Electrogenic pump current of sarcoplasmic reticulum Ca2+‐ATPase reconstituted at high lipid/protein ratio

Abstract

When Ca²⁺-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 or 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 hydrolysed at pH 7.1 can be obtained. The method is also convenient for determination of the kinetics of Ca²⁺-ATPase activation by ATP and free Ca²⁺.