Gillespie P G, Hudspeth A J
Department of Cell Biology and Neuroscience, University of Taxas Southwestern Medical Center, Dallas 75235-9039.
J Cell Biol. 1991 Feb;112(4):625-40. doi: 10.1083/jcb.112.4.625.
The small number of hair cells in auditory and vestibular organs severely impedes the biochemical characterization of the proteins involved in mechano-electrical transduction. By developing an efficient and clean "twist-off" method of hair bundle isolation, and by devising a sensitive, nonradioactive method to detect minute quantities of protein, we have partially overcome this limitation and have extensively classified the proteins of the bundles. To isolate hair bundles, we glue the saccular macula of the bullfrog to a glass coverslip, expose the tissue to a molten agarose solution, and allow the agarose to solidify to a firm gel. By rotating the gel disk with respect to the fixed macula, we isolate the hair bundles by shearing them at their mechanically weak bases. The plasma membranes of at least 80% of the stereocilia reseal. To visualize the proteins of the hair bundle, we covalently label them with biotin, separate them by SDS-PAGE, and transfer them to a charged nylon membrane. We can detect less than 500 fg of protein by probing the membrane with streptavidin-alkaline phosphatase and detecting the chemiluminescent product from the hydrolysis of the substrate 3-(4-methoxyspiro-(1,2-dioxetane-3,2'-tricyclo-[3.3.1. 1(3.7)]decan)-4-yl) phenyl phosphate (AMPPD). These techniques reveal a distinct constellation of proteins in and associated with hair bundles. Several proteins, such as calmodulin, calbindin, actin, tubulin, and fimbrin, have previously been described. A second class of proteins in the preparation appears to be derived from extracellular sources. Finally, several heretofore undescribed bundle proteins are identified and characterized by their membrane topology, subcellular localization, and glycosidase and protease sensitivities.
听觉和前庭器官中的毛细胞数量很少，这严重阻碍了对机械电转导相关蛋白质的生化特性研究。通过开发一种高效且无污染的毛束分离“拧下”方法，并设计一种灵敏的非放射性方法来检测微量蛋白质，我们部分克服了这一限制，并对毛束中的蛋白质进行了广泛分类。为了分离毛束，我们将牛蛙的球囊斑粘在玻璃盖玻片上，将组织暴露于熔融的琼脂糖溶液中，然后让琼脂糖凝固成坚固的凝胶。通过相对于固定的黄斑旋转凝胶盘，我们在毛束机械强度较弱的基部将其剪断，从而分离出毛束。至少80%的静纤毛的质膜会重新封闭。为了观察毛束中的蛋白质，我们用生物素对它们进行共价标记，通过SDS-PAGE分离它们，然后将它们转移到带电荷的尼龙膜上。我们用链霉亲和素-碱性磷酸酶探测膜，并检测底物3-(4-甲氧基螺-(1,2-二氧杂环丁烷-3,2'-三环-[3.3.1.1(3.7)]癸烷)-4-基)苯基磷酸酯(AMPPD)水解产生的化学发光产物，从而能够检测到少于500 fg的蛋白质。这些技术揭示了毛束中以及与毛束相关的一组独特蛋白质。此前已经描述了几种蛋白质，如钙调蛋白、钙结合蛋白、肌动蛋白、微管蛋白和丝束蛋白。制备物中的第二类蛋白质似乎来源于细胞外。最后，通过膜拓扑结构、亚细胞定位以及糖苷酶和蛋白酶敏感性，鉴定并表征了几种此前未描述的毛束蛋白质。
