Lindner R, Ungewickell E
Max-Planck-Institut für Biochemie, Martinsried bei München, Germany.
J Biol Chem. 1992 Aug 15;267(23):16567-73.
In search of hitherto undiscovered coat proteins, clathrin-associated proteins (APs) from coated vesicles of bovine brain were affinity-purified by one cycle of coat assembly and fractionated by gel filtration on Superose 6. Immunochemical and gel electrophoretic analysis of the fractions revealed, besides AP180, auxilin, HA1, and HA2, a component with M(r) approximately 140,000. This protein (p140) is present in coated vesicles in about equimolar proportion to auxilin. The contribution of HA1, HA2, AP180, and auxilin to the total assembly activity in the Tris-soluble coat protein fraction were quantitatively analyzed by measuring the reduction in activity when each protein was removed from the mixture by immunoaffinity chromatography. It was found that AP180 accounts for 61% and HA2 for 33% of the activity, whereas auxilin, HA1, and p140 made a negligible contribution. Based on the relative molar concentration of APs in the coat protein fraction, AP180 is about 4 times more active in promoting clathrin assembly than are HA2 or the other APs.
为了寻找迄今未被发现的被膜蛋白，通过一轮被膜组装对来自牛脑被膜小泡的网格蛋白相关蛋白（APs）进行亲和纯化，并在Superose 6上进行凝胶过滤分级分离。对这些级分的免疫化学和凝胶电泳分析显示，除了AP180、辅助蛋白、HA1和HA2外，还有一种分子量约为140,000的成分。这种蛋白质（p140）在被膜小泡中的含量与辅助蛋白的含量大致相等。通过免疫亲和色谱法从混合物中去除每种蛋白质时，测量活性的降低，定量分析了HA1、HA2、AP180和辅助蛋白对Tris可溶性被膜蛋白级分中总组装活性的贡献。结果发现，AP180占活性的61%，HA2占33%，而辅助蛋白、HA1和p140的贡献可忽略不计。根据被膜蛋白级分中APs的相对摩尔浓度，AP180在促进网格蛋白组装方面的活性比HA2或其他APs高约4倍。
