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首页> 外文期刊>Biochemistry >CA2+-BINDING STOICHIOMETRY OF CALBINDIN D-28K AS ASSESSED BY SPECTROSCOPIC ANALYSES OF SYNTHETIC PEPTIDE FRAGMENTS
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CA2+-BINDING STOICHIOMETRY OF CALBINDIN D-28K AS ASSESSED BY SPECTROSCOPIC ANALYSES OF SYNTHETIC PEPTIDE FRAGMENTS

机译：合成肽片段的光谱分析评估CALBINDIN D-28K的CA2 +结合化学计量

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摘要

Calbindin D-28k is an intracellular Ca2+-binding protein noted for its abundance and specific distribution in mammalian brain and sensory neurons. This protein contains six putative Ca2+-binding sites, referred to as EF-hands. Due to the presence of the large number of putative sites, previous studies have been unsuccessful in definitively establishing the stoichiometry of Ca2+ binding. We describe a synthetic approach to identify the number of Ca2+-binding sites in which 6 33-residue peptides, designated EF1-EF6, corresponding to the 6 EF-hand sequences of calbindin D-28k, were made. The response of each peptide to Ca2+ addition was assessed by H-1 NMR spectroscopy, circular dichroism (CD) spectroscopy, and agarose gel electrophoresis. The Ca2+ binding by CD experiments was performed at two peptide concentrations, 20 and 200 mu M, and the NMR studies at peptide concentrations ranging from 20 to 100 mu M. The CD and H-1 NMR data show that five of the six peptides bind Ca2+ as isolated peptides, namely, EF1, EF3, EF4, EF5, and EF6. The EF6 peptide appears to bind Ca2+ with lower affinity than the other four functional sites. In contrast, EF2 does not appear to bind Ca2+ under any of the spectroscopic conditions tested. The data suggest that at least five of the six putative sites in the native protein bind Ca2+, although their relative affinities cannot be deduced from studies of the isolated peptides.
机译：钙结合蛋白D-28k是一种细胞内Ca2 +结合蛋白，以其在哺乳动物脑和感觉神经元中的丰度和特异性分布而著称。该蛋白质包含六个推定的Ca2 +结合位点，称为EF手。由于存在大量推定位点，因此先前的研究未能成功地确定Ca2 +结合的化学计量。我们描述了一种合成的方法来鉴定Ca2 +结合位点的数量，在这些位点中有6个33个残基的肽，称为EF1-EF6，与calbindin D-28k的6个EF手序列相对应。通过H-1 NMR光谱，圆二色性（CD）光谱和琼脂糖凝胶电泳评估每种肽对Ca2 +添加的响应。通过CD实验进行的Ca2 +结合是在20和200μM的两个肽浓度下进行的，而NMR研究则是在20至100μM的肽浓度下进行的。CD和H-1 NMR数据显示，六个肽中有五个结合Ca2 +作为分离的肽，即EF1，EF3，EF4，EF5和EF6。 EF6肽似乎以比其他四个功能位点低的亲和力结合Ca2 +。相反，在任何测试的光谱条件下，EF2似乎都不会结合Ca2 +。数据表明，尽管不能从分离肽的研究中推论出其相对亲和力，但天然蛋白质中六个推定位点中至少有五个结合了Ca2 +。

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