Equilibrium Dialysis Measurements of the Ca2+-Binding Properties of Recombinant Radish Vacuolar Ca2+-Binding Protein Expressed inEscherichia coli | Zendy

Koji Yuasa | Zendy; Masayoshi Maeshima | Zendy

Open Access

Equilibrium Dialysis Measurements of the Ca²⁺-Binding Properties of Recombinant Radish Vacuolar Ca²⁺-Binding Protein Expressed inEscherichia coli

Author(s) -

Koji Yuasa,

Masayoshi Maeshima

Publication year - 2002

Publication title -

bioscience biotechnology and biochemistry

Language(s) - English

Resource type - Journals

SCImago Journal Rank - 0.509

H-Index - 116

eISSN - 1347-6947

pISSN - 0916-8451

DOI - 10.1271/bbb.66.2382

Subject(s) - size exclusion chromatography , raphanus , escherichia coli , recombinant dna , chemistry , affinity chromatography , ion chromatography , binding protein , chromatography , molecular mass , nitrocellulose , biochemistry , ultrafiltration (renal) , ion exchange , membrane , ion , biology , enzyme , botany , organic chemistry , gene

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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