Solubilization and Reconstitution of the Oat Root Vacuolar H+/Ca2+ Exchanger

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Abstract
Calcium is sequestered into vacuoles of oat (Avena sativa L.) root cells via a H+/Ca2+ antiporter, and vesicles derived from the vacuolar membrane (tonoplast) catalyze an uptake of calcium which is dependent on protons (pH gradient [.DELTA.pH] dependent). The first step toward purification and identification of the H+/Ca2+ antiporter is to solubilize and reconstitute the transport activity in liposomes. The vacuolar H+/Ca2+ antiporter was solubilized with octylglucoside in the presence of soybean phospholipids and glycerol. After centrifugation, the soluble proteins were reconstituted into liposomes by detergent dilution. A .DELTA.pH (acid inside) was generated in the proteoliposomes with an NH4Cl gradient (NH4+in .gtoreq. NH4+out) as determined by methylamine uptake. Fundamental properties of .DELTA.pH dependent calcium uptake such as the Km of calcium (.apprx. 15 micromolar) and the sensitivity to inhibitors such as N,N''-dicyclohexylcarbodiimide, ruthenium red, and lanthanum, were similar to those found in membrane vesicles, indicating that the H+/Ca2+ antiporter has been reconstituted in active form.