Membrane-insecticide interactions

Jones, Owen Thomas (1984) Membrane-insecticide interactions. University of Southampton, Doctoral Thesis.

Abstract

The interaction of organochlorine and pyrethroid insecticides with biological membranes was studied using multilamellar liposomes and (CaZ+ + Mgt+)-ATPase purified from rabbit muscle sarcoplasmic reticulum. The insecticides had little effect on the bulk properties of lipid as shown by a lack of effect on the leak rate of dye entrapped in liposomes or on fluidity measured using fluorescence depolarisation techniques. Binding of organochlorine insecticides to membranes was demonstratedby observing fluorescence quenching of labelled liposomes or (Ca2+ + Mgt+)ATPase, caused by isomers of hexachlorocyclohexane. Quenching profiles showed saturation but did not fit well to predictions of a saturation binding model, and light scattering showed that quenching of membranebound fluorophores was limited by saturation of the aqueous phase. The data fitted well to a partition model and lipid-water partition coefficients were similar for insecticidally-active and inactive isomers. Binding to the ATPase was stronger than to the lipid. A fluorescent pyrene analogue of the pyrethroids (PPE) was prepared to investigate pyrethroid-membrane interactions. Binding constants were evaluated from the dependence of monomer and excimer fluorescence intensities on lipid concentration for PPE and other pyrene derivatives. For the ATPase system, binding was detected to both lipid and protein components. The polarity dependence of monomer emission fine structure and fluorescence quenching experiments located the pyrene group of PPE near the bilayer surface. The effects of the insecticides and other hydrophobic molecules on ATPase activity depended on lipid structure. For ATPase reconstituted with short chain lipids, pyrethroids caused a large increase in activity; for organochlorines, a biphasic profile of activity versus concentration was noted with initial stimulation being followed by inhibition. Reconstitution of ATPase with longer chain lipids lessened the activation caused by pyrethroids and abolished that caused by organochlorines. It is suggested that inhibition of the ATPase follows from binding at the lipid-protein interface (annular sites). Activation then follows from binding to other non-annular sites on the ATPase.

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