David-Pfeuty T, Erickson H P, Pantaloni D
Proc Natl Acad Sci U S A. 1977 Dec;74(12):5372-6. doi: 10.1073/pnas.74.12.5372.
Tubulin, purified by cycles of assembly followed by phosphocellulose chromatography, exhibits a characteristic GTPase activity that is polymerization dependent and can be attributed to the tubulin itself. This activity has been observed, in a standard reassembly buffer containing low Mg2+, under three conditions that induce microtubule assembly: in the presence of microtubule-associated proteins, in the presence of DEAE-dextran, or after addition of high Mg2+ and glycerol. The phosphocellulose-purified tubulin showed no GTPase activity under the following nonpolymerizing conditions: in buffer with low Mg2+ in the absence of microtubule-associated proteins or DEAE-dextran, in buffer with high Mg2+ and glycerol at tubulin concentrations below the critical concentration, or when microtubule assembly was inhibited by vinblastine. Colchicine, on the other hand, while blocking microtubule assembly, induced a significant GTPase activity in the phosphocellulose-purified tubulin. During the process of assembly, GTP appears to be hydrolyzed as a free tubulin dimer polymerizes into a microtubule. A constant GTPase activity when polymerization equilibrium is reached apparently reflects the cyclic polymerization-depolymerization of tubulin dimers at the ends of the microtubules.
通过组装循环后进行磷酸纤维素色谱法纯化的微管蛋白，表现出一种特征性的GTP酶活性，该活性依赖于聚合作用，且可归因于微管蛋白本身。在含有低镁离子的标准重组装缓冲液中，在三种诱导微管组装的条件下观察到了这种活性：存在微管相关蛋白时、存在二乙氨基乙基葡聚糖时，或添加高镁离子和甘油后。在以下非聚合条件下，磷酸纤维素纯化的微管蛋白未显示出GTP酶活性：在无微管相关蛋白或二乙氨基乙基葡聚糖的低镁离子缓冲液中、在微管蛋白浓度低于临界浓度时的高镁离子和甘油缓冲液中，或当长春花碱抑制微管组装时。另一方面，秋水仙碱虽然会阻断微管组装，但会在磷酸纤维素纯化的微管蛋白中诱导出显著的GTP酶活性。在组装过程中，当游离的微管蛋白二聚体聚合成微管时，GTP似乎会被水解。当达到聚合平衡时，恒定的GTP酶活性显然反映了微管末端微管蛋白二聚体的循环聚合-解聚。
