Watanabe T, Flavin M
J Biol Chem. 1976 Jan 10;251(1):182-92.
Nucleotides have at least two functions in eukaryotic cilia and flagella. ATP, originating in the cells, is utilized for motility by energy-transducing protein(s) called dynein, and the binding of guanine nucleotides to tubulin, and probably certain transformations of the bound nucleotides, are prerequisites for the assembly of microtubules. Besides dynein, which can be solubulized from Chlamydomonas flagella as a heterogeneous, Mg2+ or Ca2+-activated ATPase, we have purified and characterized five other flagellar enzymes involved in nucleotide transformations. A homogeneous, low molecular weight, Ca2+-specific adenosine triphosphatase was isolated, which was inhibited by Mg2+ and was not specific for ATP. This enzyme was not formed by treating purified dynein with proteases. It was absent from extracts of Tetrahymena cilia. Its function might be an auxiliary energy transducer, or in steering or tactic responses. Two species of adenylate kinase were isolated, one of which was much elevated in regenerating flagella; the latter was also present in cell bodies. A large part of flagellar nucleoside diphosphokinase activity could not be solubilized. Two soluble enzyme species were identified, one of which was also present in cell bodies. Since these enzymes are of interest because they might function in microtubule assembly, we studied the extent to which brain nucleoside diphosphokinase co-polymerizes with tubulin purified by repeated cycles of polymerization. Arginine kinase was not detected in Chlamydomonas flagellar extracts.
核苷酸在真核生物的纤毛和鞭毛中至少具有两种功能。源自细胞的ATP被一种称为动力蛋白的能量转换蛋白用于运动，鸟嘌呤核苷酸与微管蛋白的结合以及结合核苷酸的某些转化是微管组装的先决条件。除了可以从衣藻鞭毛中以异质的、Mg2+或Ca2+激活的ATP酶形式溶解的动力蛋白外，我们还纯化并鉴定了其他五种参与核苷酸转化的鞭毛酶。分离出一种均一的、低分子量的、Ca2+特异性腺苷三磷酸酶，它被Mg2+抑制，对ATP不具有特异性。这种酶不是通过用蛋白酶处理纯化的动力蛋白形成的。它在四膜虫纤毛提取物中不存在。其功能可能是辅助能量转换器，或在转向或趋性反应中起作用。分离出两种腺苷酸激酶，其中一种在再生鞭毛中大量升高；后者也存在于细胞体中。鞭毛核苷二磷酸激酶的大部分活性不能溶解。鉴定出两种可溶性酶，其中一种也存在于细胞体中。由于这些酶可能在微管组装中起作用，我们研究了脑核苷二磷酸激酶与通过重复聚合循环纯化的微管蛋白共聚合的程度。在衣藻鞭毛提取物中未检测到精氨酸激酶。
