Reconstitution of Vesicles Capable of Energy Transformation from Phospholipids and Adenosine Triphosphatase of a Thermophilic Bacterium1

Abstract

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-anilinonaphthalene-1-sulfonate. The factors necessary for obtaining highly active vesicles were investigated. Cholate and deoxycholate were both required for solubilization of TF₀7middot;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° with soybean P-lipids and 45° with PS3 P-lipids. The optimal ratio of protein to lipid was about 1/50. 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 and an internal volume of about 0.5 μl per mg of P-lipids.