Effect of lipid peroxidation on membrane-bound Ca2+-ATPase activity of the intestinal brush-border membranes

We have studied lipid peroxidation and Ca2+-ATPase activity of the porcine intestinal brush-border membranes using a oxygen-radical-generating system consisting of dithiothreitol (DTT)/Fe2+ and tert-butyl hydroperoxide (t-BuOOH). The rates of lipid peroxidation were measured by formation of thiobarbituric acid-reactive substances (TBAR) and conjugated diene. Incubation of the membranes with DTT/Fe2+ in the absence and presence of t-BuOOH resulted in a slight (about 20%) and a marked (about 50%) inhibition of Ca2+-ATPase activity, respectively. The degree of inhibition was dependent on the hydroperoxide concentration. Addition of thiourea effectively protected Ca2+-ATPase activity but catalase and superoxide dismutase showed a slight and no effect on protection of the ATPase activity, respectively. Results of kinetic studies on the ATPase activity with varying ATP and Ca2+ concentrations revealed that the decrease in the enzyme activity by treatment with these oxidizing agents is mainly due to decrease of the Vmax value. Modification of SH groups in the membrane proteins by thiol group reagents such as N-ethylmaleimide, monoiodoacetate and monoiodacetamide did not induce the inhibition of Ca2+-ATPase activity. From these results, it is suggested that inhibition of the ATPase activity of the membranes by treatment with DTT/Fe2+ in the presence and absence of t-BuOOH is dependent on lipid peroxidation and that oxidative modification of SH groups may not be directly involved to the loss of the ATPase activity. In addition, results of the fluorescence anisotropy measurements of pyrene-labeled membranes suggested that change in the Ca2+-ATPase activity is partly related to a decrease in the membrane lipid fluidity.