GLUTAMATE-INDUCED POLYMERIZATION OF TUBULIN - CHARACTERISTICS OF THE REACTION AND APPLICATION TO THE LARGE-SCALE PURIFICATION OF TUBULIN
GLUTAMATE-INDUCED POLYMERIZATION OF TUBULIN - CHARACTERISTICS OF THE REACTION AND APPLICATION TO THE LARGE-SCALE PURIFICATION OF TUBULIN
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DOI：
10.1016/0003-9861(81)90253-8
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发表时间：
1981-01-01
影响因子：
3.9
通讯作者：
LIN, CM
中科院分区：
文献类型：
--
作者：
HAMEL, E;LIN, CM
一个3阶段的方法，提出了大规模纯化小牛脑微管蛋白，利用高浓度的谷氨酸盐的能力，稳定微管蛋白，并在GTP的存在下，诱导其聚合。纯化的微管蛋白含有1.9摩尔鸟嘌呤核苷酸/摩尔蛋白质，基本上不含核苷二磷酸激酶和ATP酶活性。谷氨酸诱导的聚合反应需要GTP，而ATP、CTP和UTP完全无效。聚合是温度依赖性和冷可逆的，并抑制秋水仙素，GDP和Ca 2+，但没有绝对的要求Mg 2+可以证明。纯化的微管蛋白具有最小的GTdR活性，除非谷氨酸存在，并且水解反应随谷氨酸浓度而变化。在更高的谷氨酸水平的水解的开始是密切相关的聚合与GTP水解的快速爆发结束聚合接近其平台，其次是一个较慢的，线性的水解速率的开始。最初水解的GTP与聚合的微管蛋白的摩尔比为约1：1。在较低的谷氨酸浓度GTP水解似乎没有被连接到微管蛋白聚合。
A 3 stage method is presented for the large-scale purification of calf brain tubulin, exploiting the ability of high concentrations of glutamate to stabilize tubulin and, in the presence of GTP, to induce its polymerization. The purified tubulin contained 1.9 mol of guanine nucleotide/mole of protein and was essentially free of nucleoside diphosphate kinase and ATPase activities. The glutamate-induced polymerization reaction required GTP, while ATP, CTP and UTP were completely ineffective. Polymerization was temperature dependent and cold reversible and was inhibited by colchicine, GDP and Ca2+, but no absolute requirement for Mg2+ could be demonstrated. The purified tubulin had minimal GTPase activity unless glutamate was present, and the hydrolytic reaction varied with the glutamate concentration. At higher glutamate levels the onset of hydrolysis was closely linked to the onset of polymerization with a rapid burst of GTP hydrolysis ending as polymerization approached its plateau, followed by a slower, linear rate of hydrolysis. Initially the molar ratio of GTP hydolyzed to tubulin polymerized was about 1:1. At lower glutamate concentrations GTP hydrolysis did not appear to be linked to tubulin polymerization.