Equilibrium dialysis measurements of the Ca2+-binding properties of recombinant radish vacuolar Ca2+-binding protein expressed in Escherichia coli |
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Authors: | Yuasa Koji Maeshima Masayoshi |
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Affiliation: | Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya 464-8601, Japan. |
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Abstract: | Vacuoles of radish (Raphanus sativus) contained a Ca2+-binding protein (RVCaB) of 43 kDa. We investigated the Ca2+-binding properties of the protein. RVCaB was expressed in Escherichia coli and was purified from an extract by ion-exchange chromatography, nitrocellulose membrane filtration, and gel-filtration column chromatography. Ca2+-binding properties of the recombinant protein were examined by equilibrium dialysis with 45Ca2+ and small dialysis buttons. The protein was estimated to bind 19Ca2+ ions per molecule with a Kd for Ca2+ of 3.4 mM. Ca2+ was bound to the protein even in the presence of high concentrations of Mg2+ or K+. The results suggested that the protein bound Ca2+ with high ion selectivity, high capacity, and low affinity. |
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