TYROSINE FLUORESCENCE OF RAM TESTIS AND OCTOPUS CALMODULINS - EFFECTS OF CALCIUM, MAGNESIUM, AND IONIC-STRENGTH
TYROSINE FLUORESCENCE OF RAM TESTIS AND OCTOPUS CALMODULINS - EFFECTS OF CALCIUM, MAGNESIUM, AND IONIC-STRENGTH
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DOI：
10.1021/bi00518a027
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发表时间：
1981-01-01
期刊：
影响因子：
2.9
通讯作者：
GERARD, D
中科院分区：
文献类型：
--
作者：
KILHOFFER, MC;DEMAILLE, JG;GERARD, D
采用内源荧光研究了Ca2+和Mg2+对钙调蛋白构象的影响。对两种钙调蛋白的酪氨酸发射特性进行了完整的研究，即公羊睾丸钙调蛋白（在第 99 和 138 位含有 2 个酪氨酸残基）和章鱼钙调蛋白（仅含有 Tyr-138）。荧光量子产率和衰减时间值的分析提供了有关无 Ca2+ 钙调蛋白中发生的猝灭机制的信息。特别是，Tyr-138 的特点是由于剧烈的静态猝灭过程而具有非常低的发射率（Φ = 0.016）。单价阳离子（150 mM Na+ 或 K+）会引起荧光的额外猝灭。相比之下，2 Ca2+/mol 钙调蛋白在结构域 I 和 II 的高亲和力位点的结合导致两个 Tyr 残基的荧光大量去猝灭。 Ca2+ 与较低亲和力位点结合后，量子产率不会进一步增加。同样，酪氨酸在 280 nm 处的摩尔椭圆率在结合 2 Ca2+/mol 后完成变化，证实了 Ca2+ 诱导的构象转变中存在 2 个步骤。在Ca 2+ 负载的蛋白质中，Tyr-99 (.PHI.=0.11)相当高的发射率可能是由α-螺旋结构附近造成的。 Mg2+ 诱导的荧光变化没有 Ca2+ 观察到的那么大。此外，150 mM KCl 或 NaCl 消除了 Mg2+ 的影响。这可能表明未刺激细胞的胞质中钙调蛋白位点未被 Mg2+ 饱和。其他指示，特别是两个 Tyr 残基暴露于溶剂的程度，也从离子猝灭剂 (Cs+) 存在下进行的实验和荧光偏振中获得。酪氨酸 99 大部分暴露，而酪氨酸 138 在不存在 Ca2+ 的情况下比存在 Ca2+ 时更多地暴露于溶剂。内在荧光和圆二色性的研究证实了负载 Ca2+ 的钙调蛋白比不含 Ca2+ 的钙调蛋白具有更高的稳定性。
The effects of Ca2+ and Mg2+ on the conformation of calmodulin were studied by intrinsic fluorescence. A complete investigation of the tryrosine emission characteristics was carried out on 2 calmodulins, ram testis calmodulin, which contains 2 tyrosine residues at positions 99 and 138, and octopus calmodulin, which contains only Tyr-138. Analysis of fluorescence quantum yield and decay time values provided information on the quenching mechanism occurring in Ca2+-free calmodulins. In particular, Tyr-138 is characterized by a very low emission yield (.PHI. = 0.016) due to a drastic static quenching process. Monovalent cations (150 mM Na+ or K+) induce an additional quenching of the fluorescence. By contrast, binding of 2 Ca2+/mol of calmodulin in the high-affinity sites of domains I and II results in a large dequenching of the fluorescence of both Tyr residues. No further increase in the quantum yield occurs upon binding of Ca2+ to the lower affinity sites. Similarly, the variation of the molar ellipticity of tyrosines at 280 nm is complete after binding of 2 Ca2+/mol, confirming the existence of 2 steps in the Ca2+-induced conformational transition. In the Ca2+-loaded protein, the rather high emission yield of Tyr-99 (.PHI. = 0.11) may perhaps be accounted for by the vicinity of .alpha.-helical structures. The Mg2+-induced fluorescence change is not as large as that observed with Ca2+. Moreover, 150 mM KCl or NaCl abolishes Mg2+ effects. This may suggest that calmodulin sites are not saturated by Mg2+ in the cytosol of unstimulated cells. Other indications, in particular on the degree of exposure to solvent of both Tyr residues, were also obtained from experiments carried out in the presence of ionic quenchers (Cs+) and from fluorescence polarization. Tyrosine-99 was largely exposed, while tyrosine-138 is more exposed to solvent in the absence than in the presence of Ca2+. The greater stability of Ca2+-loaded vs. Ca2+-free calmodulin was confirmed by the study of both intrinsic fluorescence and circular dichroism.