CYTOPLASMIC DYNEIN OF THE SEA-URCHIN EGG .2. PURIFICATION, CHARACTERIZATION AND INTERACTIONS WITH MICROTUBULES AND CA-CALMODULIN
CYTOPLASMIC DYNEIN OF THE SEA-URCHIN EGG .2. PURIFICATION, CHARACTERIZATION AND INTERACTIONS WITH MICROTUBULES AND CA-CALMODULIN
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DOI：
10.1093/oxfordjournals.jbchem.a134182
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发表时间：
1983-01-01
影响因子：
2.7
通讯作者：
SAKAI, H
中科院分区：
文献类型：
--
作者：
HISANAGA, SI;SAKAI, H
在蛋白酶抑制剂的存在下，对未受精海胆卵中的细胞质动力蛋白进行纯化，以避免整个纯化过程中的蛋白水解，该过程包括多种色谱法，包括钙调蛋白-Sepharose 4B 亲和柱色谱法。这是将细胞质动力蛋白纯化至接近均质的第一份报告。纯化的级分由单一高分子量多肽和一些次要低分子量多肽组成。高分子量多肽与鞭毛动力蛋白 A.beta 共迁移。 SDS[十二烷基硫酸钠]-聚丙烯酰胺凝胶上精子的链。纯化的细胞质动力蛋白级分中没有可用高碘酸希夫试剂 (PAS) 染色的多肽。细胞质动力蛋白表现出与轴丝动力蛋白非常相似的特征，即对 ATP 的高底物特异性和低浓度钒酸盐的抑制，尽管其 Ca-ATP 酶活性显示出与 Mg-ATP 酶活性几乎相同的对二价阳离子或 KCl 浓度的依赖性。从其性质判断，纯化的酶似乎具有功能形式：ATP酶活性的pH依赖性、ATP酶活性对MgCl2和KCl浓度的依赖性、Mg-ATP的Km以及与鞭毛双联微管的结合。仅在存在 Ca2+ 的情况下，细胞质动力蛋白才会与钙调蛋白-Sepharose 4B 结合，并用 EGTA [乙二醇-双(2-氨基乙基醚)-N,N,N'',N''-四乙酸]洗脱。此外，钙调蛋白以 Ca2+ 依赖性方式将 ATP 酶活性增强 6 倍。化学计量的三氟拉嗪可防止钙调蛋白的激活。
Purification of cytoplasmic dynein from unfertilized sea urchin eggs was performed in the presence of protease inhibitors to avoid proteolysis throughout the purification procedure, which comprised serveral chromatographies, including a calmodulin-Sepharose 4B affinity column chromatography. This is the 1st report of the purification of cytoplasmic dynein to near homogeneity. The purified fraction was composed of a single high-MW polypeptide and some minor low-MW polypeptides. The high MW polypeptide comigrated with flagellar dynein A.beta. chain from sperm on SDS[sodium dodecyl sulfate]-polyacrylamide gels. There was no polypeptide stainable with periodic acid-Schiff reagent (PAS) in the purified cytoplasmic dynein fraction. Cytoplasmic dynein showed characteristics quite similar to those of axonemal dynein, i.e., high substrate specificity for ATP and inhibition by low concentrations of vanadate, though its Ca-ATPase activity showed almost the same dependence on the concentration of either divalent cations or KCl as the Mg-ATPase activity. The purified enzyme seemed to possess functional form as judged from its properties: pH dependence of the ATPase activity, dependence of the ATPase activity on MgCl2 and KCl concentration, Km for Mg-ATP and binding to flagellar doublet microtubules. Cytoplasmic dynein bound to calmodulin-Sepharose 4B only in the presence of Ca2+ and was eluted with EGTA [ethyleneglycol-bis(2-aminoethylether)-N,N,N'',N''-tetraacetic acid]. Furthermore, the ATPase activity was enhanced 6-fold by calmodulin in a Ca2+-dependent manner. The activation by calmodulin was prevented by a stoichiometric amount of trifluoperazine.