Skerrow C J, Matoltsy A G
J Cell Biol. 1974 Nov;63(2 Pt 1):515-23. doi: 10.1083/jcb.63.2.515.
A method is reported for the isolation of desmosomes in a high yield and of a purity suitable for biochemical analysis. The procedure utilizes the selective solubilizing action of citric acid-sodium citrate (CASC) buffer, pH 2.6, on the non-cornified layers of cow nose epidermis, followed by discontinuous sucrose density gradient centrifugation. Electron microscopy with both thin sections of pellets and unfixed spread preparations reveals that after centrifugation, desmosomes are located mainly at the 55-60% sucrose interface. In the desmosome preparation thus obtained, the characteristic desmosome structure is well preserved, showing the midline, unit membranes, and dense plaques. Furthermore, removal of the epidermal filament bundles by the solubilizing action of CASC buffer has revealed a finely filamentous layer on the cytoplasmic surface of the plaques. The dimensions, location, and appearance of this layer correspond with those of the "connecting component" which has been previously suggested as being responsible for the attachment of epidermal filament bundles to the desmosome.
本文报道了一种高产且纯度适合生化分析的桥粒分离方法。该方法利用pH 2.6的柠檬酸 - 柠檬酸钠（CASC）缓冲液对牛鼻表皮非角质化层的选择性溶解作用，随后进行不连续蔗糖密度梯度离心。对沉淀的薄切片和未固定的铺展制剂进行电子显微镜观察发现，离心后，桥粒主要位于55 - 60%的蔗糖界面处。在如此获得的桥粒制剂中，特征性的桥粒结构保存良好，显示出中线、单位膜和致密斑。此外，通过CASC缓冲液的溶解作用去除表皮细丝束后，在致密斑的细胞质表面发现了一层精细的丝状层。该层的尺寸、位置和外观与先前提出的负责表皮细丝束与桥粒附着的“连接成分”一致。
