PROTEOLYTIC PROCESSING OF DELTA-ENDOTOXIN OF BACILLUS-THURINGIENSIS VAR KURSTAKI HD-1 IN INSENSITIVE INSECT, SPODOPTERA-LITURA - UNUSUAL PROTEOLYSIS IN THE PRESENCE OF SODIUM DODECYL-SULFATE

The larvae of Spodoptera litura showed a > 3600-fold lesser susceptibility to crystal δ-endotoxin of Bacillus thuringiensis var. kurstaki HD-1 than a susceptible insect Bombyx mori. To examine the ability of the gut juice from S. litura and B. mori to activate the protoxin, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and gel filtration chromatography were employed to analyze in vitro proteolysed products of the protoxin. When SDS-PAGE was employed, the protoxin treated with B. mori gut juice (≥10%, v v) was shown to be activated to the toxin with a MW of 60 kDa, while digestion with small amounts (0.1%) of S. litura gut juice yielded the 65-kDa band, but with larger amounts (≥10%) it caused complete digestion of the toxin. However, when analyzed using gel filtration chromatography under native conditions, the digest with S. litura gut juice (10%) showed a big peak of 60 kDa possessing toxicity against B. mori larvae, which was also observed at the same retention time in B. mori digest. To resolve the discrepancy of the results obtained between the two methods, various proteinase inhibitors were added to the digests and then SDS-PAGE was carried out. When one inhibitor, o-phenanthroline, was used, the 60-kDa band was detected in S. litura digest, suggesting that further degradation of the 60-kDa toxin took place during SDS treatment. This suggestion was confirmed by the observation that the complete degradation of the toxin was induced by the addition of SDS to the reaction mixture containing S. litura gut juice. From these results, it was concluded that S. litura gut juice processed the native protoxin to the 60-kDa toxin, indicating that subsequent steps to protoxin activation do not occur in S. litura larvae. Further, the data demonstrated that much attention should be paid in employing SDS-PAGE as a method of analysis of protoxin processing by gut juice. © 1992.