H+/Ca2+ exchange driven by the plasma membrane Ca2+-ATPase of Arabidopsis thaliana reconstituted in proteoliposomes after calmodulin-affinity purification

The plasma membrane Ca2+-ATPase was purified from Arabidopsis thaliana cult ured cells by calmodulin (CaM)-affinity chromatography and reconstituted in proteoliposomes by the freeze-thaw sonication procedure. The reconstituted enzyme catalyzed CaM-stimulated Ca-45(2+) accumulation and H+ ejection, mo nitored by the increase of fluorescence of the pH probe pyranine entrapped in the liposomal lumen during reconstitution. Proton ejection was immediate ly reversed by the protonophore FCCP, indicating that it is not electricall y coupled to Ca2+ uptake, but it is a primary event linked to Ca2+ uptake i n the form of countertransport. (C) 2000 Federation of European Biochemical Societies. Published by Elsevier Science B.V. All rights reserved.