CONCLUSIONS AND DISCUSSION
* We demonstrated that the alkaline pH is an essential factor for solubilization and proteolytic processing of Bacillus thuringiensis subsp. medellin Cyt1Ab1 toxin (Table 1, Fig. 1)
* The time course studies demonstrated that the appearing of Cyt1Ab1 fragments take place very quickly, but it was only completed 14 h later with trypsin and gut extracts, and 24 h later with chymotrypsin(Fig. 2).
* The in vitro proteolytic processing of Cyt1Ab1 toxin with C. quinquefasciatus midgut extracts resembles the processing of the toxin by the larvae(Fig. 3).
* The processing of the Cyt1Ab1 toxin with C. quinquefasciatus midgut extracts, trypsin and chymotrypsin showed slightly different size fragments when we compared with the results of processing of the Cyt1Aa1 toxin (1).
* The crystal inclusion of Cyt1Ab1 waslarvicidal against third instar C. quinquefasciatus larvae (Table 2); however, the soluble toxin was non-toxic but it was hemolytic. We observed hemolytic activity of the soluble toxin against sheep red blood cells and an increment of this activity was seen when the toxin was processed with proteases (Table 2).
* The changes induced by processing of Cyt1Ab1 toxin generate an increase in activity. Similar observation was also pointed out by Al-yahyaee and Ellar (1), who found that in vitro processing of Cyt1Aa1 toxin enhances hemolytic activity. The changes can modulate the activity of the toxin as we observed in the hemolytic assays with Cyt1Ab1 toxin. This observation resembles those made by other authors for Cyt-type toxins (9). Like toxicity in the Cry-type toxins, the toxicity of Cyt1Ab1 toxin is highly dependent on processing (4).
* Further studies should be done in order to characterise the processing of the toxin such as: N-terminal microsequencing of the fragments and cytotoxicity assays with purified fragments against cultured cell lines.