Analysis of the large aqueous pores produced by a Bacillus thuringiensis protein insecticide in Manduca sexta midgut-brush-border-membrane vesicles

Abstract

An osmotic swelling assay utilising carboxyfluorescein self-quenching to measure intravesicular volume changes was adapted to investigate permeability changes induced by the Bacillus thuringiensis Cry1Ac delta-endotoxin in Manduca sexta midgut-brush-border-membrane vesicles (BBMV). This assay provides a more quantitative analysis of Cry-toxin-induced BBMV permeability changes, extending our previously published protocol which employed a light-scattering signal to monitor delta-endotoxin activity [Carroll, J. & Ellar, D. J. (1993) Eur. J. Biochem. 214, 771-778]. The fluorescence signal changes, supported by electron microscopy of the BBMV, demonstrated that Cry1Ac altered the membrane permeability for large non-electrolyte solutes. With this approach Cry1Ac was observed to induce or form pores freely permeant for raffinose (1.14 nm diameter) and using non-electrolytes of increasing size the pores were estimated to have a limiting diameter of approximately 2.4-2.6 nm under alkaline pH conditions.