ISOLATION AND PARTIAL CHARACTERIZATION OF 2 TRYPSINS FROM THE LARVAL MIDGUT OF SPODOPTERA-LITTORALIS (BOISDUVAL)

Two serine-proteases (P-I and P-II) were isolated from the larval midg ut of Spodoptera littoralis by affinity chromatography. These hydrolys ed casein and BApNA and were strongly inhibited by TLCK. On SDS-PAGE, an apparent molecular weight of 27 kDa was determined for both proteas es. When mass spectrometric analysis was performed directly on the elu ted fractions, one main component per peak was observed, having a mole cular mass of 24,359 +/- 2 Da (P-I) and 24,448 +/- 2 Da (P-II). The en zymes had their maximum activity and stability at pH 10 and 4, respect ively. P-I was significantly more active than P-II at 20 degrees C or less; it was also more resistant to denaturation by heat (55 versus 50 degrees C). A breakpoint between activity and stability was recorded at 30 degrees C for both proteases. P-II was strongly inhibited by apr otinin, leupeptin and KI, and partly inactivated by LBTI and chymostat in; P-I was unaffected by LBTI and chymostatin and was also less susce ptible to aprotinin and KI, On the basis of substrate degradation and inactivation rates, it was concluded that P-I and P-II are very differ ent from the two proteases found in another strain of S. littoralis. T hough P-I and P-II were found to belong to the same mechanistic class of bovine trypsin, they were not activated by Ca2+ over the concentrat ion range 0-20 mM. It is suggested that transgenic plants harbouring g enes coding for effective P-I inhibitors should have a high level of r esistance towards this insect pest. (C) 1998 Elsevier Science Ltd. All rights reserved.