Determination of calcium-binding sites in rat brain calbindin D28K by electrospray ionization mass spectrometry.
Determination of calcium-binding sites in rat brain calbindin D28K by electrospray ionization mass spectrometry.
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通过电喷雾电离质谱法测定大鼠脑钙结合蛋白 D28K 中的钙结合位点。
DOI：
10.1021/bi9628329
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发表时间：
1997
期刊：
影响因子：
--
通讯作者：
Kumar,R
中科院分区：
文献类型：
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作者：
Veenstra,TD;Johnson,KL;Tomlinson,AJ;Naylor,S;Kumar,R
钙结合蛋白D28K是钙结合蛋白的肌钙蛋白- c超家族的一员，含有六个假定的EF-hand结构域。不同研究小组对钙结合蛋白的研究在钙结合的化学计量学方面产生了不一致的结果。据推测，该蛋白可以与3 ~ 6 mol钙/mol蛋白质结合。我们使用负离子电喷雾质谱法来确定钙结合蛋白d28k和该蛋白的两种突变形式的确切钙结合化学计量，一种缺乏EF-hand 2 （Δ2），另一种缺乏EF-hand 2和6 （Δ2,6）。全长蛋白结合4 mol钙/mol蛋白，而两个缺失突变体结合3 mol钙/mol蛋白。由于铽已被广泛用作测定钙结合蛋白的钙结合化学计量学的探针，我们也研究了在相同条件下铽与这三种蛋白的结合。全长calbindin d28k4 mol /mol的铽结合蛋白，而calbindin Δ2和Δ2,6各结合3 mol。这些结果清楚地表明，钙结合蛋白d28k4 mol钙/mol蛋白质的结合，其铽的结合化学计量与钙的相似。
Calbindin D28K, a member of the troponin-C superfamily of calcium-binding proteins, contains six putative EF-hand domains. Calcium-binding studies of the protein by different groups of investigators have yielded discordant results with respect to the stoichiometry of calcium-binding. It has been suggested that the protein binds anywhere from 3−6 mol of calcium/mol of protein. We used negative ion electrospray ionization mass spectrometry in order to definitively determine the exact calcium-binding stoichiometry of calbindin D28Kand two mutant forms of the protein, one lacking EF-hand 2 (Δ2) and the other lacking EF-hands 2 and 6 (Δ2,6). The full-length protein bound 4 mol of calcium/mol of protein, while both of the deletion mutants bound 3 mol of calcium. Since terbium has been used extensively as a probe for the determination of the calcium-binding stoichiometries of calcium-binding proteins, we also examined the binding of terbium to the three proteins under the same conditions. Full-length calbindin D28Kbound 4 mol of terbium/mol of protein, while calbindin Δ2 and Δ2,6 each bound 3 mol. These results clearly show that calbindin D28Kbinds 4 mol of calcium/mol of protein and that terbium-binding stoichiometry is similar to that of calcium.