Adoutte A, Ramanathan R, Lewis R M, Dute R R, Ling K Y, Kung C, Nelson D L
J Cell Biol. 1980 Mar;84(3):717-38. doi: 10.1083/jcb.84.3.717.
As a first step in the biochemical analysis of membrane excitation in wild-type Paramecium and its behavioral mutants we have defined the protein composition of the ciliary membrane of wild-type cells. The techniques for the isolation of cilia and ciliary membrane vesicles were refined. Membranes of high purity and integrity were obtained without the use of detergents. The fractions were characterized by electron microscopy, and the proteins of whole cilia, axonemes, and ciliary membrane vesicles were resolved by SDS polyacrylamide gel electrophoresis and isoelectric focusing in one and two dimensions. Protein patterns and EM appearance of the fractions were highly reproducible. Over 200 polypeptides were present in isolated cilia, most of which were recovered in the axonemal fraction. Trichocysts, which were sometimes present as a minor contaminant in ciliary preparations, were composed of a very distinct set of over 30 polypeptides of mol wt 11,000--19,000. Membrane vesicles contained up to 70 polypeptides of mol wt 15,000--250,000. The major vesicle species were a high molecular weight protein (the "immobilization antigen") and a group of acidic proteins with mol wt similar to or approximately 40,000. These and several other membrane proteins were specifically decreased or totally absent in the axoneme fraction. Tubulin, the major axonemal species, occurred only in trace amounts in isolated vesicles; the same was true for Tetrahymena ciliary membranes prepared by the methods described in this paper. A protein of mol wt 31,000, pI 6.8, was virtually absent in vesicles prepared from cells in exponential growth phase, but became prominent early in stationary phase in good correlation with cellular mating reactivity. This detailed characterization will provide the basis for comparison of the ciliary proteins of wild-type and behavioral mutants and for analysis of topography and function of membrane proteins. It will also be useful in future studies of trichocysts and mating reactions.
作为对野生型草履虫及其行为突变体膜兴奋进行生化分析的第一步，我们确定了野生型细胞纤毛膜的蛋白质组成。改进了纤毛和纤毛膜囊泡的分离技术。无需使用去污剂即可获得高纯度和完整性的膜。通过电子显微镜对各组分进行表征，通过SDS聚丙烯酰胺凝胶电泳以及一维和二维等电聚焦对整个纤毛、轴丝和纤毛膜囊泡中的蛋白质进行分离。各组分的蛋白质图谱和电子显微镜外观具有高度可重复性。分离出的纤毛中存在200多种多肽，其中大部分在轴丝组分中回收。刺丝泡有时作为纤毛制剂中的少量污染物存在，由一组非常独特的30多种分子量为11,000 - 19,000的多肽组成。膜囊泡含有多达70种分子量为15,000 - 250,000的多肽。主要的囊泡种类是一种高分子量蛋白质（“固定抗原”）和一组分子量与40,000相似或约为40,000的酸性蛋白质。这些以及其他几种膜蛋白在轴丝组分中特异性减少或完全缺失。微管蛋白是轴丝的主要种类，在分离的囊泡中仅微量存在；本文所述方法制备的四膜虫纤毛膜也是如此。一种分子量为31,000、pI为6.8的蛋白质在指数生长期细胞制备的囊泡中几乎不存在，但在稳定期早期变得突出，与细胞交配反应性密切相关。这种详细的表征将为比较野生型和行为突变体的纤毛蛋白以及分析膜蛋白的拓扑结构和功能提供基础。它在未来刺丝泡和交配反应的研究中也将有用。
