Human prostate CYP3A5: identification of a unique 5'-untranslated sequence and characterization of purified recombinant protein.
We have isolated a cDNA clone coding for CYP3A5 from a human prostate cDNA library. The human prostate CYP3A5 cDNA had a unique 5'-untranslated sequence, suggesting that the prostate specific regulation of CYP3A5 is different from liver. Hybridization screening using a human genomic BAC library yielded four positive clones, two of which were shown to contain the unique 5'-untranslated sequence by Southern blot analysis. The CYP3A5 recombinant protein expressed in Escherichia coli using the pCWOri expression vector was purified to an almost electrophoretically homogeneous state with a specific content of 4.4 nmol of P450/mg of protein. This P450 exhibited 6beta-hydroxylation activity toward both testosterone and progesterone. No polar metabolite of 5alpha-dihydrotestosterone (DHT) was detected. The apparent K(m) values for testosterone and progesterone 6beta-hydroxylation were 143 and 114 microM, respectively, with V(max) values of 0.48 and 0. 21 nmol/min/nmol of P450, respectively. This is the first report that a particular form of P450, CYP3A5, has been isolated from human prostate and that the purified recombinant protein of CYP3A5 has been shown to be active in the metabolism of sex hormones.
Department of Urology, Osaka City University Medical School, Osaka, 545-8585, Japan.
Y
Y
Yamakoshi
T
T
Kishimoto
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K
Sugimura
H
H
Kawashima
D020121:5' Untranslated Regions / Q000235:genetics*; D000595:Amino Acid Sequence; D001483:Base Sequence; D015139:Blotting, Southern; D003001:Cloning, Molecular; D051544:Cytochrome P-450 CYP3A; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D015723:Gene Library; D006801:Humans; D006900:Hydroxylation; D007527:Isoenzymes / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D007700:Kinetics; D008099:Liver / Q000201:enzymology; D008297:Male; D011374:Progesterone / Q000378:metabolism; D011467:Prostate / Q000201:enzymology* / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D013057:Spectrum Analysis; D013379:Substrate Specificity; D013739:Testosterone / Q000378:metabolism
D020121:5' Untranslated Regions; D007527:Isoenzymes; D011993:Recombinant Fusion Proteins; D013739:Testosterone; D011374:Progesterone; D003577:Cytochrome P-450 Enzyme System; C104464:CYP3A protein, human; C510164:CYP3A5 protein, human; D051544:Cytochrome P-450 CYP3A