Purification and characterization of four catalytically active testosterone 6 beta-hydroxylase P-450s from rat liver microsomes: comparison of a novel form with three structurally and functionally related forms.
Four microsomal cytochrome P-450s (P-450), all of which are active testosterone 6 beta-hydroxylases, were purified to electrophoretic homogeneity from livers of phenobarbital-treated (P-4506 beta-1 and P-4506 beta-3) or dexamethasone-treated adult male rats (P-4506 beta-2 and P-4506 beta-4). Purified P-4506 beta-1, P-4506 beta-2, P-4506 beta-3, and P-4506 beta-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-4506 beta-1 and P-4506 beta-3 displayed spectra of the reduced carbonyl complex with lambda max at 447 nm. P-4506 beta-2 and P-4506 beta-4 showed lambda 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-4506 beta-1 was identical to P-4506 beta-3 in the first 26 residues of the NH2-terminal amino acid sequence, but differed by 13 residues from P-4506 beta-2. The amino-terminal sequence of P-4506 beta-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.(ABSTRACT TRUNCATED AT 250 WORDS)
Nagata
K
K
Department of Pharmacology, School of Medicine, Keio University, Tokyo.
Gonzalez
F J
FJ
Yamazoe
Y
Y
Kato
R
R
D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000276:immunology; D003577:Cytochrome P-450 Enzyme System / Q000276:immunology / Q000302:isolation & purification*; D015786:Cytochromes b5 / Q000378:metabolism; D003907:Dexamethasone / Q000494:pharmacology; D006801:Humans; D006900:Hydroxylation; D007527:Isoenzymes / Q000276:immunology / Q000302:isolation & purification*; D008055:Lipids / Q000494:pharmacology; D008297:Male; D008862:Microsomes, Liver / Q000187:drug effects / Q000201:enzymology*; D008969:Molecular Sequence Data; D051381:Rats; D011919:Rats, Inbred Strains; D013250:Steroid Hydroxylases / Q000276:immunology / Q000302:isolation & purification*; D013329:Structure-Activity Relationship
D000906:Antibodies; D007527:Isoenzymes; D008055:Lipids; D003907:Dexamethasone; D015786:Cytochromes b5; D003577:Cytochrome P-450 Enzyme System; D013250:Steroid Hydroxylases; C022657:steroid hormone 6-beta-hydroxylase