Immunochemical Detection and Quantitation of Microsomal Cytochrome P-450 and Reduced Nicotinamide Adenine Dinucleotide Phosphate: Cytochrome P-450 Reductase in the Rat Ventral Prostate

Treatment with β-naphthoflavone (BNF) was found to induce 7-ethoxyresorufin O-deethylase and aryl hydrocarbon hydroxylase activities about 500-fold in the microsomal fraction of the rat ventral prostate but had no effect on aminopyrine N-demethylase or reduced nicotinamide adenine dinucleotide phosphate:cytochrome c reductase activities. Phenobarbital (PB) treatment did not alter any of these enzyme activites. Antibodies raised in rabbits against rat liver cytochrome P-450 reductase (P-450 reductase) and against P-450 BNF-B₂ and P-450 PB-B₂, the major forms of P-450 isolated from liver microsomes of BNF- and PB-treated rats, respectively, were used to characterize the P-450-dependent monooxygenase system in the rat ventral prostate. Anti-P-450 reductase immunoglobulin G inhibited reduced nicotinamide adenine dinucleotide phosphate:cytochrome c reductase activity in prostatic microsomes, and anti-P-450 BNF-B₂ but not anti-P-450 PB-B₂ immunoglobulin G inhibited the BNF-induced prostatic microsomal 7-ethoxyresorufin O-deethylase and aryl hydrocarbon hydroxylase activities. A highly sensitive immunoblotting method was used to quantitate P-450 BNF-B₂, P-450 PB-B₂, and P-450 reductase in prostatic microsomes. Using this technique, prostatic P-450 reductase with a molecular weight corresponding to that of purified liver P-450 reductase was detected at a level of 0.02 nmol/mg of microsomal protein. In the liver, the same enzyme amounts to 0.2 nmol/mg of microsomal protein. P-450 BNF-B₂ was not detected in prostatic microsomes from control or PB-treated rats, whereas a protein band with a molecular weight corresponding to that of purified liver P-450 BNF-B₂ was found in prostatic microsomes from BNF-treated rats at a level of 0.05 nmol P-450 per mg microsomal protein, P-450 PB-B₂ was not detected in prostatic microsomes from either control, PB-treated, or BNF-treated animals.