The effect of detergents on trimeric G-protein activity in isolated plasma membranes from rat brain cortex: Correlation with studies of DPH and Laurdan fluorescence

J. Sýkora, L. Bouřová, M. Hof, P. Svoboda, J. Sýkora, L. Bouřová, M. Hof, P. Svoboda

Index: Biochim. Biophys. Acta 1788(2) , 324-32, (2009)

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Abstract

The effect of non-ionic detergents on baclofen (GABA B-R agonist)-stimulated G-protein activity was measured as a [ 35S]GTPγS binding assay in the plasma membranes (PM) isolated from the brain tissue. The effect was clearly biphasic — a decrease in the activity was followed by an activation maximum and finally, at high concentrations, drastic inhibition of the G-protein activity was noticed. Contrarily, specific radioligand binding to GABA B-receptor was inhibited in the whole range of detergent concentrations step by step, i.e. it was strictly monophasic. The magnitude of both detergent effects was decreased in the same order of potency: Brij58 > Triton X-100 > Digitonin. The identical order was found when comparing detergents ability to alter fluorescence anisotropy of the membrane probe 1,6-diphenyl-1,3,5-hexatriene ( r DPH) incorporated into the hydrophobic PM interior. Decrease of r DPH, in the order of Brij58 > Triton X-100 > Digitonin, was reflected as decrease of the S-order parameter and rotation correlation time ϕ paralleled by an increase of diffusion wobbling constant D w (analysis by time-resolved fluorescence according to “ wobble-in-cone” model). The influence of the detergents on the membrane organization at the polar headgroup region was characterized by Laurdan generalized polarization (GP). As before, the effect of detergents on GP parameters proceeded in the order: Brij58 > Triton X-100 > Digitonin.