Aromatase.
Aromatase catalyzes the conversion of androgens to estrogens through a series of monooxygenations to achieve the 19-desmolation and aromatization of the neutral steroid ring-A structure. We have separated two forms of aromatase, a major (P2a) and a minor (P3) form, from human term placenta through solubilization and chromatography. Partially purified aromatase in each form was immunoaffinity chromatographed to give a single band (SDS-PAGE) cytochrome P-450 of 55 kDa, utilizing a mouse monoclonal anti-human placental aromatase cytochrome P-450 IgGi (MAb3-2C2) which is capable of suppressing placental aromatase activity. The purified cytochrome P-450 showed specific aromatase activity of 25-30 nmol/min per mg with Km of 20-30 nM for androstenedione on reconstitution with NADPH-cyt P-450 reductase and dilauroyl L-alpha-phosphatidylcholine. This one step represents a higher than 100-fold purification with maintenance of the same Km. The stability analysis showed a half-life of more than 5 yr for solubilized aromatase and 2 months for the aromatase cytochrome P-450 on storage at -90 degrees C. Contrary to the recent claim that estrogen biosynthesis by reconstituted human placental cytochrome P-450 is by trans-diaxial 1 alpha,2 beta-hydrogen elimination, all of our partially purified forms and reconstituted aromatase synthesized estrogens by cis-1 beta, 2 beta-hydrogen elimination. Use of purified aromatase and [19-3H3, 4-14C]androstenedione led us to discover a metabolic switching by aromatase to 2 beta-hydroxylation of androgen. Results of the MAb3-2C2 suppression of aromatase activity in different species and tissues including human, baboons, horses, cows, pigs and rats indicated the presence of various isozymes of aromatase.
Osawa
Y
Y
Endocrine Biochemistry Department, Medical Foundation of Buffalo, Inc., NY 14203-1196.
Higashiyama
T
T
Fronckowiak
M
M
Yoshida
N
N
Yarborough
C
C
D000728:Androgens / Q000378:metabolism; D000818:Animals; D001141:Aromatase / Q000302:isolation & purification / Q000378:metabolism; D002250:Carbon Radioisotopes; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism; D005260:Female; D006801:Humans; D007527:Isoenzymes / Q000378:metabolism; D010920:Placenta / Q000201:enzymology; D011247:Pregnancy; D014316:Tritium
D000728:Androgens; D002250:Carbon Radioisotopes; D007527:Isoenzymes; D014316:Tritium; D003577:Cytochrome P-450 Enzyme System; D001141:Aromatase