Cloning and expression of cytochrome P450(11 beta) of porcine adrenal cortex.
Four cDNA clones were isolated from a porcine adrenal gland library by using a bovine cytochrome P450(11 beta) cDNA fragment as a probe. Nucleotide sequences of the four clones overlapped with each other. The deduced amino acid sequences indicated that these clones were derived from a porcine P450(11 beta) cDNA. Consecutive alignment of these clones covered almost 70% of a coding region of the cDNA, but its 5'-terminus was missing. The adrenal mRNA was reverse-transcribed, and polymerase chain reaction was used to obtain a cDNA fragment including the 5'-terminus. A cDNA constructed from this fragment and the isolated four fragments covered the entire apparent open reading frame of the enzyme, which was thus concluded to comprise 503 amino acids including a putative extension peptide of 24 amino acids at the NH2-terminus. The amino acid sequence was 82% identical to that of bovine P450(11 beta)-3. The cDNA was transfected into COS-7 cells, and steroidogenic activity of the cells was measured. The cells not only converted 11-deoxycorticosterone to corticosterone and 18-hydroxycorticosterone, but also produced aldosterone. Thus we conclude that the primary sequence of porcine P450(11 beta) which plays a role in the biosynthesis of glucocorticoids as well as mineralocorticoids was determined.
Sun
T
T
Department of Molecular Physiological Chemistry, Osaka University Medical School, Japan.
Zhao
Y
Y
Nonaka
Y
Y
Okamoto
M
M
D015069:18-Hydroxycorticosterone / Q000378:metabolism; D000302:Adrenal Cortex / Q000201:enzymology*; D000450:Aldosterone / Q000096:biosynthesis; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002417:Cattle; D002460:Cell Line; D003001:Cloning, Molecular; D003345:Corticosterone / Q000096:biosynthesis; D019405:Cytochrome P-450 CYP11B2; D003577:Cytochrome P-450 Enzyme System / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D018076:DNA, Complementary / Q000235:genetics*; D008969:Molecular Sequence Data; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D013252:Steroid 11-beta-Hydroxylase / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D013552:Swine
D018076:DNA, Complementary; D000450:Aldosterone; D015069:18-Hydroxycorticosterone; D003577:Cytochrome P-450 Enzyme System; D019405:Cytochrome P-450 CYP11B2; D013252:Steroid 11-beta-Hydroxylase; D003345:Corticosterone