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 45Ca2+. Ca2+ uptake in the presence of ATP and bafilomycin A1, namely Ca2+-ATPase, showed a relatively low Vmax (6 nmol.min-1.mg-1 protein) and a low Km for Ca2+. The Ca2+/H+ antiporter activity driven by H+-pyrophosphatase showed a high Vmax (25 nmol.min-1.mg-1) and a relatively high Km for Ca2+. The cDNA for mung bean Ca2+/H+ antiporter (VCAX1) codes for a 444 amino-acid 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.
Ueoka-Nakanishi
H
H
Laboratory of Biochemistry, Graduate School of Bioagricultural Sciences, Nagoya University, Japan.
Nakanishi
Y
Y
Tanaka
Y
Y
Maeshima
M
M
D000595:Amino Acid Sequence; D017920:Antiporters / Q000737:chemistry* / Q000235:genetics; D001483:Base Sequence; D002135:Calcium-Binding Proteins / Q000737:chemistry* / Q000235:genetics; D000252:Calcium-Transporting ATPases / Q000378:metabolism; D027682:Cation Transport Proteins; D003001:Cloning, Molecular; D018076:DNA, Complementary; D007887:Fabaceae / Q000378:metabolism*; D007700:Kinetics; D008969:Molecular Sequence Data; D010946:Plants, Medicinal; D017386:Sequence Homology, Amino Acid
D017920:Antiporters; D002135:Calcium-Binding Proteins; D027682:Cation Transport Proteins; D018076:DNA, Complementary; C027614:calcium-hydrogen antiporters; D000252:Calcium-Transporting ATPases