Identification and partial characterization of proteases in larval preparations of the cereal leaf beetle (Oulema melanopus, Chrysomelidae, Coleoptera).

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.