Black M M, Baas P W, Humphries S
Department of Anatomy, Temple University School of Medicine, Philadelphia, Pennsylvania 19140.
J Neurosci. 1989 Jan;9(1):358-68. doi: 10.1523/JNEUROSCI.09-01-00358.1989.
The majority of the alpha-tubulin in cultured neurons is acetylated (Black and Keyser, 1987). The present studies examine the relationships of the acetylation and deacetylation reactions to tubulin assembly and disassembly in intact neurons. Extraction assays which separate assembled and unassembled tubulin pools reveal that greater than or equal to 99% of the total acetylated, as well as newly acetylated, tubulin is cytoskeletal associated. Treatment of neurons with depolymerizing drugs results in a progressive decrease in the levels of total tubulin in polymer and a corresponding increase in the levels of soluble tubulin. These drugs also cause a progressive decrease in the levels of acetylated alpha-tubulin in polymer that closely parallels in rate and extent that of total alpha-tubulin. However, there is no corresponding increase in soluble acetylated tubulin. Because the total levels of alpha-tubulin remain unchanged during drug treatment, the decrease in levels of acetylated alpha-tubulin during depolymerization must reflect its rapid conversion to nonacetylated alpha-tubulin. These findings suggest alpha-tubulin is acetylated in the polymeric form and that deacetylation is closely coupled to depolymerization. The close coupling between alpha-tubulin deacetylation and depolymerization provided a means of estimating the rate at which subunits cycle off microtubules in intact neurons. Acetate turnover on tubulin in intact neurons was determined both by pulse-chase protocols with 3H-acetate and by measuring the loss of acetylated subunits (using quantitative immunoblotting) under conditions of net microtubule depolymerization induced by colchicine. Both methods yielded similar results. Acetate turnover occurred biphasically; 30-50% of the acetate on tubulin turns over with a t1/2 of 1.5-2 hr, and the remaining half or more turns over with a t1/2 of 5-10 hr. We suggest that these kinetically distinguishable pools of acetylated alpha-tubulin reflect distinct pools of acetylated microtubules that differ in their average rates of subunit turnover.
培养的神经元中，大多数α-微管蛋白是乙酰化的（布莱克和凯泽，1987年）。本研究探讨了乙酰化和去乙酰化反应与完整神经元中微管蛋白组装和解聚的关系。分离已组装和未组装微管蛋白池的提取分析表明，总乙酰化微管蛋白以及新乙酰化微管蛋白中，大于或等于99%与细胞骨架相关。用解聚药物处理神经元会导致聚合物中总微管蛋白水平逐渐降低，可溶性微管蛋白水平相应升高。这些药物还会导致聚合物中乙酰化α-微管蛋白水平逐渐降低，其速率和程度与总α-微管蛋白密切平行。然而，可溶性乙酰化微管蛋白没有相应增加。由于药物处理期间α-微管蛋白的总水平保持不变，解聚过程中乙酰化α-微管蛋白水平的降低必然反映其迅速转化为非乙酰化α-微管蛋白。这些发现表明α-微管蛋白以聚合物形式被乙酰化，而去乙酰化与解聚密切相关。α-微管蛋白去乙酰化和解聚之间的紧密联系提供了一种估计完整神经元中亚基从微管上脱离速率的方法。通过用3H-乙酸盐进行脉冲追踪实验以及在秋水仙碱诱导的微管净解聚条件下测量乙酰化亚基的损失（使用定量免疫印迹法），确定了完整神经元中微管蛋白上乙酸盐的周转率。两种方法得出了相似的结果。乙酸盐周转呈双相性；微管蛋白上30%-50%的乙酸盐以1.5-2小时的半衰期周转，其余一半或更多以5-10小时的半衰期周转。我们认为，这些动力学上可区分的乙酰化α-微管蛋白池反映了不同的乙酰化微管蛋白池，它们的亚基平均周转率不同。
