Job D, Rauch C T, Fischer E H, Margolis R L
Biochemistry. 1982 Feb 2;21(3):509-15. doi: 10.1021/bi00532a015.
A substantial subpopulation of mammalian brain crude extract microtubules is resistant to cold-temperature disassembly. We propose here that microtubules are rendered cold stable by rare substoichiometric blocks. Mild shearing of rat brain cold-stable microtubules makes them largely cold labile. In addition, cold-stable microtubules can be destabilized by exposure to low concentrations of calmodulin (5 microM) in the presence of calcium at 0 degree C. Cold-disassembled microtubule protein, obtained from sheared or calmodulin-treated cold-stable preparations, re-forms a cold-stable subpopulation upon reassembly. These observations allow strategies for the recycling purification of cold-stable microtubules. Comparison of purified cold-labile and cold-stable material by gel electrophoresis shows enrichment for a few unique polypeptides, of 135, 70-82, and 56 kilodaltons, in the cold-stable preparation. The 64-kilodalton "switch protein", previously identified as uniquely dephosphorylated in cold-stable microtubules, is equally represented in recycled cold-stable and cold-labile microtubule preparations. Furthermore, when disassembled, cold-stable microtubule proteins are passed through a calmodulin affinity column on which the polypeptides characteristic of cold-stable microtubules are specifically retained, the breakthrough (unbound) material repolymerizes into cold-labile microtubules only. Based on the above data, a model is presented in which microtubules are rendered cold stable by the presence of substoichiometric, calmodulin-sensitive blocks that randomly reshuffle upon reassembly of cold-stable microtubules.
哺乳动物脑粗提物中的大量微管亚群对低温解聚具有抗性。我们在此提出，微管通过罕见的亚化学计量阻断而变得对冷稳定。对大鼠脑冷稳定微管进行温和剪切会使其在很大程度上变得对冷不稳定。此外，在0摄氏度的钙存在下，冷稳定微管可通过暴露于低浓度的钙调蛋白（5微摩尔）而变得不稳定。从剪切或钙调蛋白处理的冷稳定制剂中获得的冷解聚微管蛋白在重新组装后会重新形成一个冷稳定亚群。这些观察结果为冷稳定微管的循环纯化提供了策略。通过凝胶电泳对纯化的冷不稳定和冷稳定材料进行比较，结果显示冷稳定制剂中富集了一些独特的多肽，分子量分别为135、70 - 82和56千道尔顿。先前被鉴定为在冷稳定微管中唯一去磷酸化的64千道尔顿“开关蛋白”，在循环的冷稳定和冷不稳定微管制剂中含量相同。此外，当冷稳定微管蛋白解聚后通过钙调蛋白亲和柱时，冷稳定微管特有的多肽会特异性保留在柱上，而穿透（未结合）的材料只会重新聚合成冷不稳定微管。基于上述数据，我们提出了一个模型，其中微管通过亚化学计量的、对钙调蛋白敏感的阻断的存在而变得对冷稳定，这些阻断在冷稳定微管重新组装时会随机重新排列。
