A mechanical force contributes to the "osmotic swelling" of brush-border membrane vesicles.

Brush-border membrane vesicles and an osmotic swelling assay have been used extensively to monitor the pore-forming activity of Bacillus thuringiensis toxins. After a hypertonic shock, Manduca sexta midgut brush-border membrane vesicles shrink rapidly and reswell partially to a volume that depends on membrane permeability and toxin concentration rather than regaining their original volume as expected from theoretical models. Because efflux of buffer from the vesicles, as they shrink, could contribute to this phenomenon, vesicles were mixed with a hypertonic solution of the buffer with which they were loaded.

Protease inhibitors fail to prevent pore formation by the activated Bacillus thuringiensis toxin Cry1Aa in insect brush border membrane vesicles.

To investigate whether membrane proteases are involved in the activity of Bacillus thuringiensis insecticidal toxins, the rate of pore formation by trypsin-activated Cry1Aa was monitored in the presence of a variety of protease inhibitors with Manduca sexta midgut brush border membrane vesicles and by a light-scattering assay. Most of the inhibitors tested had no effect on the pore-forming ability of the toxin. However, phenylmethylsulfonyl fluoride, a serine protease inhibitor, promoted pore formation, although this stimulation only occurred at higher inhibitor concentrations than those commonly used to inhibit proteases.

Amino acid and divalent ion permeability of the pores formed by the Bacillus thuringiensis toxins Cry1Aa and Cry1Ac in insect midgut brush border membrane vesicles.

The pores formed by Bacillus thuringiensis insecticidal toxins have been shown to allow the diffusion of a variety of monovalent cations and anions and neutral solutes. To further characterize their ion selectivity, membrane permeability induced by Cry1Aa and Cry1Ac to amino acids (Asp, Glu, Ser, Leu, His, Lys and Arg) and to divalent cations (Mg(2+), Ca(2+) and Ba(2+)) and anions (SO(4)(2-) and phosphate) was analyzed at pH 7.5 and 10.