Hasko J A, Richardson G P
School of Biological Sciences, University of Sussex, Falmer, Brighton, U.K.
Hear Res. 1988 Sep 1;35(1):21-38. doi: 10.1016/0378-5955(88)90037-8.
The polyphenolic compound tannic acid and the cationic stains ruthenium red, Alcian blue and lanthanum chloride have been used to reinvestigate the ultrastructural organization of the tectorial membrane matrix. Tannic acid treatment reveals that the matrix both in between and within the Type A protofibril bundle system has a high degree of structural organization. The basic unit of this matrix is best described as a 'striated sheet'. These striated sheets are formed by alternating 'dark' and 'light' fibrils which run parallel to one another and lie within the plane of each sheet. In sodium based buffers both light and dark fibrils have diameters of approximately 7 nm and the distance between each dark fibril in a sheet varies from 30 to 46 nm. Dark and light fibrils are coupled by periodic, staggered cross-bridges which occur at approximately 12 nm intervals along the fibrils. Fibril diameters in tectorial membranes prepared and fixed in potassium based buffers are from 10-20% greater than they are in tectorial membranes prepared and fixed in sodium based buffers. Fine fibrils can also be resolved in the matrix with the cationic stains lanthanum chloride and ruthenium red, but the organization of these fibrils into a regular matrix structure is most clearly resolved with tannic acid treatment. The striated sheets are largely destroyed by treating the tectorial membranes with neutral trypsin and are insensitive to treatment with bacterial collagenase. In contrast, the Type A protofibril system is trypsin resistant and collagenase sensitive. Treatment of tectorial membranes with salt solutions containing either 5 nM EDTA or 5 mM EGTA and 2 mM MgCl2 results in a complete loss of organized striated sheets and the appearance of randomly dispersed fibrillar material and small particles. Re-addition of Ca2+ ions causes the striated sheets to reform, indicating that the structure can undergo at least one cycle of depolymerization and polymerization in vitro. Reduction of disulphide bonds with beta-mercaptoethanol causes a loss of structural organization similar to that observed after EDTA or EGTA treatment. The results demonstrate that the non-collagenous components of the tectorial form a matrix with a degree of organization that has been previously unrecognised.
多酚类化合物鞣酸以及阳离子染料钌红、阿尔辛蓝和氯化镧已被用于重新研究盖膜基质的超微结构组织。鞣酸处理显示，A型原纤维束系统之间和内部的基质都具有高度的结构组织。这种基质的基本单位最好描述为“横纹片”。这些横纹片由交替的“暗”和“亮”原纤维形成，它们相互平行排列并位于每张片的平面内。在基于钠的缓冲液中，亮原纤维和暗原纤维的直径均约为7nm，片中每条暗原纤维之间的距离在30至46nm之间变化。暗原纤维和亮原纤维通过周期性的、交错的横桥相连，这些横桥沿原纤维以约12nm的间隔出现。在基于钾的缓冲液中制备和固定的盖膜中的原纤维直径比在基于钠的缓冲液中制备和固定的盖膜中的原纤维直径大10 - 20%。用阳离子染料氯化镧和钌红也可以在基质中分辨出细原纤维，但用鞣酸处理能最清楚地分辨出这些原纤维组织成规则的基质结构。用中性胰蛋白酶处理盖膜会使横纹片大部分被破坏，且对细菌胶原酶处理不敏感。相比之下，A型原纤维系统对胰蛋白酶有抗性，对胶原酶敏感。用含有5 nM乙二胺四乙酸（EDTA）或5 mM乙二醇双乙醚二胺四乙酸（EGTA）和2 mM氯化镁的盐溶液处理盖膜会导致有组织的横纹片完全消失，并出现随机分散的纤维状物质和小颗粒。重新添加钙离子会使横纹片重新形成，这表明该结构在体外至少可以经历一个解聚和聚合循环。用β-巯基乙醇还原二硫键会导致结构组织丧失，类似于在EDTA或EGTA处理后观察到的情况。结果表明，盖膜的非胶原蛋白成分形成了一种具有先前未被认识到的组织程度的基质。
