LARGE-SCALE PURIFICATION AND CHARACTERIZATION OF CALMODULIN FROM RAM TESTIS - ITS METAL-ION-DEPENDENT CONFORMERS
LARGE-SCALE PURIFICATION AND CHARACTERIZATION OF CALMODULIN FROM RAM TESTIS - ITS METAL-ION-DEPENDENT CONFORMERS
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DOI：
10.1016/0304-4165(80)90062-8
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发表时间：
1980-01-01
期刊：
影响因子：
--
通讯作者：
DEMAILLE, JG
中科院分区：
文献类型：
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作者：
AUTRIC, F;FERRAZ, C;DEMAILLE, JG
通过简单的程序从公羊睾丸中大量分离出钙调蛋白，包括依次进行硫酸铵分级、热处理、DEAE-纤维素上的阴离子交换层析和Sephacryl S-200上的凝胶过滤。在整个纯化过程中都存在二价阳离子（Mg2+ 和/或 Ca2+），这完全是在没有螯合剂的情况下进行的。最终产量为.apprx。 90毫克/公斤睾丸。根据以下标准，公羊睾丸钙调蛋白似乎与脑同源蛋白基本相同：UV吸收光谱、显示ε-N-三甲基赖氨酸单个残基的氨基酸组成、以及通过高效液相色谱获得的胰蛋白酶肽图。火鸡砂囊肌球蛋白轻链激酶，其激活对钙调蛋白具有极高的特异性，确实在 Ca 存在下被公羊睾丸钙调蛋白激活。分离的蛋白质在十二烷基硫酸钠聚丙烯酰胺凝胶电泳时以不同的速率迁移，这取决于可能诱导不同构象的二价金属的存在或不存在。相对迁移速率为Ca2+ > Mn2+ > Mg2+ > EDTA。在二价金属存在的情况下，观察到的双峰可能归因于无离子和离子饱和形式之间的平衡，它们表现出不同的斯托克斯半径。
Calmodulin was isolated in large quantities from ram testis by a simple procedure involving sequentially ammonium sulfate fractionation, heat treatment, anion exchange chromatography on DEAE-cellulose and gel filtration on Sephacryl S-200. Divalent cations (Mg2+ and/or Ca2+) were present throughout the purification, which was entirely performed in the absence of chelators. The final yield was .apprx. 90 mg/kg testis. Ram testis calmodulin appears to be essentially identical to the brain homologous protein by the following criteria: UV absorption spectrum, amino acid composition showing a single residue of .epsilon.-N-trimethyl lysine, and tryptic peptide maps obtained by high performance liquid chromatography. Turkey gizzard myosin light-chain kinase, the activation of which is extremely specific for calmodulin was indeed activated by ram testis calmodulin in the presence of Ca. The isolated protein migrated at different rates upon sodium dodecyl sulfate polyacrylamide gel electrophoresis, depending on the absence or presence of divalent metals which probably induce different conformations. The relative migration rates were Ca2+ > Mn2+ > Mg2+ > EDTA. In the presence of divalent metals, the observed doublet may be ascribed to the equilibrium between ion-free and ion-saturated forms, which exhibit different Stokes radii.