Cloning and characterization of Syrian hamster testosterone 7 alpha-hydroxylase, CYP2A9.
We report here the cloning of a cDNA encoding testosterone 7 alpha-hydroxylase in the Syrian hamster, designated CYP2A9. Overlapping clones encoding Syrian hamster CYP2A9 were isolated by screening a liver cDNA library and by performing rapid amplification of cDNA ends polymerase chain reaction on the cDNA library. The sequence of the CYP2A9 cDNA contains an open reading frame of 1482 nucleotides encoding a polypeptide of 493 amino acids with a calculated molecular mass of 56,295 Da. The sequence is flanked by a 5'-untranslated region of 10 bp and a 3'-untranslated region of 178 bp including the poly(A) tail. The deduced amino acid sequence shares significant homology with members of CYP2A subfamily, notably with CYP2A1 and CYP2A12 which have testosterone 7 alpha-hydroxylase activity. We characterized the catalytic activity of CYP2A9 using microsomes obtained by transient expression of its cDNA in transfected COS-7 cells. CYP2A9 was found to hydroxylate testosterone at position 7 alpha. In Syrian hamster livers, a higher level of testosterone 7 alpha-hydroxylase activity as well as the mRNA of CYP2A9 in male than in female was obtained. The testosterone 7 alpha-hydroxylase activity and the mRNA level in liver were both decreased moderately by administration of 3-methylcholanthrene and slightly by administration of phenobarbital. In contrast, in kidney, both 3-methylcholanthrene and phenobarbital significantly decreased the mRNA level. These facts indicate that the regulation of the hamster testosterone 7 alpha-hydroxylase (CYP2A9) expression is different from that of the rat (CYP2A1) and hamster reported previously by other workers.
Kurose
K
K
Department of Pharmaceutical Sciences, National Institute of Public Health, Tokyo, Japan. kurose@iph.go.jp
Tohkin
M
M
Ushio
F
F
Fukuhara
M
M
D000595:Amino Acid Sequence; D000818:Animals; D001189:Aryl Hydrocarbon Hydroxylases; D001483:Base Sequence; D019556:COS Cells; D003001:Cloning, Molecular; D006224:Cricetinae; D018076:DNA, Complementary / Q000737:chemistry / Q000302:isolation & purification; D005260:Female; D015870:Gene Expression; D007668:Kidney / Q000187:drug effects / Q000201:enzymology; D008099:Liver / Q000187:drug effects / Q000201:enzymology; D008297:Male; D008647:Mesocricetus / Q000235:genetics*; D008748:Methylcholanthrene / Q000494:pharmacology; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010634:Phenobarbital / Q000494:pharmacology; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000378:metabolism; D013250:Steroid Hydroxylases / Q000737:chemistry / Q000235:genetics*; D013739:Testosterone / Q000378:metabolism; D014162:Transfection
D018076:DNA, Complementary; D012333:RNA, Messenger; D013739:Testosterone; D008748:Methylcholanthrene; D013250:Steroid Hydroxylases; D001189:Aryl Hydrocarbon Hydroxylases; C022724:testosterone 7-alpha-hydroxylase, hamster; D010634:Phenobarbital