Molecular mechanisms for changes in microtubule dynamics

一个活动，切断稳定的微管先前已被检测到M期提取物，但不是在间期提取物，非洲爪蟾卵。我们以前报道的识别和纯化从非洲爪蟾M期卵的微管切断因子。它是一种由56 kDa多肽亚基（p56）组成的同源寡聚蛋白，可缓慢切断稳定的微管。蛋白质微测序表明p56是一种以前未鉴定的蛋白质。最近，我们从非洲爪蟾M期卵中发现了微管断裂活性，并通过连续层析将其纯化至接近均一。新纯化的因子有一个单一的多肽48-kDa（p48），并切断微管非常迅速地在一个ATP-非依赖性的方式。结果发现，由p48诱导的微管切断并不伴随着微管的显著解聚。p48的这些特征与katanin的特征明显不同，katanin是一种切断和分解稳定微管的ATP酶，Vale及其同事在海胆卵中发现了这种酶。我们生产了抗p48抗体，其与总细胞裂解物中的p48特异性反应。免疫印迹结果表明，p48蛋白在非洲爪蟾和大鼠的几乎所有组织中普遍存在。此外，通过在抗p48抗体固定的珠上层析纯化的p48表现出微管切断活性，证实了p48作为切断因子的身份。当纯化的p48与从中心体发出的细胞质微管网络反应时，微管发生快速断裂。p48为EF 1-alpha。这些结果表明，EF 1-α可能在细胞中作为微管切断因子发挥作用。

An activity that severs stable microtubules has previously been detected in M phase extracts, but not in interphase extracts, of Xenopus eggs. We reported previously the identification and purification from Xenopus M phase eggs of a microtubule-severing factor. It is a homo-oligomeric protein composed of 56-kDa polypeptide subunit (p56) that can sever stable microtubules slowly. Protein microsequencing indicated that p56 is a previously unidentified protein. Recently, we found andther microtubule-severing activity from Xenopus M phase eggs and purified it to near homogeneity by sequential chromatography. The newly purified factor had a single polypeptide of 48-kDa (p48) , and severed microtubules very rapidly in an ATP-independent manner. It was found that microtubule severing induced by p48 is not accompanied by significant depolymerization of microtubules. These characteristics of p48 are clearly different from those of katanin, an ATPase that severs and disassembles stable microtubules, which has been identified in sea urchin eggs by Vale and co-workers. We produced anti-p48 antibody that reacts with p48 specifically in total cell lysates. The immunoblotting with this anti-p48 antibody revealed the universal existence of p48 in almost all tissues of Xenopus and rat. Furthermore, p48 which was purified by chromatographya on anti-p48 antibody-fixed beads exhibited a microtubule severing activity, confirming the identity of p48 as a severing factor. When purified p48 was reacted with cytoplasmic microtubule network emanating from centrosomes, rapid breaking of microtubules occurred. p48 was revealed to be EF1-alpha. These results suggest that EF1-alpha may function as a microtubule-severing factor in cells.