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.
D000251:Adenosine Triphosphatases / Q000378:metabolism*; D000817:Anilino Naphthalenesulfonates / Q000378:metabolism; D001419:Bacteria / Q000201:enzymology*; D002793:Cholic Acids / Q000494:pharmacology; D003840:Deoxycholic Acid / Q000494:pharmacology; D003911:Dextrans / Q000378:metabolism; D003956:Dialysis; D004735:Energy Transfer; D006863:Hydrogen-Ion Concentration; D008274:Magnesium / Q000494:pharmacology; D010743:Phospholipids / Q000378:metabolism*; D013025:Glycine max; D013696:Temperature; D013816:Thermodynamics
D000817:Anilino Naphthalenesulfonates; D002793:Cholic Acids; D003911:Dextrans; D010743:Phospholipids; D003840:Deoxycholic Acid; D000251:Adenosine Triphosphatases; D008274:Magnesium