Catalytic differences between porcine blastocyst and placental aromatase isozymes

Two isozymes of porcine aromatase, the placental and the blastocyst forms, were expressed in CHO cells using the mammalian cell transfection method. U sing an 'in-cell' assay (a H-3-water release method), catalytic parameters of the porcine placental aromatase were found to be very similar to those o f the human enzyme; however, the activity of the blastocyst isozyme was fou nd to be one-thirtieth that of the placental isozyme. Product isolation ass ay (using testosterone as the substrate) revealed that the major steroid pr oducts were 17 beta-estradiol and 19-nortestosterone. The product ratio of estradiol/19-nortestosterone was found to be 94 : 6 for the porcine placent al form, 6 : 94 for the porcine blastocyst form, and 92 : 8 for the human w ild-type aromatase. Therefore, the porcine blastocyst aromatase isozyme cat alyzes mainly androgen 19-desmethylation rather than aromatization. In addi tion, inhibition profile analyses on the placental and blastocyst isozymes were performed using three steroidal inhibitors [4-hydroxyandro-stenedione (4-OHA), 7 alpha-(4'-amino)phenylthio-1,4-androstandiene-3,17-dione (7 alph a-APTADD), and bridge (2,19-methyleneoxy) androstene-3,17-dione (MDL 101,00 3)], and four nonsteroidal inhibitors [aminoglutethimide (AG), CGS 20267, I CI D1033, and vorozole (R83842)]. While the two isozymes of porcine aromata se share 93% amino-acid sequence identity, our results indicate that the tw o porcine aromatase isozymes have distinct responses to various aromatase i nhibitors.