Hogan C J, Wein H, Wordeman L, Scholey J M, Sawin K E, Cande W Z
Department of Molecular and Cell Biology, University of California, Berkeley 94720.
Proc Natl Acad Sci U S A. 1993 Jul 15;90(14):6611-5. doi: 10.1073/pnas.90.14.6611.
Isolated central spindles or spindles in detergent-permeabilized cells from the diatom Cylindrotheca fusiformis can undergo ATP-dependent reactivation of spindle elongation in vitro. We have used a peptide antibody raised against a 10-amino acid portion common to the kinesin superfamily motor domain to look for kinesin-like motor activity during anaphase B of mitosis. The peptide antibody localizes to central spindles. Upon ATP reactivation of spindle elongation, antigens recognized by the antibody are associated exclusively with the central spindle midzone where antiparallel microtubules of each half-spindle overlap. The antibody recognizes several polypeptides by immunoblot using isolated spindle extracts. One of these polypeptides behaves like kinesin with respect to nucleotide-specific binding to and release from taxol-stabilized microtubules. Preincubation of the spindle model with the peptide antibody inhibits subsequent ATP reactivation of spindle elongation. Coincubation of the peptide antibody with peptide antigen rescues spindle function. These results support a role for kinesin-related protein(s) in spindle elongation (anaphase B) of mitosis and suggest that one or several polypeptides that we have identified in spindle extracts may fulfill this function.
从硅藻纺锤硅藻中分离出的中心纺锤体或经去污剂通透处理的细胞中的纺锤体，在体外可经历ATP依赖的纺锤体伸长再激活过程。我们使用了一种针对驱动蛋白超家族运动结构域共有的10个氨基酸部分产生的肽抗体，来寻找有丝分裂后期B阶段类似驱动蛋白的运动活性。该肽抗体定位于中心纺锤体。在纺锤体伸长的ATP再激活过程中，抗体识别的抗原仅与中心纺锤体中区相关，每个半纺锤体的反平行微管在该区重叠。通过使用分离的纺锤体提取物进行免疫印迹，该抗体识别几种多肽。其中一种多肽在与紫杉醇稳定的微管的核苷酸特异性结合和释放方面表现得像驱动蛋白。用肽抗体对纺锤体模型进行预孵育可抑制随后纺锤体伸长的ATP再激活。肽抗体与肽抗原共同孵育可挽救纺锤体功能。这些结果支持了驱动蛋白相关蛋白在有丝分裂纺锤体伸长（后期B）中的作用，并表明我们在纺锤体提取物中鉴定出的一种或几种多肽可能履行这一功能。
