Adrenal cytochrome P-45011β-proteoliposomes catalyzing aldosterone synthesis: Preparation and characterization

• Published in: Biochimica et biophysica acta Vol. 984; no. 1; pp. 50 - 56
• Main Authors: IKUSHIRO, S.-I, KOMINAMI, S, TAKEMORI, S
• Format: Journal Article
• Language: English
• Published: Amsterdam Elsevier 21.08.1989; North-Holland
• Subjects: Adrenal Cortex - metabolism; Aldosterone - biosynthesis; Analytical, structural and metabolic biochemistry; Animals; Biological and medical sciences; Cattle; Cytochrome P-450 Enzyme System - administration & dosage; Desoxycorticosterone - metabolism; Enzyme Stability; Enzymes and enzyme inhibitors; Fundamental and applied biological sciences. Psychology; Oxidoreductases; Proteolipids; Purification; Bovine; Enzyme; Cytochrome P450; Liposome; Biosynthesis; Biological activity; Characterization; Vertebrata; Mitochondria; Proteoliposome; Mammalia; Reconstituted system; Steroidogenesis; Artiodactyla; Mechanism of action; Ungulata
• ISSN: 0006-3002; 1878-2434

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.