Inhibition of Ca2+-induced aggregation of porcine intestinal brush-border membranes by lipid peroxidation

The effects of lipid peroxidation on Ca2+-induced aggregation of porcine intestinal brush-border membranes were examined using a system consisting of ascorbic acid/Fe2+/tertbutyl hydroperoxide (t-BuOOH). Incubation of the membranes with ascorbic acid/Fe2+/t-BuOOH resulted in inhibition of Ca2+-induced aggregation of the membranes with the formation of thiobarbituric acid-reactive substances, depending on the hydroperoxide concentration and the incubation time. The inhibition of the membrane aggregation associated with ascorbic acid/Fe2+/t-BuOOH treatment was effectively prevented by the addition of an antioxidant, 3(2)-tert-butyl-4-hydroxyanisole, to the reaction mixture. Studies with 8-anilino-1-naphthalenesulfonate (ANS) revealed that there is a linear relationship between the apparent dissociation constants (Kd) of ANS-membrane complexes and the aggregating efficiencies of the membranes with different levels of lipid peroxidation. This suggests that inhibition of the membrane aggregation by lipid peroxidation involves a change in the membrane surface charge density. Modification of the membranes with malondialdehyde also resulted in a decrease in the aggregating efficiency of the membranes with a decrease in the Kd value of ANS-membrane complex. In addition, the contribution of the lipid organization to membrane aggregation was examined by measuring the fluorescence anisotropy of diphenylhexatriene-labeled membranes in the presence of a lipid fluidizer, benzyl alcohol. The results are discussed in terms of peroxidation-induced inhibition of intramembrane interactions of the membranes.