Solubilization and reconstitution of the oat root vacuolar h/ca exchanger

Calcium is sequestered into vacuoles of oat (Avena sativa L.) root cells via a H(+)/Ca(2+) antiporter, and vesicles derived from the vacuolar membrane (tonoplast) catalyze an uptake of calcium which is dependent on protons (pH gradient [DeltapH] dependent). The first step toward purification and identification of the H(+)/Ca(2+) antiporter is to solubilize and reconstitute the transport activity in liposomes. The vacuolar H(+)/Ca(2+) 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 DeltapH (acid inside) was generated in the proteoliposomes with an NH(4)Cl gradient (NH(4) (+) (in) >> NH(4) (+) (out)) as determined by methylamine uptake. Fundamental properties of DeltapH dependent calcium uptake such as the K(m) for calcium ( approximately 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(+)/Ca(2+) antiporter has been reconstituted in active form.