Effect of cholesterol oxidase treatment on physical state of renal brush border membranes: evidence for a cholesterol pool interacting weakly with membrane lipids.

Treatment with cholesterol oxidases has shown that cholesterol is heterogeneously distributed in brush borders isolated from the apical membrane domain of the renal and intestinal epithelial cells [Bloj, B., & Zilversmit, D. B. (1982) J. Biol. Chem. 257, 7608-7614; El Yandouzi, E. H., & Le Grimellec, C. (1992) Biochemistry 31, 547-551]. To better understand the origin of cholesterol heterogeneity, the effects of cholesterol oxidation by Brevibacterium sp. cholesterol oxidase on the physical state of renal brush border membrane vesicles were determined using steady-state fluorescence polarization and differential phase fluorescence of 1,6-diphenyl-1,3,5-hexatriene (DPH). Selective quenching by trinitrobenzenesulfonate indicated that DPH distributes equally between outer and inner membrane leaflets. Oxidation of 90% of the cholesterol decreased the steady-state anisotropy of DPH, determined at 37 degrees C, by 14%. This modification corresponded to a change in the lipid order, the rotational rate of the probe being unaffected. Oxidation of the cholesterol corresponding to the readily accessible pool (30% of the total cholesterol), on the other hand, had a very limited effect on the membrane physical state. This contrasted with the linear decrease in both anisotropy and fluorescence lifetime of DPH obtained when cholesterol was replaced by cholestenone in liposomes made of phosphatidylcholine/sterol (1/1 molar ratio). These results indicate that, in brush border membranes, the cholesterol readily accessible to cholesterol oxidase interacts only weakly with the other membrane lipids.