Immunochemical Evidence for an Association of Heme Oxygenase with the Microsomal Electron Transport System

Abstract

Abstract The relationship between spleen and liver microsomal heme oxygenase activities and the microsomal electron transport system requiring NADPH and cytochrome P-450 has been demonstrated by immunochemical techniques. An antibody preparation to purified, homogeneous NADPH-cytochrome c reductase (NADPH-cytochrome c oxidoreductase, EC 1.6.2.3) was shown to inhibit concomitantly NADPH-cytochrome c reductase and heme oxygenase activities in rat liver and spleen and pig spleen microsomal preparations. Previous work demonstrated the requirement for this enzymic activity in the reduction of cytochrome P-450 associated with drug hydroxylation reactions and steroid metabolism by concomitant inhibition of these various activities with anti-NADPH-cytochrome c reductase γ-globulin. The levels of NADPH-cytochrome c reductase in both rat and pig spleen reported in the present study are less than 10% of those found in rat and pig liver. Even at these levels, however, the reductase activity is sufficient to maintain the electron flux required for heme oxygenase activity under conditions in which heme catabolism is maximal. Methemalbumin treatment of rats resulted in a 3.7-fold increase in hepatic heme oxygenase activity which was concomitantly inhibited by anti-reductase γ-globulin, indicating that the induced enzyme system maintained its requirement for the microsomal electron transport system. Comparison of electron paramagnetic resonance spectra and ethyl isocyanide difference spectra of liver microsomes from untreated and methemalbumin-treated rats failed to demonstrate the induction of a new cytochrome moiety as a result of in vivo administration of methemalbumin, a substrate for the heme oxygenase system. The administration of methemalbumin in vivo to rats did not result in an increase in the components of the hepatic microsomal electron transport system, i.e. NADPH-cytochrome c reductase activity or cytochrome P-450 content, indicating that the modulation and control mechanisms of the microsomal heme oxygenase system differ from those governing the microsomal electron transport system. Methemalbumin treatment resulted in noncompetitive inhibition of the binding of both Type I and Type II drugs in rat liver microsomal preparations. In addition to the antibody inhibition studies, the temporal relationship demonstrated among the decrease in magnitude of binding spectra of hexobarbital, the increase in heme oxygenase activity, the decrease in measurable cytochrome P-450 content, and the reversal of these parameters during the recovery phase following a single injection of methemalbumin strongly indicates the involvement of the same components of the microsomal electron transport system in both the drug metabolism and heme oxygenase systems in liver.