Detrich H W, Johnson K A, Marchese-Ragona S P
Department of Biology, Northeastern University, Boston, Massachusetts 02115.
Biochemistry. 1989 Dec 26;28(26):10085-93. doi: 10.1021/bi00452a031.
Tubulins were purified from the brain tissues of three Antarctic fishes, Notothenia gibberifrons, Notothenia coriiceps neglecta, and Chaenocephalus aceratus, by ion-exchange chromatography and one cycle of temperature-dependent microtubule assembly and disassembly in vitro, and the functional properties of the protein were examined. The preparations contained the alpha- and beta-tubulins and were free of microtubule-associated proteins. At temperatures between 0 and 24 degrees C, the purified tubulins polymerized readily and reversibly to yield both microtubules and microtubule polymorphs (e.g., "hooked" microtubules and protofilament sheets). Critical concentrations for polymerization of the tubulins ranged from 0.87 mg/mL at 0 degrees C to 0.02 mg/mL at 18 degrees C. The van't Hoff plot of the apparent equilibrium constant for microtubule elongation at temperatures between 0 and 18 degrees C was linear and gave a standard enthalpy change (delta H degree) of +26.9 kcal/mol and a standard entropy change (delta S degree) of +123 eu. At 10 degrees C, tubulin from N. gibberifrons polymerized efficiently at high ionic strength; the critical concentration increased monotonically from 0.041 to 0.34 mg/mL as the concentration of NaCl added to the assembly buffer was increased from 0 to 0.4 M. Together, the results indicate that the polymerization of tubulins from the Antarctic fishes is entropically driven and suggest that an increased reliance on hydrophobic interactions underlies the energetics of microtubule formation at low temperatures. Thus, evolutionary modification to increase the proportion of hydrophobic interactions (relative to other bond types) at sites of interdimer contact may be one adaptive mechanism that enables the tubulins of cold-living poikilotherms to polymerize efficiently at low temperatures.
通过离子交换色谱法以及体外一轮温度依赖性微管组装与拆卸，从三种南极鱼类（裸高臀南极鱼、小头南极鱼和头带冰鱼）的脑组织中纯化出微管蛋白，并检测了该蛋白的功能特性。制备物中含有α-和β-微管蛋白，且不含微管相关蛋白。在0至24摄氏度之间的温度下，纯化的微管蛋白易于且可逆地聚合，产生微管和微管多形体（例如“钩状”微管和原纤维片）。微管蛋白聚合的临界浓度范围为0摄氏度时的0.87毫克/毫升至18摄氏度时的0.02毫克/毫升。在0至18摄氏度之间温度下微管伸长的表观平衡常数的范特霍夫图呈线性，标准焓变（ΔH°）为 +26.9千卡/摩尔，标准熵变（ΔS°）为 +123熵单位。在10摄氏度时，裸高臀南极鱼的微管蛋白在高离子强度下有效聚合；随着添加到组装缓冲液中的NaCl浓度从0增加到0.4 M，临界浓度从0.041毫克/毫升单调增加到0.34毫克/毫升。这些结果共同表明，南极鱼类微管蛋白的聚合是由熵驱动的，并表明在低温下微管形成的能量学基础是对疏水相互作用的依赖性增加。因此，进化修饰以增加二聚体间接触位点处疏水相互作用（相对于其他键类型）的比例可能是一种适应性机制，使低温生活的变温动物的微管蛋白能够在低温下有效聚合。
