Cole D G, Scholey J M
Section of Molecular and Cellular Biology, University of California at Davis 95616, USA.
Biophys J. 1995 Apr;68(4 Suppl):158S-160S; discussion 160S-162S.
We have developed a biochemical screen for the identification of kinesin-related proteins (KRPs) in their natural host cells and the subsequent purification of these KRPs as native, functional multimeric complexes. The screen involves immunoblotting with pan-kinesin peptide antibodies that recognize several presumptive KRPs in cytosolic extracts; the antibodies have been used so far to monitor the purification of two bona fide kinesin-related motor protein complexes. These two KRPs were purified via AMPPNP-induced microtubule affinity binding, ATP-induced elution from AMPPNP microtubules, gel filtration fractionation, and sucrose density gradient centrifugation. KRP(85/95) from sea urchin (Strongylocentrotus purpuratus) eggs behaves as a heterotrimeric complex of 85-, 95-, and 115-kDa subunits that moves toward the plus ends of microtubule tracks at approximately 0.4 micron/s. KRP(130) from fruitfly (Drosophila melanogaster) embryos behaves as a homotetrameric complex of four 130-kDa subunits that moves toward the plus ends of microtubule tracks at approximately 0.04 micron/s. To our knowledge, KRP(85/95) and KRP(130) are the only KRPs to have been purified from native tissue as functional multimeric motor complexes.
我们开发了一种生化筛选方法，用于在天然宿主细胞中鉴定驱动蛋白相关蛋白（KRPs），并随后将这些KRPs纯化成本地功能性多聚体复合物。该筛选方法包括用泛驱动蛋白肽抗体进行免疫印迹，这些抗体可识别胞质提取物中的几种假定KRPs；到目前为止，这些抗体已用于监测两种真正的驱动蛋白相关运动蛋白复合物的纯化过程。这两种KRPs通过AMPPNP诱导的微管亲和结合、ATP诱导的从AMPPNP微管上洗脱、凝胶过滤分级分离和蔗糖密度梯度离心进行纯化。来自海胆（紫球海胆）卵的KRP(85/95)表现为一种由85 kDa、95 kDa和115 kDa亚基组成的异源三聚体复合物，以约0.4微米/秒的速度向微管轨道的正端移动。来自果蝇胚胎的KRP(130)表现为一种由四个130 kDa亚基组成的同四聚体复合物，以约0.04微米/秒的速度向微管轨道的正端移动。据我们所知，KRP(85/95)和KRP(130)是仅有的从天然组织中纯化得到的功能性多聚体运动复合物形式的KRPs。
