Stephens R E
Biochemistry. 1977 May 17;16(10):2047-58. doi: 10.1021/bi00629a001.
The membrane of both sperm flagella and gill cilia of the scallop Aequipecten irradians may be selectively solubilized in 1% Triton X-100, 30 mM tris(hydroxymethyl)-aminomethane hydrochloride (Tris-HCl), pH8, and 3 mM MgCl2, leaving the axoneme totally intact. This membrane fraction represents about 20% of the total protein of the respective organelle. Analysis of the flagellar membrane by sodium dodecyl sulfate (NaDodSO4)-polyacrylamide gel electrophoresis revealed one principal protein component, periodic acid-Schiff (PAS) positive and migrating with an apparent molecular weight of 250,000. The remaining minor proteins, none of them PAS positive, accounted for less than one-third of the total flagellar membrane fraction. Analysis of the ciliary membrane also revealed one major protein component, weakly PAS positive and migrating with an apparent molecular weight of 55,000. The remaining minor proteins represented about one-third of the total ciliary membrane fraction; two components with molecular weights of 100,000 and 40,000 predominated. The latter could be substantially reduced by purification of the cilia on a sucrose density gradient and was assumed to be actin, derived by vesiculation of the brush border during deciliation. The principal ciliary membrane protein, that of 55,000 daltons, was resolved into two equimolar components on NaDodSO4-Tris-glycine-polyacrylamide gels, comigrating with the alpha and beta chains of outer fiber tubulin. S-carboxymethylation caused increased splitting of the two components and concomitant migration with similarly treated ciliary tubulin. Preparative gel electrophoresis yielded separate components whose cyanogen bromide cleavage products were virtually identical in size distribution with those obtained from outer fiber alpha and beta chains; tryptic peptides corresponded almost exactly to those of authentic tubulin subunits but certain positional differences indicated possible side chain modification. At 25 degrees C both whole cilia and its solubilized membrane fraction bound colchicine while whole flagella and the 9 + 2 axoneme from either organelle did not. Thus certain molluscan flagellar membranes primarily contain a 250,000-dalton glycoprotein but ciliary membranes have a modified tubulin as the major protein component. At an electron microscopic level, flagellar membranes have a distinct trilamellar "unit membrane" structure while ciliary membranes appear thinner and considerably less distinct, perhaps reflecting the protein compositional differences in the membranes of these other wise morpholobically identical organelles.
海湾扇贝（Aequipecten irradians）精子鞭毛和鳃纤毛的膜可在含1% Triton X - 100、30 mM三（羟甲基）氨基甲烷盐酸盐（Tris - HCl）（pH8）和3 mM MgCl₂的溶液中被选择性溶解，而轴丝则完全保持完整。该膜部分约占相应细胞器总蛋白的20%。通过十二烷基硫酸钠（NaDodSO₄）-聚丙烯酰胺凝胶电泳对鞭毛膜进行分析，发现一种主要蛋白质成分，呈过碘酸 - 希夫（PAS）阳性，表观分子量为250,000。其余次要蛋白质均无PAS阳性反应，占鞭毛膜总部分的比例不到三分之一。对纤毛膜的分析也揭示了一种主要蛋白质成分，呈弱阳性PAS反应，表观分子量为55,000。其余次要蛋白质约占纤毛膜总部分的三分之一；分子量为100,000和40,000的两种成分占主导。通过在蔗糖密度梯度上纯化纤毛，后者可大幅减少，并被认为是肌动蛋白，是在脱纤毛过程中刷状缘泡化产生的。主要的纤毛膜蛋白，即55,000道尔顿的蛋白，在NaDodSO₄ - Tris - 甘氨酸 - 聚丙烯酰胺凝胶上可分解为两个等摩尔成分，与外纤维微管蛋白的α链和β链共同迁移。S - 羧甲基化导致这两种成分的分离增加，并与经类似处理的纤毛微管蛋白一起迁移。制备性凝胶电泳产生了单独的成分，其溴化氰裂解产物在大小分布上与从外纤维α链和β链获得的产物几乎相同；胰蛋白酶肽几乎与真实微管蛋白亚基的肽完全对应，但某些位置差异表明可能存在侧链修饰。在25℃时，整个纤毛及其溶解的膜部分结合秋水仙素，而整个鞭毛以及来自任何一种细胞器的9 + 2轴丝则不结合。因此，某些软体动物的鞭毛膜主要含有一种250,000道尔顿的糖蛋白，而纤毛膜则以一种修饰的微管蛋白作为主要蛋白质成分。在电子显微镜水平上，鞭毛膜具有独特的三层“单位膜”结构，而纤毛膜看起来更薄且明显不那么清晰，这可能反映了这些在形态上相同的细胞器的膜在蛋白质组成上的差异。
