A FLUORESCENT CALMODULIN THAT REPORTS THE BINDING OF HYDROPHOBIC INHIBITORY LIGANDS
A FLUORESCENT CALMODULIN THAT REPORTS THE BINDING OF HYDROPHOBIC INHIBITORY LIGANDS
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DOI：
10.1042/bj2110473
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发表时间：
1983-01-01
影响因子：
4.1
通讯作者：
WITTENAUER, LA
中科院分区：
文献类型：
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作者：
JOHNSON, JD;WITTENAUER, LA
在pCa 5.5-7.0范围内，Ca 2+与[牛睾丸]钙调蛋白结合，暴露了结合疏水抑制配体的疏水位点，包括钙调蛋白拮抗剂、一些Ca 2+拮抗剂和钙调蛋白结合蛋白。这些疏水性配体与钙调蛋白的结合之后可以是它们在丹磺酰化（5-二甲基氨基萘-1-磺酰化）钙调蛋白（CDRDANS）中产生的荧光增加80%。在Ca 2+存在下，钙调蛋白以约的亲和力结合钙调蛋白抑制剂R24571。2-3 nM和疏水性配体，包括三氟拉嗪（TFP）、W-7 [N-（6-氨基己基）-5-氯萘-1-磺酰胺]、芬地林、非洛地平和异戊烯胺，具有微摩尔范围内的亲和力。这种结合是强烈的Ca 2+依赖性和Mg 2+非依赖性的。钙调素在其与这些配体的结合中显示出相对于其他测试配体的相当高的特异性。因此，CDRDANS提供了一种方便和简单的方法来监测各种疏水配体与Ca 2+依赖性调节蛋白钙调蛋白的相互作用。CDRDANS与由（二肉豆蔻酰）磷脂酰胆碱或（二棕榈酰）磷脂酰胆碱制成的磷脂囊泡结合，并且仅在Ca 2+存在下和在高于其凝胶至液晶相变的温度下产生荧光增加。虽然CDRDANS中的荧光变化准确地报告了这些脂质体中的相变，但其与这些囊泡的结合较弱。钙调素可能需要一个高亲和力的脂质结合受体蛋白的高亲和力结合天然膜。
Ca2+ binding to [bovine testis] calmodulin in the pCa range 5.5-7.0 exposes hydrophobic sites that bind hydrophobic inhibitory ligands, including calmodulin antagonists, some Ca2+-antagonists and calmodulin-binding proteins. The binding of these hydrophobic ligands to calmodulin can be followed by the .apprx. 80% fluorescence increase they produce in dansylated (5-dimethylaminoaphthalene-1-sulfonylated) calmodulin (CDRDANS). In the presence of Ca2+, calmodulin binds the calmodulin inhibitor, R24571, with an affinity of .apprx. 2-3 nM and hydrophobic ligands, including trifluoperazine (TFP), W-7 [N-(6-aminohexyl)-5-chloronaphthalene-1-sulfonamide], fendiline, felodipine and prenylamine, with affinities in the micromolar range. This binding is strongly Ca2+-dependent and Mg2+-independent. Calmodulin shows a reasonably high degree of specificity in its binding of these ligands over other ligands tested. CDRDANS, therefore, provides a convenient and simple means of monitoring the interaction of a variety of hydrophobic ligands with the Ca2+-dependent regulatory protein, calmodulin. CDRDANS binds to phospholipid vesicles made of (dimyristoyl)phosphatidylcholine or (dipalmitoyl)phosphatidylcholine and produces fluorescence increases only in the presence of Ca2+ and at temperatures above their gel-to-liquid crystalline phase transition. Although the fluorescence changes in CDRDANS accurately report phase transitions in these liposomes, its binding to these vesicles is weak. Calmodulin probably requires a high-affinity lipid-bound receptor protein for its high-affinity binding to natural membranes.