Adrenal cytochrome P-45011 beta-proteoliposomes catalyzing aldosterone synthesis: preparation and characterization.
Purified cytochrome P-45011 beta from bovine adrenocortical mitochondria was successfully incorporated into the liposome membranes composed of phosphatidylcholine, phosphatidylethanolamine and cardiolipin at a molar ratio of 2:2:1. The incorporation of P-45011 beta into the liposome membranes was ascertained by the Ficoll density gradient centrifugation and the protein refractoriness to trypsin digestion. The prepared proteoliposomes containing P-45011 beta and phospholipid at a molar ratio of 1:3000 were unilamellar vesicles of about 40 nm in average diameter. The P-45011 beta embedded in the liposome membranes was found to be more stable than the detergent-solubilized form. The reconstituted system containing the P-45011 beta-proteoliposomes, adrenodoxin and NADPH-adrenodoxin reductase showed catalytic activities not only for the hydroxylation of 11-deoxycorticosterone at 11 beta- and 18-positions but also for its conversion into aldosterone with a turnover number of 2.3 nmol/min per nmol of P-45011 beta. A successive reaction without the intermediates leaving from the enzyme was suggested for the P-45011 beta-mediated conversion of 11-deoxycorticosterone to aldosterone following the result that the formation of aldosterone was linear with respect to time without the lag phase; this was confirmed by the result that radioactivity in aldosterone from 3H-labeled 11-deoxycorticosterone was scarcely decreased by the addition of unlabeled intermediates to the reactions system.
Ikushiro
S
S
Faculty of Integrated Arts and Sciences, Hiroshima University, Japan.
Kominami
S
S
Takemori
S
S
D000302:Adrenal Cortex / Q000378:metabolism*; D000450:Aldosterone / Q000096:biosynthesis*; D000818:Animals; D002417:Cattle; D003577:Cytochrome P-450 Enzyme System / Q000008:administration & dosage*; D003900:Desoxycorticosterone / Q000378:metabolism; D004795:Enzyme Stability; D011510:Proteolipids
D011510:Proteolipids; C020485:proteoliposomes; D003900:Desoxycorticosterone; D000450:Aldosterone; D003577:Cytochrome P-450 Enzyme System