MICROTUBULE REASSEMBLY INVITRO OF STRONGYLOCENTROTUS-PURPURATUS SPERM TAIL OUTER DOUBLET TUBULIN
MICROTUBULE REASSEMBLY INVITRO OF STRONGYLOCENTROTUS-PURPURATUS SPERM TAIL OUTER DOUBLET TUBULIN
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DOI：
10.1016/0022-2836(78)90371-6
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发表时间：
1978-01-01
影响因子：
5.6
通讯作者：
WILSON, L
中科院分区：
文献类型：
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作者：
FARRELL, KW;WILSON, L
用0.6 M-KCl提取精尾轴突，制备紫荆体外双线微管。外重态微管在5 mM-2-(N-morpholino)乙磺酸，1 mm -乙二醇-双-(. β。-氨基乙醚)N，N''-四乙酸，1 mM-MgSO4 (pH 6.7)，根据输入功率的不同，可溶解高达35%的外部双线蛋白，对两种亚纤维均无选择性。从超声悬浮液中获得的200,000 g上清液中，微管蛋白占总溶解蛋白的75-85%。秋水仙碱结合实验表明，微管蛋白主要以天然形式存在（KA = 106 l mol-1；在无限浓度的秋水仙碱下，每mol微管蛋白结合0.74 mol秋水仙碱）。在不添加种子或甘油的情况下，200,000 g上清液中的微管自组装通过350 nm光散射进行定量。37℃下组装的临界蛋白浓度为0.55 mg ml-1。C，在2 mM-GTP和150 mM-KCl的存在下反应最佳。溶解的外双线微管蛋白在体外条件下重组后形成单线微管。通过阴性染色和薄层电镜对微管的真实性进行了验证。秋水仙碱和鬼鬼毒素阻止了微管的聚合，并在微管冷却至0℃时迅速解聚。C.重新组装的微管对低温的敏感性表明，它们可以通过为脊椎动物大脑开发的热组装-冷拆卸程序进行回收（Borisy et al., 1974）。在十二烷基硫酸钠的存在下，通过凝胶电泳判断，两次回收的外双重微管蛋白缺乏高分子量的微管相关蛋白。在严重超载的凝胶上可见微量（< 5%）的中间分子量物质。这种物质的功能尚不确定，但它在化学上不等同于脊椎动物脑中的tau因子（Weingarten et al., 1975），因为它不能通过磷纤维素吸附从微管蛋白中分离出来。此外，经磷酸纤维素处理的微管蛋白与未经处理的微管蛋白的重组程度相同，这表明外双线微管蛋白的重组不需要脑微管相关蛋白或tau因子等蛋白质。如果外部双联微管蛋白的重组需要辅助蛋白，在所采用的条件下，它们不能被磷纤维素去除，并且必须占总蛋白的5%。
S. purpuratus outer doublet microtubules were prepared by extraction of sperm tail axonemes with 0.6 M-KCl. Sonication of the outer doublet microtubules in 5 mM-2-(N-morpholino)ethanesulfonic acid, 1 mM-ethyleneglycol-bis-(.beta.-aminoethyl ether)N,N''-tetraacetic acid, 1 mM-MgSO4 (pH 6.7) solubilized up to 35% of the outer doublet protein, depending on the power input, in a manner which was non-selective for either subfiber. Tubulin comprised 75-85% of the total solubilized protein in a 200,000 g supernatant obtained from the sonicated suspension. Colchicine-binding assays demonstrated that the tubulin was largely in a native form (KA = 106 l mol-1; 0.74 mol colchicine bound per mol of tubulin at infinite concentration of colchicine). Microtubule self-assembly from the 200,000 g supernatants in the absence of added seeds or glycerol was quantitated by light-scattering at 350 nm. The critical protein concentration for assembly was 0.55 mg ml-1 at 37.degree. C and the reaction occurred optimally in the presence of 2 mM-GTP and 150 mM-KCl. The solubilized outer doublet tubulin formed singlet microtubules upon reassembly under the in vitro conditions used. The authenticity of the microtubules was verified by negative stain and thin-section EM. Polymerization was prevented by colchicine and podophyllotoxin, and depolymerization occurred rapidly on cooling the microtubules to 0.degree. C. Susceptibility of the reassembled microtubules to low temperature suggested that they could be recycled by the warm assembly-cold disassembly procedure developed for vertebrate brain (Borisy et al., 1974). Twice recycled outer doublet tubulin was devoid of high MW microtubule-associated proteins, as judged by gel electrophoresis in the presence of sodium dodecyl sulfate. Trace amounts (< 5%) of intermediate MW material was visible on heavily overloaded gels. The function of this material is uncertain, but it is not chemically equivalent to the tau factor of vertebrate brain (Weingarten et al., 1975), since it cannot be separated from the tubulin by phosphocellulose adsorption. In addition, phosphocellulose-treated tubulin reassembled to the same extent as untreated tubulin, suggesting that the reassembly of outer doublet tubulin does not require the protein equivalents of brain microtubule-associated proteins or tau factor. If accessory proteins are required for the reassembly of outer doublet tubulin, they are not removed by phosphocellulose under the conditions employed and must comprise > 5% of the total protein.