Purification and Characterization of Four Catalytically Active Testosterone 6β-Hydroxylase P-450s from Rat Liver Microsomes: Comparison of a Novel Fprm with Three structurally and Functionally Related Forms1

Abstract

Four microsomal cytochrome P– 450s (P-450), all of which are active testosterone 6β -hydroxylases, were purified to electrophoretic homogeneity from livers of phenobarbital-treated (P- 450_6β-2 and P-450_6β-3) or dexamethasone-treated adult male rats (P-450_6β1 and P-450_6β-4). Purified P-450_6β-1, P-450_6β-2, P-450_6β-3, and P-450_6β-4 had apparent molecular weights of 52,000, 51,000, 52,000, and 52,500 as assessed by sodium dodecyl sulfate -polyacrylamide gel electrophoresis. Absolute spectra revealed that all four P-450 forms had characteristic low-spin spectral patterns in their fully oxidized states. P-450_6β-2 and P-450_6β 3 displayed spectra of the reduced carbonyl complex with λ _max at 447 nm. P-450_6β2 and P-450_6β-4 showed λ _max at 446 and 448 nm, respectively. Antibodies raised against each P-450 recognized all forms, although differences were observed with respect to the extents of cross-reactivities on Western blots. Form-specific peptide fragments were also detected among the four P-450 proteins after partial protease-digestion. P-450_6β1was identical to P-450_6β-3 in the first 26 residues of the NH₂terminal amino acid sequence, but differed by 13 residues from P-450_6β-2. The amino-terminal sequence of P-450_6β-2 was unique and was not identical with those of any rat P-450 previously reported. This P-450 form was detected in the livers of untreated male rats and was induced by treatment with dexamethasone, but not with phenobarbital. All four purified preparations catalyzed testosterone 6/9-hydroxylation in a reconstituted system containing cytochrome £%, extracted microsomal lipids and sodium cholate. Rates of hydroxylation were 35.3, 23.7, 13.8, and 0. 6 nmol/min/ nmol P-450, respectively, with P-450_6β-1, P-450_6β -2, P-450_6β-3, and P-450_6β-4. In addition, all antibodies against each of the four P-450_6β-2, forms inhibited more than 90% of the formation of 6/ 5-hydroxytestosterone in hepatic microsomes of untreated rats. These results indicate that at least four distinct yet structurally related forms of P-450, including a new form, P– 4505β -2> exist in rat livers, and all of them catalyze testosterone 6β -hydroxylation.