Novel properties of human placental aromatase as cytochrome P-450: purification and characterization of a unique form of aromatase.
Aromatase has been purified to homogeneity from human placental microsomes based on detection of its catalytic activities in the eluates from columns of octylamino-Sepharose 4B, hydroxylapatite, Mono S, hydroxylapatite HCA, and Mono Q. The purified preparation shows only one band corresponding to the apparent subunit molecular weight of 51,000 daltons on sodium dodecyl sulfate-polyacrylamide gel. The aromatase in the presence of NADPH and NADPH-cytochrome P-450 reductase converts testosterone to 17 beta-estradiol with the high specific activity of 103 nmol/min/mg of protein. However, whether the preparation is reduced by sodium dithionate chemically or by NADPH and the reductase enzymatically, its reduced, CO-difference spectrum has no peak at about 450 nm and has only a small peak at about 420 nm, probably due to its inactivation in spite of the catalytically full activity in the same preparation. The absolute spectrum of the aromatase exhibits a Soret peak at 423 nm in the absence of testosterone and addition of testosterone to the aromatase sample makes its absorption peak shift gradually from 423 to 393 nm (high spin type peak), which is a usual characteristic in the spectrum of cytochrome P-450. The reconstituted aromatase system efficiently catalyzes aromatization of 4-androstenedione, 19-hydroxy-4-androstenedione as well as testosterone. 16 alpha-Hydroxy-4-androstenedione and 16 alpha-hydroxytestosterone are also aromatized less efficiently and 19-nortestosterone is aromatized least efficiently. The reconstituted aromatase could scarcely oxidize various xenobiotics examined, suggesting a strict and narrow substrate specificity of this enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)
D001141:Aromatase / Q000302:isolation & purification / Q000378:metabolism*; D003577:Cytochrome P-450 Enzyme System / Q000302:isolation & purification / Q000378:metabolism*; D005260:Female; D006801:Humans; D005779:Immunodiffusion; D008861:Microsomes / Q000201:enzymology*; D008970:Molecular Weight; D010084:Oxidation-Reduction; D010920:Placenta / Q000201:enzymology*; D011247:Pregnancy; D013379:Substrate Specificity
D003577:Cytochrome P-450 Enzyme System; D001141:Aromatase