Functional Reconstitution of an ATP-Driven Ca-Transport System from the Plasma Membrane of Commelina communis L.
The protein(s) that constitute(s) the ATP-driven Ca(2+)-translocator of plasma membrane enriched vesicles obtained by aqueous two-phase partitioning from leaves of Commelina communis L. has/have been solubilized and reincorporated into tightly sealed liposomes. The reconstituted Ca(2+)-transport system was studied using ATP-driven (45)Ca(2+) import into the proteoliposomes as a measure of activity. The detergent, 3-[(3-cholamidopropyl) dimethylammonio]-1-propane-sulfonate proved to be the most suitable and was used at 10 millimolar concentration, i.e. just above its critical micellar concentration. The presence of additional phospholipid (2 milligrams phosphatidylcholine per milliliter) and ATP (5 millimolar) improved the solubilization and/or reconstitution. The characteristics of the reconstituted system were similar to those of the plasma membrane-bound activity, including the apparent K(m) for Ca(2+) (5.2 micromolar), inhibition by relatively high levels of vanadate (IC(50) = 500 micromolar) and lacking response to added calmodulin. The reconstituted transport system was very strongly inhibited by erythrosine B (IC(50) = 0.01 micromolar) and had a low apparent K(m) for ATP (11.4 micromolar). As in the plasma membrane vesicles, the protonophore carbonylcyanide m-chlorophenyl hydrazone did not affect Ca(2+)-transport detectably in the reconstituted system. However, low levels of the Ca(2+)-ionophore A 23187 instantaneously discharged 90% of the Ca(2+) associated with the vesicles, proving that it had been accumulated in the intravesicular volume in soluble, freely exchangeable form. Ca(2+)-transport in the reconstituted system was thus primary active, through a Ca(2+)-translocating ATPase. The system reported here may serve as a valuable tool for purifying the Ca(2+)-ATPase and for studying structural and functional aspects of the purified enzyme.
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Lehrstuhl für Pflanzenphysiologie, Ruhr-Universität Bochum, Postfach 102148, D-4630 Bochum, Federal Republic of Germany.
Weiler
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