TURBIDIMETRIC STUDIES OF INVITRO ASSEMBLY AND DISASSEMBLY OF PORCINE NEUROTUBULES
TURBIDIMETRIC STUDIES OF INVITRO ASSEMBLY AND DISASSEMBLY OF PORCINE NEUROTUBULES
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DOI：
10.1016/0022-2836(74)90048-5
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发表时间：
1974-01-01
影响因子：
5.6
通讯作者：
SHELANSKI, ML
中科院分区：
文献类型：
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作者：
GASKIN, F;CANTOR, CR;SHELANSKI, ML
浊度测量已被用于研究猪神经管的体外组装和拆解。所有测定均采用纯度大于80%的微管蛋白。这种蛋白质在体外组装时形成的小管很长，浑浊度对长度不敏感，只是高分子量材料总质量的函数。在最佳条件下，猪微管蛋白的组装临界浓度约为0.2 mg/ml，pH 6.6，0.1M-2-(N-吗啉)乙烷磺酸，26~37℃。在这些条件下，在过量GTP存在的情况下，组装和拆解基本上是完全可逆的。组装动力学表现出强烈的温度依赖性的初始滞后和初始速率。我们的样品显示出不超过二阶的浓度依赖关系。组装反应的表观活化热为25kcal/mol，链传播步骤的组装反应表观反应热为21kcal/mol。分解动力学显示明显的负活化热为28kcal/−。它们与小管长度无关，意味着缓慢的活化步骤，随后是快速解聚。在20℃时，聚合和解聚的循环在组装动力学中表现出滞后效应，尽管不是在分解速率或最终状态下。这可以通过假设小管组装的起始复合体在4℃时缓慢可逆失活来解释。报道了产生微管蛋白的条件，该状态除非添加核苷酸，否则不能在水溶液缓冲液中组装。GTP、ATP和ADP对小管组装有促进作用，而GDP无明显作用。我们制剂中的腺苷酸激酶杂质可能是造成这种不寻常效果的原因。它是否真的与微管蛋白或小管有关尚不清楚。
Turbidity measurements have been used to study thein vitroassembly and disassembly of porcine neurotubules. All measurements were carried out with tubulin with a purity higher than 80%. Tubules formed byin vitroassembly of this protein are so long that the turbidity is insensitive to length and is a function only of the total mass of high molecular weight material. Porcine tubulin shows a critical concentration for assembly of about 0.2 mg/ml under optimal conditions, pH 6.6, 0.1m-2-(N-morpholino)ethane sulfonic acid, 26 to 37 °C. Under these conditions assembly and disassembly are essentially fully reversible in the presence of excess GTP. The kinetics of assembly show an initial lag and initial rates which are strongly temperature dependent. Our samples show a concentration dependence of no more than second order. The apparent activation enthalpy of assembly is 25 kcal/mol; the apparent reaction enthalpy of assembly for the chain propagation step is 21 kcal/mol. Disassembly kinetics show an apparent negative activation enthalpy of −28 kcal/mol. They are independent of tubule length implying a slow activation step followed by rapid depolymerization. At 20 °C, cycles of polymerization and depolymerization show hysteresis effects in the assembly kinetics though not in disassembly rates or final states. This is most easily explained by postulating a slow reversible inactivation at 4 °C of the initiation complex for tubule assembly. Conditions are reported for producing tubulin in a state which cannot assemble in aqueous buffer unless nucleotides are added. GTP, ATP and ADP, but not GDP, are effective in promoting tubule assembly. An adenylate kinase impurity in our preparation may be the cause of this unusual effect. Whether or not it is actually associated with tubulin or tubules is unknown.