Hamel E, Lin C M
Biochemistry. 1984 Aug 28;23(18):4173-84. doi: 10.1021/bi00313a026.
A new method for separating microtubule-associated proteins (MAPs) and tubulin, appropriate for relatively large-scale preparations, was developed. Most of the active tubulin was separated from the MAPs by centrifugation after selective polymerization of the tubulin was induced with 1.6 M 2-(N-morpholino)ethanesulfonate (Mes) and GTP. The MAPs-enriched supernatant was concentrated and subsequently clarified by prolonged centrifugation. The supernatant (total soluble MAPs) contained almost no tubulin, most of the nucleosidediphosphate kinase activity of the microtubule protein, good activity in promoting microtubule assembly in 0.1 M Mes, and proteins with the electrophoretic mobility of MAP-1, MAP-2, and tau factor. The pellet, inactive in supporting microtubule assembly, contained denatured tubulin, most of the ATPase activity of the microtubule protein, and significant amounts of protein with the electrophoretic mobility of MAP-2. Insoluble material at this and all previous stages, including the preparation of the microtubule protein, could be heat extracted to yield soluble protein active in promoting microtubule assembly and containing MAP-2 as a major constituent. The total soluble MAPs were further purified by DEAE-cellulose chromatography into bound and unbound components, both of which induced microtubule assembly. The bound component (DEAE-MAPs) contained proteins with the electrophoretic mobility of MAP-1, MAP-2, and tau factor. The polymerization reaction induced by the unbound component (flow-through MAPs) produced very high turbidity readings. This was caused by the formation of bundles of microtubules. Although the flow-through MAPs contained significantly more ATPase, tubulin-independent GTPase, and, especially, nucleosidediphosphate kinase activity than the DEAE-MAPs, preparation of a MAPs fraction without these enzymes required heat treatment.
开发了一种适用于相对大规模制备的分离微管相关蛋白（MAPs）和微管蛋白的新方法。在用1.6M 2-（N-吗啉代）乙磺酸盐（Mes）和GTP诱导微管蛋白选择性聚合后，通过离心将大部分活性微管蛋白与MAPs分离。富含MAPs的上清液被浓缩，随后通过长时间离心进行澄清。上清液（总可溶性MAPs）几乎不含微管蛋白，含有微管蛋白的大部分核苷二磷酸激酶活性，在0.1M Mes中促进微管组装的活性良好，以及具有MAP-1、MAP-2和tau因子电泳迁移率的蛋白质。沉淀在支持微管组装方面无活性，含有变性的微管蛋白、微管蛋白的大部分ATP酶活性以及大量具有MAP-2电泳迁移率的蛋白质。在这个阶段以及之前所有阶段的不溶性物质，包括微管蛋白的制备物，都可以通过热提取得到在促进微管组装方面有活性且以MAP-2为主要成分的可溶性蛋白质。总可溶性MAPs通过DEAE-纤维素色谱进一步纯化成分离的结合和未结合组分，两者都能诱导微管组装。结合组分（DEAE-MAPs）含有具有MAP-1、MAP-2和tau因子电泳迁移率的蛋白质。未结合组分（流过MAPs）诱导的聚合反应产生非常高的浊度读数。这是由微管束的形成引起的。尽管流过MAPs比DEAE-MAPs含有明显更多的ATP酶、不依赖微管蛋白的GTP酶，尤其是核苷二磷酸激酶活性，但制备不含这些酶的MAPs组分需要热处理。
