Aromatase cytochrome P450 (P450arom), a membrane-associated enzyme is expressed in diverse microsomal environments which include NADPH- cytochrome P450 reductase (reductase), an essential microsomal component of the active enzyme complex responsible for androgen metabolism to estrogen. Hypothesis: The catalytic properties of the aromatase complex are dependent on P450arom structure and components are dependent environment. Objectives: 1) To compare and contrast the catalytic properties of porcine gonadal, porcine gonadal, porcine placental and human P450arom activity in ovary and placenta. 3) To investigate competition between P450arom and P450c17 affecting ovarian androgen and estrogen synthesis. 4) To define the structural domains and important amino acid residues determining catalytic properties of P450arom. Health Relatedness: Balanced androgen metabolism and estrogen synthesis is essential for normal development, reproduction, fertility and general health. Research Design: Functional differences between unique porcine isozymes of P450arom will be exploited to elucidate biochemical function, physiological significance and structure/function relationships. Methods: Recombinant wild-type and chimeric P450arom enzymes will be used in reconstituted assays in parallel with microsomes from porcine and human ovarian and placental tissues. Levels of P450s and reductase, determined by immunoblot, will be used to assess the microsomal environment in vivo. Enzyme activities, steroid products and kinetic estimates will be examined under varying redox environments. Mutational analysis, based on chimeric P450 enzyme function will be used to determine residues and sequence motifs influencing P450arom activity and reductase interactions. This approach is novel in utilizing structural conservation and functional divergence between human and porcine P450arom isoforms to probe molecular interactions with reductase and essential questions concerning the integration of ovarian steroidogenesis.
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