INVITRO POLYMERIZATION OF FLAGELLAR AND CILIARY OUTER FIBER TUBULIN INTO MICROTUBULES
INVITRO POLYMERIZATION OF FLAGELLAR AND CILIARY OUTER FIBER TUBULIN INTO MICROTUBULES
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DOI：
10.1093/oxfordjournals.jbchem.a131247
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发表时间：
1976-01-01
影响因子：
2.7
通讯作者：
KURIYAMA, R
中科院分区：
文献类型：
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作者：
KURIYAMA, R
海胆、花球海胆、粉刺海胆和梨形四膜虫的纤毛外纤维部分的总蛋白中，apprx.在含有5 mM MES[2-(N-吗啉基)乙磺酸]、0.5 mM硫酸镁、1.0 mM EGTA[ethyleneglycol-bis(.beta.-aminoethylether)-tetraacetic酸、1.0 mM GTP和0或50 mM KCl的溶液中，在pH 6.7下超声溶解10-20%。分析离心法表明，该提取物主要由6-S组分(微管蛋白二聚体)组成，凝胶过滤测得其相对分子质量为103,000，对秋水仙碱的结合活性为0.8mol/微管蛋白二聚体。微管蛋白组分本身不能聚合成微管。然而，加入少量的纤毛外纤维碎片或重组的短脑微管可诱导聚合，流动双折射的粘度变化以及光学显微镜或EM观察表明。用电子显微镜观察外源性微管蛋白与DEAE-葡聚糖修饰的脑微管混合后异质微管的生长情况。当一个浓缩的微管蛋白组分在35℃升温时，微管由不添加任何核组分的外纤维微管蛋白重组而成。C.在许多单线微管之间可观察到几条双线状微管或成对平行的纵向排列紧密的单线微管。与外纤维微管不同，重组聚合物可通过钙离子、高或低离子强度、秋水仙碱、低温或SH试剂解聚。在此条件下未组装微管。
Of the total protein in the outer fiber fraction [of flagella from spermatozoa of the sea-urchins Pseudocentrotus depressus, Anthocidaris crassispina, Hemicentrotus pulcherrimus, and cilia from Tetrahymena pyriformis], .apprx. 10-20% was solubilized by sonication in a solution containing 5 mM MES [2-(N-morpholino)ethanesulfonic acid], 0.5 mM MgSO4, 1.0 mM EGTA [ethyleneglycol-bis(.beta.-aminoethylether)-tetraacetic acid], 1.0 mM GTP, and 0 or 50 mM KCl at pH 6.7. The sonicated extract was shown by analytical centrifugation to consist largely of a 6 S component (tubulin dimer), having a MW of 103,000 as determined by gel filtration, and possessing a colchicine-binding activity of 0.8 mol/tubulin dimer. The tubulin fraction failed to polymerize into microtubules by itself. Addition of a small amount of the ciliary outer fiber fragments or reconstituted short brain microtubules, however, induced polymerization, as demonstrated by viscosity of flow birefringence changes as well as light microscopic or EM observations. The growth of heterogeneous microtubules upon mixing outer fiber tubulin with DEAE-dextran-decorated brain microtubules was observed by EM. Microtubules were reconstituted from outer fiber tubulin without addition of any nuclear fraction when a concentrated tubulin fraction was warmed at 35.degree. C. A few doublet-like microtubules or pairs of parallel singlet microtubules, closely aligned longitudinally, could be observed among many singlet microtubules. Unlike outer fiber microtubules the reconstituted polymers were depolymerized by exposure to Ca2+, high or low ionic strength, colchicine, low temperature or SH reagents. No microtubules were assembled under these conditions.