Solubilization and reconstitution of ca pump from corn leaf plasma membrane

The Ca(2+) transport system of corn (Zea mays) leaf plasma membrane is composed of Ca(2+) pump and Ca(2+)/H(+) antiporter driven by H(+) gradient imposed by a H(+) pump (M Kasai, S Muto [1990] J Membr Biol 114: 133-142). It is necessary for characterization of these Ca(2+) transporters to establish the procedure for their solubilization, isolation, and reconstitution into liposomes. We attempted to solubilize and reconstitute the Ca(2+) pump in the present study. A nonionic detergent octaethyleneglycol monododecyl ether (C(12)E(8)) was the most effective detergent for a series of extraction and functional reconstitution of the Ca(2+) pump among seven detergents examined. This was judged from activities of ATP-dependent (45)Ca(2+) uptake into liposomes reconstituted with the respective detergent-extract of the plasma membrane by the detergent dilution method. C(12)E(8)-extract of the plasma membrane was subjected to high performance liquid chromatography using a DEAE anion exchange column. Ca(2+)-ATPase was separated from VO(4) (3-)-sensitive Mg(2+)-ATPase. These ATPases were separately reconstituted into liposomes, and their ATP-dependent Ca(2+) uptake was measured. The liposomes reconstituted with the Ca(2+)-ATPase, but not with the VO(4) (3-)-sensitive Mg(2+)-ATPase, showed ATP-dependent Ca(2+) uptake. Nigericin-induced pH gradient (acid inside) caused only a little Ca(2+) uptake into liposomes reconstituted with the Ca(2+)-ATPase, suggesting that the Ca(2+)/H(+) antiporter was not present in the preparation. These results indicate that the Ca(2+)-ATPase actually functions as Ca(2+) pump in the corn leaf plasma membrane.