Dell'Angelica E C, Schleicher C H, Santomé J A
Instituto de Química y Fisicoquímica Biológicas, Facultad de Farmacia y Bioquímica, Universidad de Buenos Aires, Argentina.
J Biol Chem. 1994 Nov 18;269(46):28929-36.
In this paper we report the biochemical characterization of calgranulin C, a new member of the S100 protein family. The protein is highly abundant in the cytosol of pig granulocytes, with relatively small amounts in lymphocytes. A simple protocol for the rapid purification of calgranulin C is described. The purified protein migrates as a single entity on SDS-polyacrylamide gel electrophoresis while it has two isoforms focusing at pH 5.8 and 5.5. Gel filtration and cross-linking experiments indicate that calgranulin C is capable of dimerization. The complete amino acid sequence was determined by Edman degradation of peptides generated by trypsin and V8 protease digestion. Calgranulin C consists of 91 residues and has a calculated molecular mass of 10,614 daltons. This value is virtually identical to that obtained by electrospray mass spectrometry. Sequence analysis predicts two EF-hand calcium-binding motifs, the first having an extended loop that is distinctive of the S100 protein family. The metal-binding properties were studied by means of a direct 45Ca(2+)-binding assay and by tyrosine fluorescence titration. Calgranulin C binds not only calcium but also zinc ions. A single high affinity Zn(2+)-binding site per monomer was evidenced by fluorimetric titration. Zinc binding to calgranulin C induces a remarkable increase in the protein affinity for calcium; in the absence of zinc, the protein binds 1 Ca2+/monomer with a binding constant of about 2 x 10(4) M-1, whereas the Zn(2+)-loaded form binds 2 Ca2+/monomer with Ka values of approximately 3 x 10(7) and 6 x 10(4) M-1. Circular dichroism analysis showed that the binding of calcium to calgranulin C induces a 15% decrease in the apparent alpha-helix content. This result and the calcium-dependent binding of the protein to a phenyl-Superose column strongly suggest that calgranulin C undergoes a gross conformational change upon calcium binding, thus supporting the idea that this protein may be involved in Ca(2+)-dependent signal transduction events.
在本文中，我们报告了钙粒蛋白C（S100蛋白家族的一个新成员）的生化特性。该蛋白在猪粒细胞的胞质溶胶中含量极高，在淋巴细胞中的含量相对较少。本文描述了一种快速纯化钙粒蛋白C的简单方法。纯化后的蛋白在SDS-聚丙烯酰胺凝胶电泳中以单一实体迁移，而在pH 5.8和5.5聚焦时有两种异构体。凝胶过滤和交联实验表明钙粒蛋白C能够二聚化。通过对胰蛋白酶和V8蛋白酶消化产生的肽进行埃德曼降解确定了完整的氨基酸序列。钙粒蛋白C由91个残基组成，计算分子量为10,614道尔顿。该值与通过电喷雾质谱法获得的值几乎相同。序列分析预测有两个EF-手型钙结合基序，第一个具有独特的延伸环，这是S100蛋白家族所特有的。通过直接的45Ca(2+)结合测定和酪氨酸荧光滴定研究了金属结合特性。钙粒蛋白C不仅结合钙，还结合锌离子。荧光滴定证明每个单体有一个高亲和力的Zn(2+)结合位点。锌与钙粒蛋白C的结合导致该蛋白对钙的亲和力显著增加；在没有锌的情况下，该蛋白以约2×10(4) M-1的结合常数结合1个Ca2+/单体，而锌负载形式以约3×10(7)和6×10(4) M-1的Ka值结合2个Ca2+/单体。圆二色性分析表明，钙与钙粒蛋白C的结合导致表观α-螺旋含量下降15%。这一结果以及该蛋白与苯基-超琼脂糖柱的钙依赖性结合强烈表明，钙粒蛋白C在结合钙后会发生总体构象变化，从而支持了该蛋白可能参与Ca(2+)依赖性信号转导事件的观点。
