Properties and molecular cloning of Ca2+/H+ antiporter in the vacuolar membrane of mung bean

Kinetic and molecular properties of the Ca2+/H+ antiporter in the vacuolar membrane of mung bean hypocotyls were examined and compared with Ca2+-ATPase. Ca2+ transport activities of both transporters were assayed separately by the filtration method using vacuolar membrane vesicles and Ca-45(2+). Ca2+ uptake in the presence of ATP and bafilomycin A(1), namely Ca2+-ATPase, showed a relatively low V-max (6 nmol.min(-1).mg(-1) protein) and a low K-m for Ca2+. The Ca2+/H+ antiporter activity driven by H+-pyrophosphatase showed a high V-max (25 nmol.min(-1).mg(-1)) and a relatively high K-m for Ca2+. The cDNA for mung bean Ca2+/H+ antiporter (VCAX1) codes for a 444 aminoacid polypeptide. Two peptide-specific antibodies of the antiporter clearly reacted with a 42-kDa protein from vacuolar membranes and a cell lysate from a Escherichia coli transformant in which VCAX1 was expressed. These observations directly demonstrate that a low-affinity, high-capacity Ca2+/H+ antiporter and a high-affinity Ca2+-ATPase coexist in the vacuolar membrane. It is likely that the Ca2+/H+ antiporter removes excess Ca2+ in the cytosol to lower the Ca2+ concentration to micromolar levels after stimuli have increased the cytosolic Ca2+ level, the Ca2+-ATPase then acts to lower the cytosolic Ca2+ level further.