Release of exchangeably bound guanine nucleotides from tubulin in a magnesium-free buffer.
Release of exchangeably bound guanine nucleotides from tubulin in a magnesium-free buffer.
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在无镁缓冲液中从微管蛋白中释放可交换结合的鸟嘌呤核苷酸。
DOI：
10.1021/bi00324a035
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发表时间：
1985
期刊：
影响因子：
2.9
通讯作者：
WilliamsJr,RC
中科院分区：
文献类型：
--
作者：
Croom,HB;Correia,JJ;Baty,LT;WilliamsJr,RC
Department of Molecular Biology，范德比尔特大学，纳什维尔，田纳西州37235接收1983年11月16日;修订版Mandarin pt接收1984年7月11日摘要：鸟嘌呤核苷酸的摩尔数（表示为GXP）、鸟苷5 '-三磷酸（GTP）或鸟苷5 H-二磷酸（GDP），已经测量了在凝胶过滤到各种无核苷酸缓冲液中后与一摩尔磷酸纤维素纯化的微管蛋白结合的量（. B。Croom、JJ Correia和R. C.小威廉姆斯，未公布的结果）。我们研究的所有缓冲液，促进结合核苷酸的数量减少到每个微管蛋白二聚体少于两个，也最终导致蛋白质活性的不可逆损失。然而，在0.11，4-哌嗪二乙磺酸（pH 6.9）和2 mM二硫代硫酸钠（不含Mg 2+）中，微管蛋白在两次连续的凝胶过滤过程中快速释放约0.4mol结合的核苷酸，所需时间少于0.5小时，并且当立即将镁和GTP加入缓冲液中时，微管蛋白恢复了溶解能力。圆二色性或沉降速度检测构象没有变化伴随着这个可逆的过程。(Upon然而，在缓冲液中长时间孵育，微管蛋白根据表观一级动力学经历不可逆变化，半衰期为8小时。这些变化包括核苷酸的不可逆释放，聚合能力的丧失，以及210和240 nm之间摩尔椭圆率的降低。在该缓冲液中可逆释放的核苷酸来自在体外容易与[3 H] GTP交换的群体。当用磷酸纤维素层析法纯化的微管蛋白在含Mg 2+的缓冲液中与浓度为70 μ g-1 mM的[3 H] GTP在0 - 24 ℃温育30分钟时，结合的总GXP和通过交换结合的[3 H] GTP之间的差异始终为1.2±0.1/微管蛋白二聚体。这些观察结果表明，磷酸纤维素纯化的微管蛋白由两种类型的二聚体组成：一种（约占总量的80%）容易在其“可交换”位点交换鸟嘌呤核苷酸，另一种（约占总量的20%）交换核苷酸较差。在含Mg ~（2+）缓冲液中容易交换GTP的二聚体似乎与在无Mg ~（2+）缓冲液中快速释放结合核苷酸的二聚体相同。鸟嘌呤核苷酸（GXP）1和微管蛋白之间相互作用的性质仍不完全清楚（Jacobs，1979; Timasheff和Grisham，1980）。已经确定，两个核苷酸分子（GDP或GTP）与在GTP存在下分离的微管蛋白紧密结合（Weisenberg et al.，1968; Berry & Shelanski，1972）。结合位点已被区分的基础上，他们的能力，结合[3 H] GTP在体外孵育时间短的时间间隔。不可交换位点（N位点）可能含有GTP，该GTP从不在体外交换游离核苷酸，并且只有当蛋白质变性时才能从该位点释放（Spiegelman et al.，1977年）。可交换位点（E位点）含有GDP或GTP的分子，其被认为在体外与游离核苷酸快速交换（Weisenberg et al.，1968年）。在微管蛋白聚合过程中，E位点的GTP水解为GDP;这种水解滞后于二聚体掺入微管（Penningroth & Kirschner，1977; Carlier & Pantaloni，1981）。为了更好地理解GTP结合和水解在微管组装过程中的作用，首先了解核苷酸与微管蛋白E位点之间的结合平衡的性质是有帮助的。方法f这项工作是.
Department of Molecular Biology, Vanderbilt University, Nashville, Tennessee 37235 Received November 16, 1983; Revised Manuscript Received July 11, 1984 abstract: The number of moles of guanine nucleotides (denoted GXP), eitherguanosine 5'-triphosphate (GTP) or guanosine 5/-diphosphate (GDP), bound to a mole of phosphocellulose-purified tubulin after gel filtration into a variety of nucleotide-free buffers has been measured (. B. Croom, JJ Correia, and R. C. Williams, Jr., unpublished results). All buffers we have studied that promote reduction of the number of bound nucleotides to fewer than two per tubulin dimer also eventually cause irreversible loss of activity of the protein. However, in 0.1 1, 4-piperazinediethanesulfonic acid (pH 6.9) and 2 mM dithioerythritol (with no Mg2+), tubulin rapidly releases approximately 0.4 mol of bound nucleotides during two successive gel filtrations requiring less than 0.5 h and regainsthe ability to polymerize when magnesium and GTP are immediately added to the buffer. No change in conformation detectable by circular dichroism or sedimentation velocity accompanies this reversibleprocess.(Upon prolonged incubation in the buffer, however, tubulin undergoes irreversible changes according to apparent first-order kinetics with a half-life of ap-proximately 8 h. These changes include the irreversible release of nucleotide, a loss of the ability to polymerize, and a decrease in molar ellipticity between 210 and 240 nm.) The nucleotide which is reversibly released in this buffer comes from that population which exchanges readily with [3H] GTP in vitro. When tubulin purified byphosphocellulose chromatography is incubated in a Mg2+-containing buffer for 30 min with [3H] GTP at concentrations from 70 µ to 1 mM at temperatures from 0 to 24 C, the differencebetween the total GXP bound and the [3H] GTP that becomes boundby exchange is consistently 1.2±0.1 per tubulin dimer. These observations indicate that phosphocellulose-purifiedTubulin is composed of two types of dimers: one (approximately 80% of the total) which readily exchanges guanine nucleotides at its “exchangeable” site and one (approximately 20% of the total) which exchanges nucleotides poorly. Those dimers that exchange GTP readily in Mg2+-containing buffersappear to be the same dimers that rapidly release bound nucleotide in Mg2+-free buffers. e nature of the interaction between guanine nucleotides (GXP) 1 and tubulin is still not fully understood (Jacobs, 1979; Timasheff & Grisham, 1980). It is well established that two molecules of nucleotide (eitherGDP or GTP) are tightly bound to tubulin that has been isolated in the presence of GTP (Weisenberg et al., 1968; Berry & Shelanski, 1972). The binding sites have been distinguished on the basis of their ability to bind [3H] GTP when incubated in vitro for short intervals. The nonexchangeable site (N site) probably contains GTP that never exchanges for free nucleotide invitro and that can be released from that site only when the protein is dena-tured (Spiegelman et al., 1977). The exchangeable site (E site) contains a molecule of either GDP or GTP which is thought to exchange rapidly with free nucleotide in vitro (Weisenberg et al., 1968). During polymerization of tubulin, GTP at the E site is hydrolyzed to GDP; this hydrolysis lags behind the incorporation of a dimer into a microtubule (Penningroth & Kirschner, 1977; Carlier & Pantaloni, 1981). In order to understand better the role of GTP binding and hydrolysis during the assembly of microtubules, it is helpful first to understand the nature of the binding equilibrium be-tween the nucleotide and the E site of tubulin. Approaches f This work was …
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体外微管组装中的核苷酸结合和磷酸化。
影响因子：
5.6
作者：
S. Penningroth;M. Kirschner
通讯作者：
M. Kirschner
微管蛋白与核苷酸类似物的组装。
影响因子：
4.8
作者：
M. Kirsch;L. Yarbrough
通讯作者：
L. Yarbrough
GTP 水解和微管蛋白聚合的化学计量。
DOI：
--
发表时间：
1977
影响因子：
11.1
作者：
R. Maccioni;N. Seeds
通讯作者：
N. Seeds
DOI：
10.1016/s0021-9258(19)43386-3
发表时间：
1973
期刊：
The Journal of biological chemistry
影响因子：
--
作者：
James C. Lee;R. Frigon;S. N. Timasheff
通讯作者：
S. N. Timasheff
铬 (III)-核苷酸复合物作为鸟苷 5-三磷酸诱导的微管组装的探针。
影响因子：
3.9
作者：
R. MacNeal;D. Purich
通讯作者：
D. Purich