Patzelt C, Singh A, Marchand Y L, Orci L, Jeanrenaud B
J Cell Biol. 1975 Sep;66(3):609-20. doi: 10.1083/jcb.66.3.609.
Colchicine-binding activity of mouse liver high-speed supernate has been investigated. It has been found to be time and temperature dependent. Two binding activities with different affinities for colchicine seem to be present in this high-speed supernate, of which only the high-affinity binding site (half maximal binding at 5 x 10(-6) M colchicine) can be attributed to microtubular protein by comparison with purified tubulin. Vinblastine interacted with this binding activity by precipitating it when used at high concentrations (2 x 10(-3) M), and by stabilizing it at low concentrations (10(-5) M). Lumicolchicine was found not to compete with colchicine. The colchicine-binding activity was purified from liver and compared with that of microtubular protein from brain. The specific binding activity of the resulting preparation, its electrophoretic behavior, and the electron microscope appearance of the paracrystals obtained upon its precipitation with vinblastine permitted its identification as microtubular protein (tubulin). Electrophoretic analysis of the proteins from liver supernate that were precipitated by vinblastine indicated that this drug was not specific for liver tubulin. Preincubation of liver supernate with 5 mM EGTA resulted in a time-dependent decrease of colchicine-binding activity, which was partly reversed by the addition of Ca++. However, an in vitro formation of microtubules upon lowering the Ca++ concentration could not be detected. Finally, a method was developed enabling that portion of microtubular protein which was present as free tubulin to be measured and to be compared with the total amount of this protein in the tissue. This procedure permitted demonstration of the fact that, under normal conditions, only about 40% of the tubulin of the liver was assemled as microtubules. It is suggested that, in the liver, rapid polymerization and depolymerization of microtubules occur and may be an important facet of the functional role of the microtubular system.
对小鼠肝脏高速上清液的秋水仙碱结合活性进行了研究。发现其具有时间和温度依赖性。在这种高速上清液中似乎存在两种对秋水仙碱具有不同亲和力的结合活性，通过与纯化的微管蛋白比较，其中只有高亲和力结合位点（在5×10⁻⁶ M秋水仙碱时达到半数最大结合）可归因于微管蛋白。长春花碱在高浓度（2×10⁻³ M）使用时通过沉淀作用与这种结合活性相互作用，在低浓度（10⁻⁵ M）时则使其稳定。发现光秋水仙碱不与秋水仙碱竞争。从肝脏中纯化了秋水仙碱结合活性，并与脑微管蛋白的秋水仙碱结合活性进行了比较。所得制剂的特异性结合活性、其电泳行为以及用长春花碱沉淀后获得的副晶体的电子显微镜外观使其被鉴定为微管蛋白（微管球蛋白）。对长春花碱沉淀的肝脏上清液中的蛋白质进行电泳分析表明，这种药物对肝脏微管球蛋白不具有特异性。肝脏上清液与5 mM乙二醇双四乙酸（EGTA）预孵育导致秋水仙碱结合活性随时间下降，加入钙离子（Ca++）后部分逆转。然而，未检测到降低钙离子浓度时微管在体外的形成。最后，开发了一种方法，能够测量以游离微管球蛋白形式存在的微管蛋白部分，并将其与组织中该蛋白的总量进行比较。该程序证明了这样一个事实，即在正常情况下，肝脏中只有约40%的微管球蛋白组装成微管。有人提出，在肝脏中，微管会快速聚合和解聚，这可能是微管系统功能作用的一个重要方面。
