二价阳离子与钙调节蛋白结合诱导的构象变化。

Conformational changes induced by binding of divalent cations to calregulin.

作者信息

Khanna N C, Tokuda M, Waisman D M

出版信息

J Biol Chem. 1986 Jul 5;261(19):8883-7.

Abstract

Scatchard analysis of equilibrium dialysis studies have revealed that in the presence of 3.0 mM MgCl2 and 150 mM KCl, calregulin has a single binding site for Ca2+ with an apparent dissociation constant (apparent Kd) of 0.05 microM and 14 binding sites for Zn2+ with apparent Kd(Zn2+) of 310 microM. Ca2+ binding to calregulin induces a 5% increase in the intensity of intrinsic fluorescence and a 2-3-nm blue shift in emission maximum. Zn2+ binding to calregulin causes a dose-dependent increase of about 250% in its intrinsic fluorescence intensity and a red shift in the emission maximum of about 11 nm. Half-maximal wavelength shift occurs at 0.4 mol of Zn2+/mol of calregulin, and 100% of the wavelength shift is complete at 2 mol of Zn2+/mol of calregulin. In the presence of Zn2+ and calregulin the fluorescence intensity of the hydrophobic fluorescent probe 8-anilino-1-napthalenesulfonate (ANS) was enhanced 300-400% with a shift in emission maximum from 500 to 480 nm. Half-maximal Zn2+-induced shift in ANS emission maximum occurred at 1.2 mol of Zn2+/mol of calregulin, and 100% of this shift occurred at 6 mol of Zn2+/mol of calregulin. Of 12 cations tested, only Zn2+ and Ca2+ produced changes in calregulin intrinsic fluorescence, and none of these metal ions could inhibit the Zn2+-induced red shift in intrinsic fluorescence emission maximum. Furthermore, none of these cations could inhibit or mimic the Zn2+-induced blue shift in ANS emission maximum. These results suggest that calregulin contains distinct and specific ligand-binding sites for Ca2+ and Zn2+. While Ca2+ binding results in the movement of tryptophan away from the solvent, Zn2+ causes a movement of tryptophan into the solvent and the exposure of a domain with considerable hydrophobic character.

摘要

对平衡透析研究的斯卡查德分析表明，在存在3.0 mM MgCl₂ 和150 mM KCl的情况下，钙调节蛋白对Ca²⁺ 有一个结合位点，其表观解离常数（表观Kd）为0.05 μM，对Zn²⁺ 有14个结合位点，表观Kd(Zn²⁺)为310 μM。Ca²⁺ 与钙调节蛋白结合会导致固有荧光强度增加5%，发射最大值发生2 - 3纳米的蓝移。Zn²⁺ 与钙调节蛋白结合会使其固有荧光强度呈剂量依赖性增加约250%，发射最大值发生约11纳米的红移。最大波长位移的一半发生在0.4摩尔Zn²⁺/摩尔钙调节蛋白时，在2摩尔Zn²⁺/摩尔钙调节蛋白时波长位移达到100%。在存在Zn²⁺ 和钙调节蛋白的情况下，疏水荧光探针8 - 苯胺基 - 1 - 萘磺酸盐（ANS）的荧光强度增强了300 - 400%，发射最大值从500纳米移至480纳米。最大波长位移的一半在1.2摩尔Zn²⁺/摩尔钙调节蛋白时发生，在6摩尔Zn²⁺/摩尔钙调节蛋白时达到100%的位移。在测试的12种阳离子中，只有Zn²⁺ 和Ca²⁺ 会引起钙调节蛋白固有荧光的变化，并且这些金属离子均不能抑制Zn²⁺ 诱导的固有荧光发射最大值的红移。此外，这些阳离子均不能抑制或模拟Zn²⁺ 诱导的ANS发射最大值的蓝移。这些结果表明钙调节蛋白含有针对Ca²⁺ 和Zn²⁺ 的独特且特异性的配体结合位点。虽然Ca²⁺ 结合导致色氨酸远离溶剂移动，但Zn²⁺ 会使色氨酸向溶剂移动，并暴露出具有相当疏水特性的结构域。