Sandoval I V, MacDonald E, Jameson J L, Cuatrecasas P
Proc Natl Acad Sci U S A. 1977 Nov;74(11):4881-5. doi: 10.1073/pnas.74.11.4881.
Incubation of 48,000 X g rat brain supernatants for 30 min at 37 degrees with 1-2 mM guanylyl 5'-methylenediphosphonate [Gmp(CH2)pp] results in polymerization of 95-98% of the tubulin present. This is considerably more than the 50% polymerization that can be achieved with the natural nucleotide, GTP, under optimal conditions. Gmp(CH2)pp is also much more effective than GTP in inducing polymerization of purified tubulin. Assembly of microtubules with Gmp(CH2)pp occurs at tubulin concentrations one-third of those possible with GTP. Furthermore, with Gmp(CH2)pp, microtubule assembly does not require the high molecular weight basic proteins needed with GTP. Polymerization of tubulin by Gmp(CH2)pp is neither prevented nor reversed by concentrations of calcium (2 mM) that can either prevent microtubule assembly or disrupt already formed microtubules if the nucleotide used is GTP or guanylyl imidodiphosphate. When Ca2+ is added before or after microtubule assembly, electron microscopy of the Gmp(CH2)pp preparations reveals normal microtubules turning into twisted ribbons. Low temperature (4 degrees) can both prevent and disrupt the tubulin assembled Gmp(CH2)pp although disruption proceeds much more slowly when GTP is used.
将48,000倍重力离心得到的大鼠脑上清液在37摄氏度下与1 - 2 mM鸟苷酰5'-亚甲基二磷酸酯[Gmp(CH2)pp]温育30分钟，会使95 - 98%的微管蛋白发生聚合。这比在最佳条件下使用天然核苷酸GTP所能实现的50%聚合率要高得多。在诱导纯化的微管蛋白聚合方面，Gmp(CH2)pp也比GTP有效得多。用Gmp(CH2)pp组装微管时，所需的微管蛋白浓度仅为使用GTP时的三分之一。此外，使用Gmp(CH2)pp时，微管组装不需要GTP所需的高分子量碱性蛋白。如果使用的核苷酸是GTP或鸟苷酰亚氨基二磷酸，2 mM的钙浓度能够阻止微管组装或破坏已形成的微管，但Gmp(CH2)pp介导的微管蛋白聚合既不会被其阻止，也不会被其逆转。当在微管组装之前或之后添加Ca2+时，Gmp(CH2)pp制剂的电子显微镜观察显示正常的微管会变成扭曲的带。低温（4摄氏度）既能阻止也能破坏由Gmp(CH2)pp组装的微管蛋白，不过当使用GTP时，破坏过程要慢得多。
