Electrogenic pump current of sarcoplasmic reticulum Ca(2+)-ATPase reconstituted at high lipid/protein ratio

Abstract

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+.