Cornelius F, Møller J V
Institute of Biophysics, University of Aarhus, Denmark.
FEBS Lett. 1991 Jun 17;284(1):46-50. doi: 10.1016/0014-5793(91)80758-u.
When Ca(2+)-ATPase from sarcoplasmic reticulum was reconstituted with excess phospholipid (at a 1:800 weight ratio) in a monomeric state and activated by Ca2+ and ATP a transmembrane potential developed which could be continuously recorded by the fluorochrome oxonol VI. The results demonstrate the electrogenicity of active Ca2+ transport during continuous turnover. The fluorescence signal can be quantified in terms of net current electrical flow through the vesicular membranes and compared to the ATP hydrolysis to give the number of electrostatic charges transferred during Ca2+ transport. From such measurements a stoichiometry of 1.8 +/- 0.4 Ca2+ per ATP hydrolyzed at pH 7.1 can be obtained. The method is also convenient for determination of the kinetics of Ca(2+)-ATPase activation by ATP and free Ca2+.
当肌浆网的Ca(2+)-ATP酶以单体状态与过量磷脂（重量比为1:800）重组，并由Ca2+和ATP激活时，会产生跨膜电位，该电位可用荧光染料氧杂萘酚VI连续记录。结果表明，在连续周转过程中，活性Ca2+转运具有电生性。荧光信号可以根据通过囊泡膜的净电流进行量化，并与ATP水解进行比较，以得出Ca2+转运过程中转移的静电荷数量。通过此类测量，在pH 7.1条件下，每水解1个ATP可得到1.8±0.4个Ca2+的化学计量关系。该方法也便于测定ATP和游离Ca2+对Ca(2+)-ATP酶的激活动力学。
