Abstract
The effects of lipid peroxidation on the SH reactivity of the proteins in porcine intestinal brush-border membranes were examined using a fluorogenic thiol reagent, N- [7-dimethylamino-4-methylcoumarinyl] maleimide (DACM) in relation to lipid organization. Changes in the lipid organization were assessed by measurement of the rate of incorporation of 1, 6-diphenyl-1, 3, 5-hexatriene (DPH) into the membrane lipids and the fluorescence anisotropy of DPH-labeled membranes. Treatment of the membranes with an oxygen-radicalgenerating system, i.e., ascorbicacid/Fe2+/tert-butyl hydroperoxide (t-BuOOH), resulted in decrease in the rate of DACM incorporation into the SH groups of the membrane proteins (DACM-labeling) and the amount of DACM labeled to the SH groups with a decrease in the lipid fluidity, depending on the formation of thiobarbituric acid-reactive substances and conjugated diene. Pretreatment of the membranes with diphenylamine effectively prevented the ascorbic acid/Fe2+/ t-BuOOH-induced decreases in the DACM-labeling and DPH incorporation rates, whereas neither superoxide dismutase, catalase, sodium benzoate, nor mannitol showed a protective effect. The contribution of the lipid fluidity to the SH reactivity to DACM of the proteins in the membranes with different levels of lipid peroxidation was further examined using a lipid fluidizer, benzyl alcohol. The results showed that the DPH incorporation rate increased in proportion to increasing concentrations of the alcohol regardless of the peroxidation level of the membranes, whereas the susceptibility of the SH reactivity of the membrane proteins as to benzyl alcohol transitionally changed as the membranes were peroxidized to levels greater than 400 nmol conjugated diene/mg protein. Based on these results, we proposed the possibility that the SH reactivity of the membrane proteins changes in a different manner before and after a critical level of lipid peroxidation, and that the SH reactivity of the membrane proteins in highly peroxidized membranes is not dependent on the lipid fluidity per se.
