Monovalent cation-insensitive hydrophobic region on calmodulin facilitates the rapid isolation and quantitation of calmodulin free from other Ca2+-dependent hydrophobic proteins.
Monovalent cation-insensitive hydrophobic region on calmodulin facilitates the rapid isolation and quantitation of calmodulin free from other Ca2+-dependent hydrophobic proteins.
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钙调蛋白上的单价阳离子不敏感疏水区域有利于钙调蛋白与其他 Ca2 依赖性疏水蛋白的快速分离和定量。
DOI：
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发表时间：
1985
期刊：
影响因子：
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通讯作者：
W. Anderson
中科院分区：
文献类型：
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作者：
R. Gopalakrishna;W. Anderson
钙调素通过钙离子诱导的疏水结合区与苯基琼脂糖定量结合。肌钙蛋白C和S-100蛋白，以及存在于大鼠组织中的其他几种蛋白，也以钙离子依赖的方式与苯基琼脂糖结合。虽然单价阳离子对钙调蛋白与苯基琼脂糖凝胶结合的钙依赖性影响不大，但它们似乎确实竞争钙离子与包括S-100蛋白在内的大多数其他蛋白质的结合，这些蛋白质表现出钙离子诱导与苯基琼脂糖凝胶的相互作用。在低浓度(100微米)的Ca~(2+)存在下，用0.5M的单价阳离子如K~+、Na~+和NH_4~+可以选择性地将这些蛋白质从苯基-琼脂糖柱上洗脱出来。粗细胞提取物中与钙调蛋白相关的钙调蛋白结合蛋白可以阻止钙调蛋白与苯基琼脂糖凝胶的相互作用，导致这些组织中钙调素的回收率较低。这些干扰蛋白中的大多数是热不稳定的，因此对细胞提取物进行有限时间(5分钟)的加热(沸腾)可以消除这些蛋白与钙调蛋白的任何结合，并允许通过疏水相互作用层析定量回收钙调蛋白。该方法可以快速、定量地从各种大鼠组织制备的胞浆和Triton X-100溶解颗粒组分中回收高纯度的钙调蛋白。用考马斯亮蓝染料或荧光胺直接测定蛋白质或通过环核苷酸磷酸二酯酶刺激试验可以准确、可靠地定量以这种方式分离的钙调蛋白。
Calmodulin binds quantitatively to phenyl-Sepharose through its Ca2+-induced hydrophobic binding region. Troponin C and S-100 protein, as well as several other proteins present in rat tissues, also bind to phenyl-Sepharose in a Ca2+-dependent manner. While the Ca2+-dependent binding of calmodulin to phenyl-Sepharose is not altered appreciably by monovalent cations, they do appear to compete for Ca2+ binding to most of the other proteins, including S-100 protein, which exhibit Ca2+-induced interaction with phenyl-Sepharose. The selective elution of these proteins from the phenyl-Sepharose column can be achieved with a 0.5 M concentration of monovalent cations such as K+, Na+, and NH4+ in the presence of a low (100 microM) Ca2+ concentration. Calmodulin-binding proteins associated with calmodulin in crude cell extracts can prevent the interaction of calmodulin with the phenyl-Sepharose, resulting in low recoveries of calmodulin from these tissues. The majority of these interfering proteins are heat labile so that heat treatment (boiling) of the cell extract for a limited time (5 min) negates any binding of these proteins to calmodulin and allows the quantitative recovery of calmodulin by hydrophobic interaction chromatography. This procedure allows the rapid and quantitative recovery of highly purified calmodulin from both cytosolic and Triton X-100-solubilized particulate fractions prepared from various rat tissues. Calmodulin isolated in this manner can be accurately and reliably quantitated by direct protein determination with Coomassie brilliant blue dye or fluorescamine or by the cyclic nucleotide phosphodiesterase stimulation assay.