ENDOPROTEASES FROM THE MIDGUT OF LARVAL SPODOPTERA-LITTORALIS INCLUDE A CHYMOTRYPSIN-LIKE ENZYME WITH AN EXTENDED BINDING-SITE

Endoproteases from the lumen of the midgut of larvae of Spodoptera littoralis were characterised with respect to their substrate specificity and pH optimum. Alkaline serine protease activities corresponding to trypsin and chymotrypsin were detected. Cysteine, aspartic or metalloprotease activities were not found, nor was serine protease activity associated with microbes. Chymotrypsin hydrolysed SA(2)PPpNA, SA(2)PLpNA, BTEE, SA(3)pNA and casein, but not BTpNA, ALpNA or SPpNA, indicating an extended binding site. It was effectively inhibited by chymostatin, PMSF, antipain, SBTI, BBTI, LBTI, CEOI and TEW, but unaffected by TPCK. Trypsin hydrolysed BApNA, TGPLpNA, TAME and BAEE. It was effectively inhibited by TLCK, benzamidine, leupeptin, chymostatin, antipain, CEOI, aprotinin, pCMPS and E-64. Chymostatin, antipain and CEOI were the only three inhibitors which were effective against both endopeptidases. Trypsin and chymotrypsin have a molecular weight of 24,000 and 19,000, respectively, as determined by gel filtration. The optimal pH for both enzymes was pH 10.0 using p-nitroanilide substrates, but lower for esterase substrates (between pH 8.0 and 9.5) and higher for casein hydrolysis by chymotrypsin (pH 11.0).