INTERACTION OF CALCIUM AND CALMODULIN IN THE PRESENCE OF SODIUM DODECYL-SULFATE
INTERACTION OF CALCIUM AND CALMODULIN IN THE PRESENCE OF SODIUM DODECYL-SULFATE
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DOI：
10.1016/0005-2795(80)90254-8
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发表时间：
1980-01-01
期刊：
影响因子：
--
通讯作者：
KRETSINGER, RH
中科院分区：
文献类型：
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作者：
BURGESS, WH;JEMIOLO, DK;KRETSINGER, RH
钙调素被纯化到均匀使用改进的程序，允许快速处理几公斤牛麸皮。在十二烷基硫酸钠（SDS）存在下，钙调蛋白的电泳迁移率发生了钙依赖性变化。新鲜制备的钙调素或冷冻的钙调素，储存于-80度。当样品，凝胶和流动缓冲液在EDTA中制作0.1 mM时，C在1-7 mo时以单波段迁移，表观MW为21,000。当0.1 mM CaCl2取代EDTA时，新分离的钙调蛋白以单波段迁移，表观MW为15,000。当储存的蛋白质在相同条件下电泳时，除了15,000 MW的波段外，还观察到更慢的迁移带。钙结合实验表明，在0.1 mM CaCl2中，在0.1% SDS的存在下，新制备的钙调蛋白结合了4 mol Ca/mol的蛋白质。骨骼肌肌钙蛋白C、鲤鱼小白蛋白和牛脑S-100b不表现出这种流动性变化。钙依赖性迁移率变化可用于粗蛋白制剂中钙调素的鉴定。从紫圆海胆（Strongylocentrotus purpuratus）的精子中鉴定并纯化了钙调素。海胆钙调蛋白激活环核苷酸磷酸二酯酶的程度与脑钙调蛋白相同。几个标准被用来确定钙调素不存在作为可溶性蛋白在大肠杆菌。
Calmodulin was purified to homogeneity using an improved procedure that allows rapid processing of several kilograms of bovine bran. A Ca-dependent change in the electrophoretic mobility of calmodulin in the presence of sodium dodecyl sulfate (SDS) was observed. Freshly prepared calmodulin or lyophilized calmodulin stored at -80.degree. C for 1-7 mo. migrates as a single band with an apparent MW of 21,000 when the sample, gel and running buffer are made 0.1 mM in EDTA. When 0.1 mM CaCl2 is substituted for EDTA, freshly isolated calmodulin migrates as a single band with an apparent MW of 15,000. More slowly migrating bands, in addition to the 15,000 MW band, are observed when the stored protein is electrophoresed under the same conditions. Ca binding experiments show that freshly prepared calmodulin binds 4 mol of Ca/mol of protein in the presence of 0.1% SDS in 0.1 mM CaCl2. Skeletal muscle troponin C, carp parvalbumin and bovine brain S-100b do not show this mobility change. The Ca-dependent mobility change can be used to identify calmodulin in crude protein preparations. Calmodulin was identified in the sperm of the sea urchin, Strongylocentrotus purpuratus, and purified. The urchin calmodulin activates cyclic nucleotide phosphodiesterase to the same extent as does brain calmodulin. Several criteria were used to determine that calmodulin is not present as a soluble protein in Escherichia coli.