The Journal of Biochemistry · 1990 · 63 citations · 0 references
Life SciencesBiochemistryMolecular BiologyEnzymologyMetabolismSteroid BiochemistryCytochrome P450 EnzymesMitochondrial Function11 Beta-hydroxylaseCdna CloningCytochrome P450 Enzyme ActivityCytochrome P450 IsoenzymesCytochrome P450-mediated Steroid MetabolismImmunofluorescence StainingMicroscopyRecombinant Cytochrome P450 Enzymes
Expression plasmids were constructed using two cDNA clones of P-450(11 beta), pcP-450-(11 beta)-2, and pcP-450(11 beta)-3 (Morohashi et al. (1987) J. Biochem. 102, 559-568 and Kirita et al. (1988) J. Biochem. 104, 683-686), and introduced into COS-7 cells by electroporation. The expression of P-450(11 beta) proteins and their localization in the mitochondria were demonstrated by immunoblotting, immunofluorescence microscopy, and immunoelectron microscopy. The enzymatic activities of the expressed P-450(11 beta)s were determined using deoxycorticosterone (DOC), deoxycortisol, and corticosterone as substrates. Though the activities of the two P-450(11 beta)s for 11-, 18-, and 19-hydroxylation of DOC were almost equal, the production of 18-hydroxycorticosterone and aldosterone from corticosterone by P-450(11 beta)-3 was greater than that by P-450(11 beta)-2.