FUNCTIONAL RECONSTITUTION OF AN ATP-DRIVEN CA-2+-TRANSPORT SYSTEM FROM THE PLASMA-MEMBRANE OF COMMELINA-COMMUNIS L

The protein(s) that constitute(s) the ATP-driven Ca2+-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 Ca2+-transport system was studied using ATP-driven 45Ca2+ 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 Km for Ca2+ (5.2 micromolar), inhibition by relatively high levels of vanadate (IC50 = 500 micromolar) and lacking response to added calmodulin. The reconstituted transport system was very strongly inhibited by erythrosine B (IC50 = 0.01 micromolar) and had a low apparent Km for ATP (11.4 micromolar). As in the plasma membrane vesicles, the protonophore carbonylcyanide m-chlorophenyl hydrazone did not affect Ca2+-transport detectably in the reconstituted system. However, low levels of the Ca2+-ionophore A 23187 instantaneously discharged 90% of the Ca2+ associated with the vesicles, proving that it had been accumulated in the intravesicular volume in soluble, freely exchangeable form. Ca2+-transport in the reconstituted system was thus primary active, through a Ca2+-translocating ATPase. The system reported here may serve as a valuable tool for purifying the Ca2+-ATPase and for studying structural and functional aspects of the purified enzyme.