EVIDENCE THAT RAT-LIVER MICROSOMAL GLUTATHIONE TRANSFERASE IS RESPONSIBLE FOR GLUTATHIONE-DEPENDENT PROTECTION AGAINST LIPID-PEROXIDATION

Evidence that rat liver microsomal glutathione transferase is responsi ble for the glutathione-dependent inhibition of lipid peroxidation in liver microsomes has been obtained. Activation of the microsomal gluta thione transferase in microsomes by cystamine renders this organelle e ven more resistant to lipid peroxidation in the presence of glutathion e compared with untreated microsomes. Upon examining the effect of sev en glutathione analogues on lipid peroxidation, it was found that only those that serve as good substrates for the microsomal glutathione tr ansferase (Glutaryl-L-Cys-Gly and alpha-L-Glu-L-Cys-Gly) can inhibit l ipid 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, a lpha-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 muM) and phospholipid hydroperoxide glutathione peroxidase (iodoacetat e, 1 mM + glutathione, 0.5 mM) do not inhibit the glutathione-dependen t protection of rat liver microsomes against lipid peroxidation. Purif ied microsomal glutathione transferase, NADPH-cytochrome P450 reductas e and cytochrome P450 were reconstituted in microsomal phospholipid ve sicles by cholate dialysis. The resulting membranes contained function al enzymes and did display enzymic lipid peroxidation induced by 75 mu M NADPH and 10 muM Fe-EDTA (2:1). This model system was used to invest igate whether microsomal glutathione transferase could inhibit lipid p eroxidation in a glutathione-dependent manner. The results show that 5 mM glutathione did inhibit lipid peroxidation when functional microso mal glutathione transferase was included. This was not the case when t he enzyme had been pre-inactivated with diethylpyrocarbonate. Furtherm ore, the protective effect of glutathione could be partly reversed by an inhibitor (100 muM bromosulphophtalein) of the enzyme. Apparently, rat liver microsomal glutathione transferase has the capacity to inhib it lipid peroxidation in a reconstituted system.