Ramanathan R, Adoutte A, Dute R R
Biochim Biophys Acta. 1981 Mar 6;641(2):349-65. doi: 10.1016/0005-2736(81)90491-0.
The swimming behavior of Paramecium is regulated by an excitable membrane that covers the body and cilia of the protozoan. In order to obtain information on the topology and function of ciliary membrane proteins, Paramecia were treated with trypsin, chymotrypsin or pronase and the effects of these proteases were analyzed using electron microscopy, gel electrophoresis of ciliary fractions and behavioral tests. At the concentrations used, trypsin and chymotrypsin had little or no effect on the cells while pronase removed the cell surface coat, visible as fuzzy material covering the cell membrane. The same pronase treatment caused the specific removal of a high molecular weight protein (250 000), as judged by sodium dodecyl sulfate polyacrylamide gel electrophoresis. This protein, the 'immobilization antigen', constitutes the major protein of the ciliary membrane. Although the immobilization antigen was removed (or markedly decreased), no marked and reproducible difference was observed in the swimming behavior of the treated cells. We also determined the effects of proteases on isolated ciliary fractions to explore the sidedness of ciliary membrane proteins. A set of proteins relatively resistant to protease digestion was identified; they may be intrinsic membrane proteins.
草履虫的游动行为受覆盖其身体和纤毛的可兴奋膜调控。为获取有关纤毛膜蛋白拓扑结构和功能的信息，用胰蛋白酶、胰凝乳蛋白酶或链霉蛋白酶处理草履虫，并通过电子显微镜、纤毛组分的凝胶电泳及行为测试分析这些蛋白酶的作用。在所使用的浓度下，胰蛋白酶和胰凝乳蛋白酶对细胞几乎没有影响，而链霉蛋白酶去除了细胞表面被膜，在电子显微镜下可见其为覆盖细胞膜的模糊物质。通过十二烷基硫酸钠聚丙烯酰胺凝胶电泳判断，相同的链霉蛋白酶处理导致一种高分子量蛋白（250 000）被特异性去除。这种蛋白，即“固定抗原”，是纤毛膜的主要蛋白。尽管固定抗原被去除（或显著减少），但在处理过的细胞的游动行为中未观察到明显且可重复的差异。我们还测定了蛋白酶对分离的纤毛组分的作用，以探究纤毛膜蛋白的方向性。鉴定出一组对蛋白酶消化相对抗性的蛋白；它们可能是内在膜蛋白。
