Equilibrium dialysis measurements of the Ca2+-binding properties of recombinant radish vacuolar Ca2+-binding protein expressed in Escherichia coli.
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.
Yuasa
Koji
K
Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya 464-8601, Japan.
Maeshima
Masayoshi
M
D000595:Amino Acid Sequence; D002118:Calcium / Q000737:chemistry / Q000378:metabolism*; D002135:Calcium-Binding Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D003101:Collodion / Q000737:chemistry; D003956:Dialysis / Q000295:instrumentation / Q000379:methods; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D011485:Protein Binding; D031224:Raphanus / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D020449:Repetitive Sequences, Amino Acid; D013379:Substrate Specificity; D014617:Vacuoles / Q000737:chemistry / Q000378:metabolism
D002135:Calcium-Binding Proteins; D011994:Recombinant Proteins; D003101:Collodion; D002118:Calcium