Immunochemical Studies on Electron Transport Chains Involving Cytochrome P-450: I. EFFECTS OF ANTIBODIES TO PIG LIVER MICROSOMAL REDUCED TRIPHOSPHOPYRIDINE NUCLEOTIDE-CYTOCHROME c REDUCTASE AND THE NON-HEME IRON PROTEIN FROM BOVINE ADRENOCORTICAL MITOCHONDRIA

Abstract

Abstract An antibody preparation to purified, homogeneous TPNH-cytochrome c reductase from pig liver microsomes was shown to inhibit concomitantly TPNH-cytochrome c reductase (NADPH-cytochrome c oxidoreductase, EC 1.6.2.3) and ethylmorphine N-demethylase activities in pig liver microsomes. The antibody also inhibited TPNH-cytochrome c reductase and TPNH-cytochrome P-450 reductase activities in rat and pig liver microsomes and bovine adrenocortical microsomes, but it did not inhibit either of these activities in bovine adrenocortical mitochondria. In addition, an antibody to adrenodoxin, the non-heme iron protein isolated from bovine adrenocortical mitochondria, inhibited both TPNH-cytochrome c reductase and TPNH-cytochrome P-450 reductase activities in bovine adrenocortical mitochondria but not in any of the microsomal preparations, including those isolated from adrenal cortex. The results presented establish these antibodies as specific inhibitors which can be utilized in studying hydroxylation reactions mediated by cytochrome P-450. With the use of these specific antibodies in each system studied, either the same or immunochemically similar enzymes were shown to mediate the TPNH-dependent reduction of both cytochrome c and cytochrome P-450, and the flavoprotein functional in the mixed-function oxidations of the mitochondria of adrenal cortex was shown to differ from that found in the microsomes of liver and adrenal cortex. Furthermore, the lack of inhibition of TPNH-dependent electron transport activities in liver and adrenal cortex microsomes, by antibody to adrenodoxin, which strongly inhibits bovine adrenal cortical mitochondrial TPNH-cytochrome c and cytochrome P-450 reductase activities, is further evidence that a non-heme iron protein similar to adrenodoxin is not involved in cytochrome P-450-mediated reactions in microsomes.