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H + /Ca 2+ exchange driven by the plasma membrane Ca 2+ ‐ATPase of Arabidopsis thaliana reconstituted in proteoliposomes after calmodulin‐affinity purification
Author(s) -
Luoni Laura,
Bonza Maria Cristina,
De Michelis Maria Ida
Publication year - 2000
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/s0014-5793(00)02065-2
Subject(s) - protonophore , chemistry , calmodulin , atpase , arabidopsis thaliana , pyranine , membrane , chaotropic agent , biochemistry , biophysics , p type atpase , diaphragm pump , enzyme , biology , mutant , gene , materials science , micropump , nanotechnology
The plasma membrane Ca 2+ ‐ATPase was purified from Arabidopsis thaliana cultured cells by calmodulin (CaM)‐affinity chromatography and reconstituted in proteoliposomes by the freeze‐thaw sonication procedure. The reconstituted enzyme catalyzed CaM‐stimulated 45 Ca 2+ accumulation and H + ejection, monitored by the increase of fluorescence of the pH probe pyranine entrapped in the liposomal lumen during reconstitution. Proton ejection was immediately reversed by the protonophore FCCP, indicating that it is not electrically coupled to Ca 2+ uptake, but it is a primary event linked to Ca 2+ uptake in the form of countertransport.