Analysis of the properties of Bacillus thuringiensis insecticidal toxins using a potential-sensitive fluorescent probe.
A potential-sensitive fluorescent probe, 3,3'-dipropylthiadicarbocyanine iodide, was used to analyze, at pH 7.5 and 10.5, the effects of Bacillus thuringiensis toxins on the membrane potential generated by the efflux of K(+) ions from brush border membrane vesicles purified from the midgut of the tobacco hornworm, Manduca sexta. Fluorescence levels were strongly influenced by the pH and ionic strength of the media. Therefore, characterization of the effects of the toxins was conducted at constant pH and ionic strength. Under these conditions, the toxins had little effect on the fluorescence levels measured in the presence or absence of ionic gradients, indicating that the ionic selectivity of their pores is similar to that of the intact membrane. Valinomycin greatly increased the potential generated by the diffusion of K(+) ions although membrane permeability to the other ions used to maintain the ionic strength constant also influenced fluorescence levels. In the presence of valinomycin, active toxins (Cry1Aa, Cry1Ab, Cry1Ac, Cry1C and Cry1E) efficiently depolarized the membrane at pH 7.5 and 10.5.
Kirouac
M
M
Université de Montréal, P.O. Box 6128, Centre Ville Station, Montreal, Quebec, H3C 3J7, Canada.
Vachon
V
V
Rivest
S
S
Schwartz
J-L
JL
Laprade
R
R
D000818:Animals; D001413:Bacillus thuringiensis / Q000737:chemistry*; D001427:Bacterial Toxins / Q000633:toxicity*; D002463:Cell Membrane Permeability / Q000187:drug effects / Q000502:physiology; D004225:Dithiazanine; D004563:Electrochemistry / Q000295:instrumentation / Q000379:methods; D005456:Fluorescent Dyes; D006863:Hydrogen-Ion Concentration; D007313:Insecta / Q000737:chemistry / Q000187:drug effects; D007413:Intestinal Mucosa / Q000378:metabolism*; D007422:Intestines / Q000737:chemistry / Q000166:cytology / Q000187:drug effects* / Q000648:ultrastructure; D018402:Manduca / Q000737:chemistry / Q000187:drug effects / Q000502:physiology / Q000648:ultrastructure; D008564:Membrane Potentials / Q000187:drug effects*; D008871:Microvilli / Q000737:chemistry / Q000187:drug effects / Q000502:physiology; D011188:Potassium / Q000378:metabolism; D013050:Spectrometry, Fluorescence / Q000295:instrumentation / Q000379:methods*; D018675:Toxicity Tests / Q000295:instrumentation / Q000379:methods*; D014634:Valinomycin / Q000737:chemistry / Q000494:pharmacology
D001427:Bacterial Toxins; D005456:Fluorescent Dyes; D014634:Valinomycin; D004225:Dithiazanine; D011188:Potassium