Analysis of the Large Aqueous Pores Produced by a Bacillus Turingiensis Protein Insecticide in Manduca Sexta Midgut-Brush-Border-Membrane Vesicles


D. J. Ellar, Department of Biochemistry, University of Cambridge, Tennis Court Road, Cambridge, England CB2 1QW
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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 -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 -endotoxin activity [Carroll, J. & Ellar, D. J. (1993)Eur. J. Biochem. 214, 771–7781, The fluorescence signal changes, supported by electron microscopy of the BBMV, demonstrated that CrylAc altered the membrane permeability for large non-electrolyte solutes. With this approach CrylAc 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.