H(+)/Ca(2+) exchange driven by the plasma membrane Ca(2+)-ATPase of Arabidopsis thaliana reconstituted in proteoliposomes after calmodulin-affinity purification.
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 45Ca(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.
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Dipartimento di Biologia 'L. Gorini', Università di Milano, Centro di Studio del CNR per la Biologia Cellulare e Molecolare delle Piante, via G. Celoria 26, 20133, Milan, Italy.
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De Michelis
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D017920:Antiporters / Q000378:metabolism*; D017360:Arabidopsis / Q000201:enzymology* / Q000378:metabolism; D001692:Biological Transport; D002118:Calcium / Q000378:metabolism; D002135:Calcium-Binding Proteins / Q000378:metabolism*; D000252:Calcium-Transporting ATPases / Q000302:isolation & purification / Q000378:metabolism*; D002147:Calmodulin / Q000737:chemistry; D027682:Cation Transport Proteins; D002462:Cell Membrane / Q000201:enzymology / Q000378:metabolism; D002846:Chromatography, Affinity; D011510:Proteolipids / Q000378:metabolism*; D017494:Proton Pumps / Q000378:metabolism
D017920:Antiporters; D002135:Calcium-Binding Proteins; D002147:Calmodulin; D027682:Cation Transport Proteins; D011510:Proteolipids; D017494:Proton Pumps; C027614:calcium-hydrogen antiporters; C020485:proteoliposomes; D000252:Calcium-Transporting ATPases; D002118:Calcium