Veenstra T D, Johnson K L, Tomlinson A J, Naylor S, Kumar R
Department of Medicine, Mayo Clinic Foundation, Rochester, Minnesota 55905, USA.
Biochemistry. 1997 Mar 25;36(12):3535-42. doi: 10.1021/bi9628329.
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 D28K and two mutant forms of the protein, one lacking EF-hand 2 (delta2) and the other lacking EF-hands 2 and 6 (delta2,6). 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 D28K bound 4 mol of terbium/mol of protein, while calbindin delta2 and delta2,6 each bound 3 mol. These results clearly show that calbindin D28K binds 4 mol of calcium/mol of protein and that terbium-binding stoichiometry is similar to that of calcium.
钙结合蛋白D28K是肌钙蛋白-C超家族中钙结合蛋白的一员，含有六个假定的EF手结构域。不同研究小组对该蛋白进行的钙结合研究在钙结合化学计量方面得出了不一致的结果。有人提出该蛋白每摩尔蛋白结合3至6摩尔钙。我们使用负离子电喷雾电离质谱法来明确确定钙结合蛋白D28K及其两种突变形式（一种缺乏EF手2（δ2），另一种缺乏EF手2和6（δ2,6））的确切钙结合化学计量。全长蛋白每摩尔蛋白结合4摩尔钙，而两种缺失突变体均结合3摩尔钙。由于铽已被广泛用作测定钙结合蛋白钙结合化学计量的探针，我们还在相同条件下研究了铽与这三种蛋白的结合。全长钙结合蛋白D28K每摩尔蛋白结合4摩尔铽，而钙结合蛋白δ2和δ2,6均结合3摩尔铽。这些结果清楚地表明，钙结合蛋白D28K每摩尔蛋白结合4摩尔钙，且铽结合化学计量与钙相似。
