Molecular mechanisms for changes in microtubule dynamics

以前在爪蟾卵的M期提取物中检测到切断稳定微管的活性，但在间期提取物中没有检测到。我们以前报道过从爪蟾M期卵中鉴定和纯化微管切断因子。它是一种由56-kDa多肽亚基（p56）组成的同源寡聚蛋白，可以缓慢切断稳定的微管。蛋白微测序表明p56是一种以前未被发现的蛋白。近年来，我们从爪蟾M相卵中发现了两种微管切断活性，并通过序列色谱法对其进行了纯化。新纯化的因子具有一个48 kda （p48）的单多肽，并且以不依赖atp的方式非常迅速地切断微管。发现p48诱导的微管断裂不伴有微管的显著解聚。p48的这些特征与katanin明显不同，katanin是一种切断和分解稳定微管的atp酶，Vale和同事已经在海胆卵中发现了katanin。我们制备了抗p48抗体，该抗体在总细胞裂解物中特异性地与p48反应。免疫印迹分析显示，p48在爪蟾和大鼠的几乎所有组织中普遍存在。此外，经p48抗体固定珠层析纯化的p48显示出微管切断活性，证实了p48作为切断因子的身份。纯化后的p48与源自中心体的细胞质微管网络反应时，微管发生快速断裂。p48为EF1-alpha。这些结果表明，ef1 - α可能在细胞中起微管切断因子的作用。

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.