Structure of a gene and cDNA of a major constitutive form of testosterone 6β-hydroxylase (P450/6βA) encoding CYP3A2: Comparison of the cDNA with P450PCN2

Masaaki Miyata, Kiyoshi Nagata, Miki Shimada, Yasushi Yamazoe, Ryuichi Kato

    Research output: Contribution to journalArticlepeer-review

    31 Citations (Scopus)

    Abstract

    P450/6βA gene was isolated from a rat genomic library and its encoding protein was characterized by the expression of the corresponding cDNA in COS-1 cells. Between exon regions of P450/6βA gene and P450PCN2 cDNA (F. J. Gonzalez, B.-J. Song, and J. P. Hardwick (1986) Mol. Cell. Biol. 6, 2969-2976), 12 nucleotide differences were observed, involving two amino acid changes, His → Asp [429] and Asp → Gly [445], respectively. A cDNA (6β-A cDNA), whose nucleotide sequence was completely identical with the corresponding exon of P450/6βA gene, was isolated from a rat cDNA library. Northern blotting using specific oligonucleotide probes showed that 6β-A mRNA, but not P450PCN2 mRNA, was a major form in livers of the male rats. 6β-A protein expressed in COS-1 cells (at about 0.1 to 0.3% of total microsomal protein) catalyzed testosterone 6β-hydroxylation. The reaction was enhanced by the addition of NADPH-P450 reductase (2.5-fold) and by the simultaneous addition of cytochrome b5; and NADPH-P450 reductase (13.7-fold). Catalytic properties of 6β-A for typical CYP3A substrates were consistent with those of purified rat P450(6β-1) and P450(6β-3), corresponding, respectively, to CYP3A2 or the variant form (K. Nagata, F. J. Gonzalez, Y. Yamazoe, and R. Kato (1990) J. Biochem. 107, 718725). Apparent Michaelis constants (K(m)) of 6β-A for testosterone 6β-hydroxylation and a O-dealkylation ratio of propoxycoumarin to pentoxycoumarin, however, showed higher degrees of similarity to those of purified P4506β-3 than those of P4506β-1.

    Original languageEnglish
    Pages (from-to)351-359
    Number of pages9
    JournalArchives of Biochemistry and Biophysics
    Volume314
    Issue number2
    DOIs
    Publication statusPublished - 1994 Jan 1

    ASJC Scopus subject areas

    • Biophysics
    • Biochemistry
    • Molecular Biology

    Fingerprint Dive into the research topics of 'Structure of a gene and cDNA of a major constitutive form of testosterone 6β-hydroxylase (P450/6βA) encoding CYP3A2: Comparison of the cDNA with P450PCN2'. Together they form a unique fingerprint.

    Cite this