STRUCTURE AND BIOCHEMICAL COMPOSITION OF DESMOSOMES AND TONOFILAMENTS ISOLATED FROM CALF MUZZLE EPIDERMIS
STRUCTURE AND BIOCHEMICAL COMPOSITION OF DESMOSOMES AND TONOFILAMENTS ISOLATED FROM CALF MUZZLE EPIDERMIS
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DOI：
10.1083/jcb.79.2.427
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发表时间：
1978-01-01
影响因子：
7.8
通讯作者：
FRANKE, WW
中科院分区：
文献类型：
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作者：
DROCHMANS, P;FREUDENSTEIN, C;FRANKE, WW
通过低离子强度的中等碱性缓冲液和机械均质化，从小牛鼻口棘层细胞中分离出质膜片段与桥粒和附着的张力细丝的复合物。通过使用各种处理，包括超声处理、蔗糖梯度离心和用含有高浓度盐、尿素、柠檬酸或洗涤剂的缓冲液提取，进一步分级分离这些结构。亚组分富含桥粒张力丝复合物和张力丝片段进行了详细研究。桥粒结构，即，中线的三层膜轮廓和桥粒斑块似乎保存良好，并且对所采用的各种处理具有显著抗性。含有桥粒-张力丝复合物的组分总是由张力丝的非膜蛋白占主导地位，其表现为以约2：1：1：2：2的摩尔比存在的5个主要多肽条带（表观MW：48，000; 51，000; 58，000; 60，000; 68，000）。这些多肽条带（4）的电泳迁移率与牛蹄前角蛋白多肽的电泳迁移率相似。最大的多肽（68，000 MW）在聚丙烯酰胺凝胶中的迁移显著小于蹄前角蛋白的最大组分（. 63，000兆瓦）。鉴定了一系列次要条带，包括含碳水化合物的蛋白质。它们代表桥粒膜的成分。蛋白质结合的碳水化合物（在富含桥粒的亚级分中总共270 μ g/mg磷脂）的分析显示相对高量的葡糖胺、甘露糖、半乳糖和唾液酸。这些数据和脂质组成（例如，高胆固醇与磷脂的比率、相对高含量的鞘磷脂和神经节苷脂以及脂肪酸模式）表明桥粒膜在蛋白质和脂质组成方面是复杂的，并且具有典型的质膜特征。桥粒相关张力丝的前角蛋白丝和其他形式的中等大小的长丝的相似性进行了讨论。
Complexes of plasma membrane segments with desmosomes and attached tonofilaments were separated from the stratum spinosum cells of calf muzzle by means of moderately alkaline buffers of low ionic strength and mechanical homogenization. These structures were further fractionated by using various treatments including sonication, sucrose gradient centrifugation and extraction with buffers containing high concentrations of salt, urea, citric acid or detergents. Subfractions enriched in desmosome-tonofilament complexes and tonofilament fragments were studied in detail. The desmosome structures, i.e., of the midline, the trilaminar membrane profile and the desmosomal plaque appeared well preserved and were notably resistant to various treatments employed. Fractions containing desmosome-tonofilament complexes were invariably dominated by the nonmembranous proteins of tonofilaments which appeared as 5 major polypeptide bands (apparent MW: 48,000; 51,000; 58,000; 60,000; 68,000) present in molar ratios of approximately 2:1:1:2:2. These polypeptide bands (4) showed electrophoretic mobilities similar to those of prekeratin polypeptides from bovine hoof. The largest polypeptide (68,000 MW) migrated significantly less in polyacrylamide gels than the largest component of the hoof prekeratin (.apprx. 63,000 MW). A series of minor bands, including carbohydrate-containing proteins, were identified. They represent constituents of the desmosomal membrane. The analysis of protein-bound carbobydrates (total 270 .mu.g/mg phospholipid in desmosome-enriched subfractions) showed relatively high amounts of glucosamine, mannose, galactose and sialic acids. These data and the lipid composition (e.g., high ratio of cholesterol to phospholipids, relatively high contents of sphingomyelin and gangliosides and fatty acid pattern) indicate that the desmosomal membrane is complex in protein and lipid composition and has a typical plasma membrane character. The similarity of the desmosome-associated tonofilaments to prekeratin filaments and other forms of intermediate-sized filaments was discussed.