Differences in the catalytic activity of the placental and gonadal isozymes of porcine aromatase cytochrome P450 (P450arom) were examined in cell lines exhibiting stable expression of recombinant enzyme. Cell lines were selected that expressed high, but similar, immunodetectable levels of each isozyme based on Western analysis. Aromatase activity varied with growth in culture, decreasing at confluence from a peak reached between 50-80% cell density. Cells expressing the placental isozyme had 3-5 times higher catalytic activity (per mg protein) than those expressing the gonadal isozyme. The P450arom inhibitor fadrazole (1 μM) inhibited more than 97% of this activity, whereas another imidazole, etomidate (1 μM), selectively inhibited gonadal P450arom activity by 92%. Estrogen synthesis from androstenedione and testosterone was determined by RIA and confirmed by HPLC analysis, which also identified the accumulation of the 19-hydroxy and 19-oxo intermediates of the respective substrates. There was no evidence of other steroid metabolites accumulating in the media of cell lines expressing either isozyme. Tritiated water formed during aromatization of substrates 3H labeled at the C1 and C2 positions was stereo-selective for the β orientation, but less so for testosterone than androstenedione during metabolism by the porcine placental (and human) isozyme than the gonadal isozyme. Testosterone showed a higher affinity for the porcine placental P450arom than the gonadal P450arom, but both isozymes had similar affinities for androstenedione. Testosterone was also aromatized more slowly than androstenedione by the porcine gonadal P450arom. These data suggest that catalytic differences have arisen in the substrate binding pocket during the evolution of isozymes of porcine P450arom that affect androgen metabolism, particularly the aromatization of testosterone. The physiological significance of these differences to the reproductive biology of the pig remains to be determined.
|Original language||English (US)|
|Number of pages||9|
|State||Published - 1999|
ASJC Scopus subject areas
- Endocrinology, Diabetes and Metabolism