Xu S, Gaskin F
Department of Psychiatric Medicine, University of Virginia School of Medicine, Charlottesville 22908, USA.
Biochim Biophys Acta. 1998 Mar 3;1383(1):111-22. doi: 10.1016/s0167-4838(97)00193-3.
Tubulin assembly studies with GTP alpha S diastereoisomers have shown that there is stereoselectivity at the alpha-phosphate binding region of tubulin. GTP alpha S(Sp) bound tighter than GTP alpha S(Rp) and promoted nucleation and assembly better than GTP and GTP alpha S(Rp). ATP and dATP have been reported to bind weakly to tubulin and to be less effective than GTP and dGTP in promoting tubulin assembly. This study was done to learn if ATP alpha S(Sp) and dATP alpha S(Sp) are good promoters of tubulin assembly and to compare these ATP thiotriphosphate analogues to the corresponding GTP analogues in tubulin assembly. Studies were also done with ATP alpha S(Rp), GTP, ATP beta S(Sp) and ATP gamma S. At least three cycles of tubulin (25 microM) assembly-disassembly were found with 1 mM ATP alpha S(Sp) and dATP alpha S(Sp) and both nucleotides were incorporated and hydrolyzed in the polymers. Less dATP alpha S(Sp) (25 microM) than ATP alpha S(Sp) (100 microM) promoted assembly to 50% of the maximum value. The critical concentrations (Cc) for assembly with 1 mM nucleotide were low for ATP alpha S(Sp) (3 microM) and dATP alpha S(Sp) (2 microM) and compared favorably with GTP (5 microM), GTP alpha S(Sp) (2 microM) and dGTP alpha S(Sp) (1 microM). Both 1 mM ATP and dATP were poor promoters of tubulin assembly and were not detected in the polymers. The predominant structures induced by 1 mM (ATP alpha S(Sp) and dATP alpha S(Sp) were bundles of sheets and microtubules, which were more stable to the cold and to Ca(II) than microtubules assembled with GTP, ATP or dATP. ATP alpha S(Rp) (1 mM) did not promote assembly suggesting that there is stereoselectivity at the ATP alpha S alpha-phosphate binding region of tubulin as there is with GTP alpha S diastereoisomers. ATP alpha S(Sp) and dATP alpha S(Sp) mimic GTP alpha S(Sp) and dGTP alpha S(Sp) in tubulin assembly since all four nucleotides promote bundles of tubulin in buffer with glycerol, and the deoxy nucleotides have lower Cc, shorter lags and faster rates for tubulin assembly.
利用GTPαS非对映异构体进行的微管蛋白组装研究表明，在微管蛋白的α-磷酸结合区域存在立体选择性。GTPαS(Sp)比GTPαS(Rp)结合更紧密，并且比GTP和GTPαS(Rp)能更好地促进成核和组装。据报道，ATP和dATP与微管蛋白的结合较弱，在促进微管蛋白组装方面不如GTP和dGTP有效。本研究旨在了解ATPαS(Sp)和dATPαS(Sp)是否是微管蛋白组装的良好促进剂，并将这些三磷酸硫代腺苷类似物与微管蛋白组装中相应的GTP类似物进行比较。还对ATPαS(Rp)、GTP、ATPβS(Sp)和ATPγS进行了研究。发现1 mM ATPαS(Sp)和dATPαS(Sp)时，微管蛋白（25 μM）至少经历了三个组装-解聚循环，并且两种核苷酸都被掺入聚合物中并发生水解。与ATPαS(Sp)（100 μM）相比，较少的dATPαS(Sp)（25 μM）能将组装促进到最大值的50%。对于1 mM核苷酸的组装，ATPαS(Sp)（3 μM）和dATPαS(Sp)（2 μM）的临界浓度（Cc）较低，与GTP（5 μM）、GTPαS(Sp)（2 μM）和dGTPαS(Sp)（1 μM）相比具有优势。1 mM ATP和dATP都是微管蛋白组装的不良促进剂，并且在聚合物中未检测到。1 mM（ATPαS(Sp)和dATPαS(Sp)诱导的主要结构是片层束和微管，与用GTP、ATP或dATP组装的微管相比，它们对冷和Ca(II)更稳定。1 mM ATPαS(Rp)不促进组装，这表明在微管蛋白的ATPαSα-磷酸结合区域存在立体选择性，就像GTPαS非对映异构体一样。ATPαS(Sp)和dATPαS(Sp)在微管蛋白组装中模拟GTPαS(Sp)和dGTPαS(Sp)，因为所有四种核苷酸在含有甘油的缓冲液中都能促进微管蛋白束的形成，并且脱氧核苷酸的Cc较低、滞后时间较短且微管蛋白组装速率较快。
