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)
Department of Pharmacology, School of Medicine, Keio University, Tokyo.
K
K
Nagata
F J
FJ
Gonzalez
Y
Y
Yamazoe
R
R
Kato
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