A new sensitive method for determining the toxicity of a highly purified fraction from δ-endotoxin produced by Bacillus thuringiensis var. entomocidus on isolated larval midgut of Spodoptera littoralis (Lepidoptera, Noctuidae)

The purification procedure of the toxic fraction from the δ-endotoxin produced by Bacillus thuringiensis var. entomocidus was considerably shortened and simplified by combining the solubilization in a high pH (10.0) solution with glycine, opening SS bonds with dithiothreitol, and releasing hydrophobic connections with the detergents Triton N-101 and sodium cholate. The subsequent fractionation was immediately continued (without dialysis or concentration) on a Sepharose 6B column, yielding the active fraction designated C. Fraction C was then passed through an octyl Sepharose 4B column, yielding the active fraction designated I, followed by gel filtration on a Sepharose 6B column yielding the active fraction designated CI. All treatments were done with the same buffer solution at 4°C. Column elution was with the same buffer but with 0.075% detergents. The purity of the fraction CI was apparent by its appearance as a well-defined band on polyacrylamide gel isoelectric focusing at pH 6.1, accompanied by a small faint band. A sensitive method for evaluating the toxicity of endotoxin fractions on an isolated midgut system (from larvae of Spodoptera littoralis) was developed. The system is based on measuring the activity of the enzyme, reduced glutathione S-transferase, released to the medium from epithelial cells ruptured by the toxin. Fraction CI, M_r 64,000, was toxic to the epithelial cells of the isolated midgut in the absence of the peritrophic membrane. When the detergents were removed by dialysis, the active protein formed high molecular weight aggregates due to hydrophobic interactions. The passage of those aggregates to the sensitive epithelial cells of the midgut was prevented by the peritrophic membrane.