Immunoaffinity purification of aromatase cytochrome P-450 from human placental microsomes, metabolic switching from aromatization to 1 beta and 2 beta-monohydroxylation, and recognition of aromatase isozymes.
Microsomal estrogen synthetase (aromatase) cytochrome P-450 was purified from fresh human placental microsomes by monoclonal anti-aromatase P-450 antibody-Sepharose 4B chromatography. The purified P-450 showed a single band of 55 kDa on SDS-polyacrylamide gel electrophoresis and the aromatase specific activity on reconstitution was 70 nmol/min/mg protein. The purified P-450 was stable with a t 1/2 of approximately 2 years on storage at -90 degrees C and showed Km = 43 nM for androstenedione aromatization. However, it was unstable under spectral measurement conditions in the presence of sodium dithionite and carbon monoxide and the carbon monoxide difference spectra showed a maximum at 450 nm and a specific content of 9.1 nmol of P-450/mg protein, giving a turnover number of approximately 7.7 per min for the purified aromatase. The one-step immunochemical purification method gave a 490-fold increase of specific activity with 55% yield of aromatase activity of the original microsomes. Analysis of androgen metabolism by the purified aromatase and an apparent large kinetic isotope effect found at the secondary positions when using [19(-3)H3, 4(-14)C] androgens revealed metabolic switching from the first 19-hydroxylation to 1 beta- and 2 beta- monohydroxylation by aromatase. Substrate specificity for [19(-3)H3]androstenedione and testosterone was indicated by differences in the extent of metabolic switching (18% and 30%) and in the 2 beta/1 beta ratio (60/40 and 10/90, respectively). The mouse monoclonal antibody used for immunoaffinity purification suppresses aromatase activity of human placenta, but was totally ineffective for aromatase in goldfish brain and rat ovary. Rabbit polyclonal antibodies to human placental aromatase P-450 suppressed both human placental and rat ovarian aromatase but were ineffective for goldfish brain aromatase. The study indicates that they are isozymes of aromatase based on different structures of P-450.
D000735:Androstenedione / Q000378:metabolism; D000818:Animals; D000911:Antibodies, Monoclonal / Q000302:isolation & purification; D001141:Aromatase / Q000032:analysis / Q000302:isolation & purification*; D002846:Chromatography, Affinity; D005260:Female; D006801:Humans; D006900:Hydroxylation; D007120:Immunochemistry; D007527:Isoenzymes / Q000032:analysis*; D051379:Mice; D008861:Microsomes / Q000201:enzymology*; D009251:NADPH-Ferrihemoprotein Reductase / Q000302:isolation & purification; D010920:Placenta / Q000201:enzymology*; D011247:Pregnancy; D011487:Protein Conformation; D013379:Substrate Specificity; D013739:Testosterone / Q000378:metabolism
D000911:Antibodies, Monoclonal; D007527:Isoenzymes; D013739:Testosterone; D000735:Androstenedione; D001141:Aromatase; D009251:NADPH-Ferrihemoprotein Reductase