The reconstituted alpha 3 beta 3 delta complex of the thermostable F1-ATPase.
Previously we reported that ATPase activity was recovered when the subunit alpha + beta + gamma or alpha + beta + delta of the F1-ATPase from the thermophilic bacterium PS3 were combined under appropriate conditions. Unlike that of holoenzyme (TF1) and the alpha + beta + gamma mixture, ATPase activity of the alpha + beta + delta mixture was heat labile and insensitive to azide inhibition (Yoshida, M., Sone, N., Hirata, H., and Kagawa, Y. (1977) J. Biol. Chem. 252, 3480-3485). Here, the properties of purified subunit complexes were compared in detail with those of native TF1. The subunit stoichiometries of the complexes were determined to be alpha 3 beta 3 gamma 1 and alpha 3 beta 3 delta 1. In general, the properties of the alpha 3 beta 3 gamma complex are very similar to those of TF1, whereas those of the alpha 3 beta 3 delta complex are significantly different. ATPase activity of the alpha 3 beta 3 delta complex is cold labile. The alpha 3 beta 3 delta complex showed a less stringent specificity for substrate and divalent cation than TF1 and the alpha 3 beta 3 gamma complex. Two Km values for ATP were exhibited by the alpha 3 beta 3 delta complex with the lower one being in the range of 0.1 microM. Equilibrium dialysis experiments revealed that the alpha 3 beta 3 delta complex cannot specifically bind ADP in the absence of Mg2+, while TF1 and the alpha 3 beta 3 gamma complex bind about 1 and 3 mol of ADP/mol of enzyme, respectively. ADP-dependent inactivation of the alpha 3 beta 3 delta complex by dicyclohexylcarbodiimide was not observed. The alpha 3 beta 3 gamma complex was readily formed when the gamma subunit was added to the alpha 3 beta 3 delta complex, suggesting that the alpha 3 beta 3 delta complex is not a "dead-end" complex. The cause of thermolability of the alpha 3 beta 3 delta complex appears to be the low stability of the complex itself at high temperature and not due to an unusually low thermostability of the delta subunit.
Yokoyama
K
K
Department of Life Science, Tokyo Institute of Technology, Midori-ku Yokohama, Japan.
Hisabori
T
T
Yoshida
M
M
D000244:Adenosine Diphosphate / Q000378:metabolism; D001419:Bacteria / Q000201:enzymology*; D002413:Cations, Divalent; D004024:Dicyclohexylcarbodiimide / Q000494:pharmacology; D004795:Enzyme Stability; D006358:Hot Temperature; D007700:Kinetics; D046911:Macromolecular Substances; D008274:Magnesium / Q000494:pharmacology; D011485:Protein Binding; D006180:Proton-Translocating ATPases / Q000302:isolation & purification / Q000378:metabolism*
D002413:Cations, Divalent; D046911:Macromolecular Substances; D004024:Dicyclohexylcarbodiimide; D000244:Adenosine Diphosphate; D006180:Proton-Translocating ATPases; D008274:Magnesium