Rao R, Al-Shawi M K, Senior A E
Department of Biochemistry, University of Rochester Medical Center, New York 14642.
J Biol Chem. 1988 Apr 25;263(12):5569-73.
The stoichiometry of nucleotide binding to the isolated alpha- and beta-subunits of Escherichia coli F1-ATPase was investigated using two experimental techniques: (a) titration with fluorescent trinitrophenyl (TNP) derivatives of AMP, ADP, and ATP and (b) the centrifuge column procedure using the particular conditions of Khananshvili and Gromet-Elhanan (Khananshvili, D., and Gromet-Elhanan, Z. (1985) FEBS Lett. 178, 10-14). Both procedures showed that alpha-subunit contains one nucleotide-binding site, confirming previous work. TNP-ADP and TNP-ATP bound to a maximal level of 1 mol/mol beta-subunit, consistent with previous equilibrium dialysis studies which showed isolated beta-subunit bound 1 mol of ADP or ATP per mol (Issartel, J. P., and Vignais, P. V. (1984) Biochemistry 23, 6591-6595). However, binding of only approximately 0.1 mol of ATP or ADP per mol of beta-subunit was detected using centrifuge columns. Our results are consistent with the conclusion that each of the alpha- and beta-subunits contains one nucleotide-binding domain. Because the subunit stoichiometry is alpha 3 beta 3 gamma delta epsilon, this can account for the location of the six known nucleotide-binding sites in E. coli F1-ATPase. Studies of in vitro assembly of isolated alpha-, beta-, and gamma- subunits into an active ATPase showed that ATP, GTP, and ITP all supported assembly, with half-maximal reconstitution of ATPase occurring at concentrations of 100-200 microM, whereas ADP, GDP, and IDP did not. Also TNP-ATP supported assembly and TNP-ADP did not. The results demonstrate that (a) the nucleotide-binding site on beta-subunit has to be filled for enzyme assembly to proceed, whereas occupancy of the alpha-subunit nucleotide-binding site is not required, and (b) that enzyme assembly requires nucleoside triphosphate.
运用两种实验技术研究了核苷酸与大肠杆菌F1 - ATP酶分离出的α亚基和β亚基的化学计量关系：（a）用AMP、ADP和ATP的荧光三硝基苯基（TNP）衍生物进行滴定；（b）采用Khananshvili和Gromet - Elhanan的特定条件（Khananshvili, D., and Gromet - Elhanan, Z. (1985) FEBS Lett. 178, 10 - 14）的离心柱法。两种方法均表明α亚基含有一个核苷酸结合位点，这证实了先前的研究工作。TNP - ADP和TNP - ATP与β亚基的最大结合量为1摩尔/摩尔，这与先前的平衡透析研究一致，该研究表明分离出的β亚基每摩尔结合1摩尔ADP或ATP（Issartel, J. P., and Vignais, P. V. (1984) Biochemistry 23, 6591 - 6595）。然而，使用离心柱检测到每摩尔β亚基仅结合约0.1摩尔的ATP或ADP。我们的结果与α亚基和β亚基各含有一个核苷酸结合结构域的结论一致。由于亚基化学计量比为α3β3γδε，这可以解释大肠杆菌F1 - ATP酶中六个已知核苷酸结合位点的位置。对分离出的α、β和γ亚基体外组装成活性ATP酶的研究表明，ATP、GTP和ITP均支持组装，ATP酶半数最大重组时的浓度为100 - 200微摩尔，而ADP、GDP和IDP则不能。此外，TNP - ATP支持组装，TNP - ADP则不能。结果表明：（a）β亚基上的核苷酸结合位点必须被占据，酶组装才能进行，而α亚基核苷酸结合位点的占据并非必需；（b）酶组装需要核苷三磷酸。
