Quest A F, Shapiro B M
Department of Biochemistry, University of Washington, Seattle 98195.
J Biol Chem. 1991 Oct 15;266(29):19803-11.
The flagellar creatine kinase (TCK) of Strongylocentrotus purpuratus sperm is both a principal component of sperm tail membrane preparations and a cytosolic enzyme. An improved purification scheme identified three pools of TCK, termed TCK I, TCK II, and TCK III. TCK I and II were essentially homogeneous protein preparations, while TCK III was heavily contaminated with other flagellar proteins, predominantly guanylate cyclase, and alpha- and beta-tubulin. The three TCK species are roughly present in a 1:10:1 ratio as assessed by activity measurements. TCK I and II are similar proteins as shown by two-dimensional gel electrophoresis, partial proteolytic fragmentation, and cellulose polyacetate electrophoresis and have the same pH-dependent specific activity. However, they are functionally distinct with respect to their capacity to associate with lipids. TCK II associated readily with phospholipid liposomes and detergent micelles, while TCK I did not. Association of TCK II was as a protein monomer with an apparent Kd of approximately 1-2 mM at a 10(4):1 lipid or detergent to protein ratio. Whereas the Kd estimates were pH independent, the rate of association increased 2-3-fold between pH 6.5 and 8. The data are consistent with membrane-association of TCK II being a two-step process, involving a pH-dependent, intramolecular, TCK-specific step and a charge-facilitated, but pH-independent, membrane association step. Membrane association of TCK may, together with microtubule association (Tombes, R.M., Farr, A., and Shapiro, B.M. (1988) Exp. Cell Res. 178, 307-317) represent a mechanism required for specific accumulation of the enzyme within the flagellum.
紫海胆精子的鞭毛肌酸激酶（TCK）既是精子尾部膜制剂的主要成分，也是一种胞质酶。一种改进的纯化方案鉴定出了TCK的三个组分，分别称为TCK I、TCK II和TCK III。TCK I和II基本上是纯蛋白质制剂，而TCK III则被其他鞭毛蛋白严重污染，主要是鸟苷酸环化酶以及α和β微管蛋白。通过活性测量评估，这三种TCK组分的比例大致为1:10:1。二维凝胶电泳、部分蛋白水解片段化以及纤维素醋酸酯电泳表明，TCK I和II是相似的蛋白质，并且具有相同的pH依赖性比活性。然而，就它们与脂质结合的能力而言，它们在功能上是不同的。TCK II很容易与磷脂脂质体和去污剂胶束结合，而TCK I则不然。TCK II以蛋白质单体的形式结合，在脂质或去污剂与蛋白质的比例为10(4):1时，其表观解离常数（Kd）约为1-2 mM。虽然Kd估计值与pH无关，但在pH 6.5至8之间，结合速率增加了2-3倍。这些数据与TCK II的膜结合是一个两步过程一致，该过程涉及一个pH依赖性的分子内TCK特异性步骤和一个电荷促进但pH无关的膜结合步骤。TCK的膜结合可能与微管结合（Tombes, R.M., Farr, A., and Shapiro, B.M. (1988) Exp. Cell Res. 178, 307-317）一起，代表了该酶在鞭毛内特异性积累所需的一种机制。
