Abstract
Cytochrome oxidase, QH2-cytochrome c reductase, and the oligomycin-sensitive adenosine triphosphatase were incorporated into liposomes by a new procedure which yielded unidirectional orientation of the proteins. Cytochrome oxidase was reconstituted in the mitochrondrial orientation and the adenosine triphosphatase in the submitochondrial orientation. Reconstitutions were achieved by incubating the proteins at room temperature with liposomes which contained phosphatidylcholine, phosphatidylethanolamine, and an acidic phospholipid (cardiolipin, phosphatidylinositol, or phosphatidylserine). The incorporation occurred without added detergent or sonication. This incorporation procedure may serve as a model for the insertion of proteins in vivo.
Highlights
Cytochrome oxidase, QH,cytochrome c reductase, and the oligomycin-sensitive adenosine triphosphatase were incorporated into liposomes by a new procedure which yielded unidirectional orientation of the proteins
Respiratory control ratio of 5 implies that at least 80% of the cytochrome oxidase molecules were incorporated into the vesicles
High respiratory control ratios could be obtained at phospholipid: protein ratios considerably lower than those required for cytochrome oxidase incorporation
Summary
Materials-Cytochrome oxidase was prepared according to Yonetani [22] as modified by Eytan et al [23]. In some experiments the presence of Emasol was avoided by preparing the cytochrome oxidase with cholate according to Yu et al [24]. Purified cytochrome oxidase (1 mg) was added and the mixture was incubated at room temperature. Incorporation-A solution of phospholipids in chloroform:methanol (2:l) was dried down in a test tube under a stream of nitrogen, dissolved in ether, and dried again They were resuspended at a concentration of 25 mM in either 50 mM KP, (pH 7.0) or 10 mM Hepes’. The resulting liposomes were incubated with the protein (1 mg/ml) for 30 min at room temperature in the presence of 10 mM MgCl, or as specified in the legends to the figures
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