Evidence that rat liver microsomal glutathione transferase is responsible for glutathione-dependent protection against lipid peroxidation

Evidence that rat liver musomal glutathione transferase is responsible for the glutathione-dependent inhibition of lipid peroxidation in liver musomes has been obtained. Activation of the musomal glutathione transferase in musomes by cystamine renders this organelle even more resistant to lipid peroxidation in the presence of glutathione compared with untreated musomes. Upon examining the effect of seven glutathione analogues on lipid peroxidation, it was found that only those that serve as good substrates for the musomal glutathione transferase (Glutaryl- l-Cys-Gly and α- l-Glu- l-Cys-Gly) can inhibit lipid peroxidation. The lack of inhibition by the other five analogues (α d-Glu- l-Cys-Gly, γ- d-Glu- l-Cys-Gly, β- l-Asp- l-Cys-Gly, α- l-Asp- l-Cys-Gly and α- 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 μM) and phospholipid hydroperoxide glutathione peroxidase (iodoacetate, 1 mM + glutathione, 0.5 mM) do not inhibit the glutathione-dependent protection of rat liver musomes against lipid peroxidation. Purified musomal glutathione transferase, NADPH-cytochrome P450 reductase and cytochrome P450 were reconstituted in musomal phospholipid vesicles by cholate dialysis. The resulting membranes contained functional enzymes and did display enzymic lipid peroxidation induced by 75 μM NADPH and 10 μM Fe-EDTA (2: 1). This model system was used to investigate whether musomal glutathione transferase could inhibit lipid peroxidation in a glutathione-dependent manner. The results show that 5 mM glutathione did inhibit lipid peroxidation when functional musomal 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 (100μM bromosulphophtalein) of the enzyme. Apparently, rat liver musomal glutathione transferase has the capacity to inhibit lipid peroxidation in a reconstituted system.