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. 1991 Jun;96(2):565–570. doi: 10.1104/pp.96.2.565

Solubilization and Reconstitution of Ca2+ Pump from Corn Leaf Plasma Membrane 1

Minobu Kasai 1,2, Shoshi Muto 1
PMCID: PMC1080807  PMID: 16668222

Abstract

The Ca2+ transport system of corn (Zea mays) leaf plasma membrane is composed of Ca2+ pump and Ca2+/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 Ca2+ transporters to establish the procedure for their solubilization, isolation, and reconstitution into liposomes. We attempted to solubilize and reconstitute the Ca2+ pump in the present study. A nonionic detergent octaethyleneglycol monododecyl ether (C12E8) was the most effective detergent for a series of extraction and functional reconstitution of the Ca2+ pump among seven detergents examined. This was judged from activities of ATP-dependent 45Ca2+ uptake into liposomes reconstituted with the respective detergent-extract of the plasma membrane by the detergent dilution method. C12E8-extract of the plasma membrane was subjected to high performance liquid chromatography using a DEAE anion exchange column. Ca2+-ATPase was separated from VO43−-sensitive Mg2+-ATPase. These ATPases were separately reconstituted into liposomes, and their ATP-dependent Ca2+ uptake was measured. The liposomes reconstituted with the Ca2+-ATPase, but not with the VO43−-sensitive Mg2+-ATPase, showed ATP-dependent Ca2+ uptake. Nigericin-induced pH gradient (acid inside) caused only a little Ca2+ uptake into liposomes reconstituted with the Ca2+-ATPase, suggesting that the Ca2+/H+ antiporter was not present in the preparation. These results indicate that the Ca2+-ATPase actually functions as Ca2+ pump in the corn leaf plasma membrane.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

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