Inhibition of placental aromatase activity in a cell free assay by ovarian protein.
Previously we identified a fraction of follicular fluid (follicle regulatory protein: FRP) which inhibits granulosa cell aromatase activity. During the course of these studies the question of FRP acting via autocrine as well as paracrine mechanisms arose in addition to the need for a more efficient method of screening for aromatase inhibitory activity during the purification of FRP. Accordingly, we assessed the effects of FRP on aromatase activity in a microsomal assay. Placental microsome preparations were preincubated for 20 minutes with or without FRP prior to a 20 minute incubation with testosterone. Significantly less conversion of testosterone to estrogen occurred with FRP compared to control preincubation. When follicular protein was added without pre-incubation, there was no apparent change in microsomal aromatase activity, whereas after a 20 minute pre-incubation with the follicular protein fraction, significantly less testosterone was converted into estrogen. When various concentrations of FRP were assayed in the placental aromatase assay, a dose-response curve demonstrated a 50% inhibitory dose (ID50) of approximately 400 micrograms/ml. To further purify the aromatase inhibitory activity, 5 mg of the crude follicular fluid preparation was eluted through an anion exchange column via HPLC using a sodium acetate gradient. The fractions in the central elution peak contained aromatase inhibitor activity with ID50 values of 25-160 micrograms/ml. Thus Fractions were further purified by elution through a gel exclusion column via HPLC which demonstrated inhibition of cell free placental aromatase activity in the 15,000-18,000 molecular weight range with an ID50 of 5 micrograms/ml.(ABSTRACT TRUNCATED AT 250 WORDS)
D047072:Aromatase Inhibitors; D002474:Cell-Free System; D005260:Female; D006801:Humans; D066298:In Vitro Techniques; D036341:Intercellular Signaling Peptides and Proteins; D008861:Microsomes / Q000201:enzymology; D008970:Molecular Weight; D010455:Peptides / Q000302:isolation & purification / Q000494:pharmacology*; D010920:Placenta / Q000201:enzymology*; D011247:Pregnancy
D047072:Aromatase Inhibitors; D036341:Intercellular Signaling Peptides and Proteins; D010455:Peptides; C018355:intraovarian peptides