Evidence that rat liver microsomal glutathione transferase is responsible for glutathione-dependent protection...

Evidence that rat liver microsomal glutathione transferase is responsible for the glutathione-dependent inhibition of lipid peroxidation in liver microsomes has been obtained. Activation of the microsomal glutathione transferase in microsomes by cystamine renders this organelle even more resistant to lipid peroxidation in the presence of glutathione compared with untreated microsomes. Upon examining the effect of seven glutathione analogues on lipid peroxidation, it was found that only those that serve as good substrates for the microsomal glutathione transferase (Glutaryl-L-Cys-Gly and alpha-L-Glu-L-Cys-Gly) can inhibit lipid peroxidation. The lack of inhibition by the other five analogues (alpha-D-Glu-L-Cys-Gly, gamma-D-Glu-L-Cys-Gly, beta-L-Asp-L-Cys-Gly, alpha-L-Asp-L-Cys-Gly and alpha-D-Asp-L-Cys-Gly) shows the specificity of the protection and rules out any non-enzymic component. Inhibitors of selenium-dependent glutathione peroxidase (mercaptosuccinate at 50 microM) and phospholipid hydroperoxide glutathione peroxidase (iodoacetate, 1 mM + glutathione, 0.5 mM) do not inhibit the glutathione-dependent protection of rat liver microsomes against lipid peroxidation. Purified microsomal glutathione transferase, NADPH-cytochrome P450 reductase and cytochrome P450 were reconstituted in microsomal phospholipid vesicles by cholate dialysis. The resulting membranes contained functional enzymes and did display enzymic lipid peroxidation induced by 75 microM NADPH and 10 microM Fe-EDTA (2:1). This model system was used to investigate whether microsomal glutathione transferase could inhibit lipid peroxidation in a glutathione-dependent manner. The results show that 5 mM glutathione did inhibit lipid peroxidation when functional microsomal glutathione transferase was included. This was not the case when the enzyme had been pre-inactivated with diethylpyrocarbonate. Furthermore, the protective effect of glutathione could be partly reversed by an inhibitor (100 microM bromosulphophtalein) of the enzyme. Apparently, rat liver microsomal glutathione transferase has the capacity to inhibit lipid peroxidation in a reconstituted system.