Leung I K, Mani R S, Kay C M
Biochem J. 1986 Aug 1;237(3):757-64. doi: 10.1042/bj2370757.
The brain-specific S-100 protein is a mixture of two predominant components, S-100a and S-100b, with subunit compositions of alpha beta and beta beta respectively. In the present study, the alpha-subunit, isolated from S-100a by using anion-exchange chromatography in the presence of 8 M-urea, was homogeneous by the criteria of SDS/polyacrylamide-gel, urea/SDS/polyacrylamide-gel and non-SDS/polyacrylamide-gel electrophoresis. The alpha-subunit underwent a conformational change upon binding Ca2+ and Zn2+ at pH 7.5, as revealed by u.v. difference spectroscopy, c.d. and fluorescence measurements. Far-u.v. c.d. studies indicated the apparent alpha-helical content to fall when the protein bound either Ca2+ or Zn2+. Addition of Ca2+ to the alpha-subunit resulted in exposing to the solvent the single tryptophan residue and one or more tyrosine and phenylalanine residues. Zn2+ induced only a small conformational change, and among the aromatic chromophores only tyrosine residues were affected to a small extent. Ca2+ was able to bind to the alpha-subunit in the presence of Zn2+, and the two metal-ion-binding sites appeared to be different. When the apoprotein was excited at 280 nm, the fluorescence emission maximum was located at 337 nm. In the presence of Ca2+, the emission maximum occurred at 340 nm and was accompanied by a nearly 25% increase in fluorescence intensity. Fluorescence titration with Ca2+ at pH 7.5 revealed only one class of binding site, with a Kd value of 1.26 X 10(-4) M. The effect of K+ on the protein was slightly antagonistic to that of Ca2+, as indicated by u.v. difference spectroscopy and fluorescence titration.
脑特异性S-100蛋白是两种主要成分S-100a和S-100b的混合物，其亚基组成分别为αβ和ββ。在本研究中，通过在8M尿素存在下使用阴离子交换色谱法从S-100a中分离出的α亚基，根据SDS/聚丙烯酰胺凝胶、尿素/SDS/聚丙烯酰胺凝胶和非SDS/聚丙烯酰胺凝胶电泳标准，其为均一的。紫外差光谱、圆二色光谱和荧光测量结果表明，α亚基在pH 7.5时与Ca2+和Zn2+结合后发生了构象变化。远紫外圆二色光谱研究表明，当该蛋白与Ca2+或Zn2+结合时，其表观α螺旋含量下降。向α亚基中添加Ca2+会导致单个色氨酸残基以及一个或多个酪氨酸和苯丙氨酸残基暴露于溶剂中。Zn2+仅诱导了较小的构象变化，并且在芳香发色团中只有酪氨酸残基受到了较小程度的影响。在Zn2+存在的情况下，Ca2+能够与α亚基结合，并且两个金属离子结合位点似乎不同。当脱辅基蛋白在280nm处被激发时，荧光发射最大值位于337nm。在Ca2+存在的情况下，发射最大值出现在340nm处，并且荧光强度增加了近25%。在pH 7.5时用Ca2+进行荧光滴定仅显示出一类结合位点，Kd值为1.26×10^(-4)M。紫外差光谱和荧光滴定表明，K+对该蛋白的作用与Ca2+的作用略有拮抗。
