Buffer conditions and non-tubulin factors critically affect the microtubule dynamic instability of sea urchin egg tubulin.
Buffer conditions and non-tubulin factors critically affect the microtubule dynamic instability of sea urchin egg tubulin.
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缓冲条件和非微管蛋白因素严重影响海胆卵微管蛋白的微管动态不稳定性。
DOI：
10.1002/cm.970210102
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发表时间：
1992
影响因子：
--
通讯作者：
Salmon,ED
中科院分区：
文献类型：
--
作者：
Simon,JR;Parsons,SF;Salmon,ED
使用视频增强微分干涉（VE-DIC）光学显微镜检查细胞质提取物中或由高度纯化的海胆卵微管蛋白组装而成的单个微管（Mts）的动态不稳定性。提取物 Mts（内源性微管蛋白 = 12.1 μM）仅显示正向生长。在切换（灾难）到快速缩短之前，伸长速度为 7.8 μm/min，平均持续时间为 1.3 分钟，在切换（救援）回伸长阶段之前，伸长速度为 13.0 μm/min，平均持续时间为 0.5 分钟。这些参数是相间 Mt 动态不稳定性的典型参数。令人惊讶的是，由纯化的海胆卵微管蛋白在标准缓冲液中组装而成的 Mts 的动态性低于报道的纯化脑微管蛋白或提取物中的 Mts 的动态。更改了缓冲区参数以尝试模拟提取 Mt 结果。 pH 缓冲剂本身（Hepes 或 Pipes）极大地改变了 Mt 动力学，但无法在高灾难频率下实现高伸长速度。 1 μM 钙的影响可以忽略不计，而将 pH 从 6.9 增加到 7.2 会刺激伸长速度。最后，在细胞质提取物的超滤液（MW cut-off <30 kD）中测定了纯化卵微管蛋白（11.9 μM）的 Mt 动力学。 Mts 以 1.2 μm/min 的速度缓慢伸长 26 分钟，然后以 11.8 μm/min 的速度快速缩短。救援频率低于未过滤的提取物，观察到负端生长，并且在微管蛋白浓度稍高时发生自组装。因此，鸡蛋提取物和细胞质必须含有非缓冲因子，能够在不自组装的情况下将伸长速度提高 6.5 倍，使灾难频率增加 20 倍，并阻止负端生长。
The dynamic instability of individual microtubules (Mts) in cytoplasmic extracts or assembled from highly purified sea urchin egg tubulin was examined using video‐enhanced, differential‐interference contrast (VE‐DIC) light microscopy. Extract Mts (endogenous tubulin = 12.1 μM) displayed only plus‐ended growth. The elongation velocity was 7.8 μm/min for an average duration of 1.3 min before switching (catastrophe) to rapid shortening, which occurred at 13.0 μm/ min for an average duration of 0.5 min before switching (rescue) back to the elongation phase. These parameters are typical of interphase Mt dynamic instability. Surprisingly, Mts assembled from purified urchin egg tubulin in standard buffers were less dynamic that those reported for purified brain tubulin or Mts in the extract. Buffer parameters were changed in an attempt to mimic the extract Mt results. The pH buffer itself, Hepes or Pipes, drastically altered Mt dynamics but could not achieve high elongation velocity with high catastrophe frequencies. Calcium at 1 μM had negligible effects, while increasing pH from 6.9 to 7.2 stimulated elongation velocity. Finally, Mt dynamics of purified egg tubulin (11.9 μM) were assayed in ultrafiltiates (MW cut‐off <30 kD) of the cytoplasmic extracts. Mts elongated slowly at 1.2 μm/min for 26 min before a catastrophe and rapid shortening at 11.8 μm/min. Rescue was less frequent than unfiltered extracts, minus‐ended growth was observed, and self‐assembly occurred at slightly higher tubulin concentrations. Therefore, the egg extracts and cytoplasm must contain non‐buffer factors which stimulate elongation velocity by 6.5‐fold without self‐assembly, increase catastrophe frequency by 20‐fold, and block minus‐ended growth.