Reconstitution of vesicles capable of energy transformation from phospholipids and adenosine triphosphatase of a thermophilic bacterium.
1. A stable ATPase [EC 3.6.1.3] complex (TF0-F1) from the thermophilic bacterium PS3 was reconstituted into vesicles capable of energy transformation,measured as ATP-dependent enhancement of fluorescence of 8-anilinonoaphthalene-1-sulfonate. 2. The factors necessary for obtaining highly active vesicles were investigated. Cholate and deoxycholate were both required for solubilization of TF0-F1 and P-lipids, and removal of the detergents by dialysis resulted in vesicle formation. Medium of around pH 8 and low ionic strength containing 2.5 mM MgSO4 was found suitable for dialysis. The optimal temperature for reconstitution was 30 degrees with soybean P-lipids and 45 degree with PS3 P-lipids. The optimal ratio of protein to lipid was about 1/50. 3. The vesicles obtained under these conditions were mainly 100-200 nm in diameter, covered with 9.5 nm spheres, and had a bouyant density of 1.06 in sucrose andan internal volume of about 0.5 mul per mg of P-lipids.
Reconstitution of vesicles capable of energy transformation from phospholipids and adenosine triphosphatase of a thermophilic bacterium. 1. A stable ATPase [EC 3.6.1.3] complex (TF0-F1) from the thermophilic bacterium PS3 was reconstituted into vesicles capable of energy transformation,measured as ATP-dependent enhancement of fluorescence of 8-anilinonoaphthalene-1-sulfonate. 2. The factors necessary for obtaining highly active vesicles were investigated. Cholate and deoxycholate were both required for solubilization of TF0-F1 and P-lipids, and removal of the detergents by dialysis resulted in vesicle formation. Medium of around pH 8 and low ionic strength containing 2.5 mM MgSO4 was found suitable for dialysis. The optimal temperature for reconstitution was 30 degrees with soybean P-lipids and 45 degree with PS3 P-lipids. The optimal ratio of protein to lipid was about 1/50. 3. The vesicles obtained under these conditions were mainly 100-200 nm in diameter, covered with 9.5 nm spheres, and had a bouyant density of 1.06 in sucrose andan internal volume of about 0.5 mul per mg of P-lipids.