Purification and characterization of human placental aromatase cytochrome P-450.
Aromatase cytochrome P-450, which catalyzes the conversion of androgens to estrogens, was purified from human placental microsomes. The enzyme was extracted with sodium cholate, fractionated by ammonium sulfate precipitation, and subjected to column chromatography in the presence of its substrate, androstenedione, and the nonionic detergent, Nonidet P-40. The preparation exhibits a single major band when analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and has a specific content of 11.5 nmol of P-450/mg of protein. The purified enzyme displays spectroscopic properties typical of the ferric and ferrous forms of cytochrome P-450. Full enzymatic activity can be reconstituted with rabbit liver microsomal cytochrome P-450 reductase and Nonidet P-40. Purified aromatase cytochrome P-450 displays catalytic characteristics similar to the enzyme in intact microsomes in the aromatization of androstenedione, 19-hydroxyandrostenedione and 19-oxoandrostenedione. Testosterone and 16 alpha-hydroxytestosterone are aromatized at maximal rates similar to androstenedione, and all substrates exhibit relative affinities corresponding to those observed in microsomes. We have raised rabbit antibodies to the purified enzyme which show considerable specificity and sensitivity on immunoblots.
D000728:Androgens / Q000378:metabolism; D000818:Animals; D001141:Aromatase / Q000302:isolation & purification* / Q000378:metabolism; D002845:Chromatography; D003577:Cytochrome P-450 Enzyme System / Q000302:isolation & purification* / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D005590:Fractional Precipitation; D006801:Humans; D007159:Immunologic Tests; D007700:Kinetics; D008862:Microsomes, Liver / Q000201:enzymology; D009251:NADPH-Ferrihemoprotein Reductase / Q000378:metabolism; D017830:Octoxynol; D010920:Placenta / Q000201:enzymology*; D011092:Polyethylene Glycols; D011247:Pregnancy; D011817:Rabbits; D013053:Spectrophotometry
D000728:Androgens; D011092:Polyethylene Glycols; D017830:Octoxynol; D003577:Cytochrome P-450 Enzyme System; C010615:Nonidet P-40; D001141:Aromatase; D009251:NADPH-Ferrihemoprotein Reductase