Purification and characterization of a calmodulin-dependent kinase from rat brain cytosol able to phosphorylate tubulin and microtubule-associated proteins.
Purification and characterization of a calmodulin-dependent kinase from rat brain cytosol able to phosphorylate tubulin and microtubule-associated proteins.
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DOI：
10.1016/s0021-9258(17)44223-2
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发表时间：
1983-10
期刊：
影响因子：
--
通讯作者：
J. Goldenring;B. González;J. McGuire;R. Delorenzo
中科院分区：
文献类型：
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作者：
J. Goldenring;B. González;J. McGuire;R. Delorenzo
微管蛋白是突触细胞质中内源性Ca 2 +-钙调蛋白依赖性磷酸化的主要底物。本研究详细说明了纯化到明显的同质性和表征的脑细胞溶质钙调蛋白依赖性激酶磷酸化微管蛋白和微管相关蛋白作为主要底物。通过磷酸纤维素树脂、钙调蛋白亲和树脂和Fractogel TSK HW-55上的连续色谱法纯化的细胞溶质激酶系统在Sephacryl S-300上色谱为约600，000 Da的均质复合物。这种钙调蛋白依赖性激酶具有一组特异性表征该酶系统的性质：1）该酶含有两个钙调蛋白结合双联体，rho和sigma，分别约为52，000和63，000 Da; 2）rho和sigma亚基均显示等电点在6.7和7.2之间; 3）rho和sigma亚基均显示自磷酸化; 4）通过胰蛋白酶肽指纹评估，rho和σ亚基都显示出显著的同源性; 5）在不存在底物的情况下，rho和σ亚基都表现出较低的迁移率的自磷酸化种类; 6）激酶在苏氨酸和丝氨酸残基上同等地磷酸化β-微管蛋白。底物特异性，动力学参数，钙调蛋白结合特性，亚基组成和亚基等电点清楚地区分这种酶从其他先前报道的钙调蛋白依赖性激酶。
Tubulin is a major substrate for endogenous Ca2+-calmodulin-dependent phosphorylation in synaptic cytoplasm. The present study details the purification to apparent homogeneity and characterization of a brain cytosolic Ca2+-calmodulin-dependent kinase which phosphorylates tubulin and microtubule-associated proteins as major substrates. The cytosolic kinase system, purified by sequential chromatography on phosphocellulose resin, calmodulin-affinity resin, and Fractogel TSK HW-55, chromatographs as a homogeneous complex of approximately 600,000 Da on Sephacryl S-300. This calmodulin-dependent kinase possesses a group of properties which specifically characterize this enzyme system: 1) the enzyme contains two calmodulin-binding doublets, rho and sigma, of approximately 52,000 and 63,000 Da, respectively; 2) both the rho and the sigma subunits demonstrate isoelectric points between 6.7 and 7.2; 3) both the rho and sigma subunits demonstrate autophosphorylation; 4) both the rho and sigma subunits show significant homologies as assessed by tryptic peptide fingerprints; 5) in the absence of substrate, both the rho and sigma subunits manifest lower mobility autophosphorylated species; 6) the kinase phosphorylates beta-tubulin equally on threonine and serine residues. Substrate specificity, kinetic parameters, calmodulin-binding properties, subunit composition, and subunit isoelectric points clearly differentiate this enzyme from other previously reported calmodulin-dependent kinases.