Purification, subunit structure, and kinetics of the chloroform-released F1ATPase complex from Rhodospirillum rubrum and its comparison with F1ATPase forms isolated by other methods.
A stable and homogeneous adenosine-5'-triphosphatase (ATPase, EC 3.6.1.3) has been solubilized from Rhodospirillum rubrum (R. rubrum) chromatophores by chloroform extraction. Purification of the Ca2+-dependent ATPase activity was 200-fold. Ca2+ can be replaced by Mg2+, Cd2+, and Mn2+. The Km for Ca-ATP (0.17 mM) is increased about 5-fold during solubilization of the enzyme, whereas the Km values for Mg-ATP (0.029 mM) and Cd-ATP (0.014 mM) are not affected. The chloroform-released ATPase has a molecular weight of 400,000 +/- 30,000 and consists of the following subunits (molecular weights in parenthesis): alpha(58,000), beta(53,500), gamma(39,000), delta(18,500), and epsilon(14,000). The amino acid composition and the fluorescence spectra are presented. Besides the chloroform-released ATPase complex three other Ca2+-dependent ATPase forms have been isolated from R. rubrum chromatophores by other methods for comparison. Ultrasonication of the membranes leads to the release of an ATPase complex which is mainly composed of alpha, beta, and gamma-subunits. From an acetone powder extract an ATPase complex could be purified by affinity chromatography which is composed of four kinds of subunits (alpha, beta, gamma, delta). The same acetone powder yields an ATPase consisting of only three different types of subunits (alpha, beta, gamma) if the final purification step is preparative disc electrophoresis on 6% polyacrylamide gels instead of affinity chromatography.
D000251:Adenosine Triphosphatases / Q000302:isolation & purification / Q000378:metabolism; D000596:Amino Acids / Q000032:analysis; D020130:Bacterial Chromatophores / Q000201:enzymology; D002725:Chloroform; D007700:Kinetics; D046911:Macromolecular Substances; D008970:Molecular Weight; D010086:Oxidative Phosphorylation Coupling Factors / Q000302:isolation & purification / Q000378:metabolism; D012247:Rhodospirillum rubrum / Q000201:enzymology*; D012997:Solvents; D013050:Spectrometry, Fluorescence
D000596:Amino Acids; D046911:Macromolecular Substances; D010086:Oxidative Phosphorylation Coupling Factors; D012997:Solvents; D002725:Chloroform; D000251:Adenosine Triphosphatases