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Electrogenic pump current of sarcoplasmic reticulum Ca 2+ ‐ATPase reconstituted at high lipid/protein ratio
Author(s) -
Cornelius Flemming,
Møller Jesper V.
Publication year - 1991
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/0014-5793(91)80758-u
Subject(s) - chemistry , endoplasmic reticulum , atp hydrolysis , atpase , biophysics , phospholipid , membrane , biochemistry , analytical chemistry (journal) , chromatography , enzyme , biology
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 Ca 2+ and ATP a transmembrane potential developed which could be continuously recorded by the fluorochrome oxonol VI. The results demonstrate the electrogenicity or active Ca 2+ 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 Ca 2+ transport. From such measurements a stoichiometry of 1.8±0.4 Ca 2+ per ATP hydrolysed 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 Ca 2+ .