001517:Coleoptera / Q000201:enzymology* / Q000254:growth & development; D041981:Gastrointestinal Tract / Q000201:enzymology*; D006863:Hydrogen-Ion Concentration; D007814:Larva / Q000201:enzymology / Q000254:growth & development; D045726:Metalloproteases; D010447:Peptide Hydrolases / Q000302:isolation & purification / Q000378:metabolism*; D011480:Protease Inhibitors / Q000494:pharmacology
chemical_list: D011480:Protease Inhibitors; D045726:Metalloproteases; D010447:Peptide Hydrolases
keywords: aspartyl protease; cereal leaf beetle (Oulema melanopus); cysteine protease; metalloprotease; protease inhibitors; proteases; serine protease