BUNDLE BLOT PURIFICATION AND INITIAL PROTEIN CHARACTERIZATION OF HAIR CELL STEREOCILIA
BUNDLE BLOT PURIFICATION AND INITIAL PROTEIN CHARACTERIZATION OF HAIR CELL STEREOCILIA
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DOI：
10.1073/pnas.86.13.4973
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发表时间：
1989-07-01
影响因子：
11.1
通讯作者：
COREY, DP
中科院分区：
文献类型：
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作者：
SHEPHERD, GMG;BARRES, BA;COREY, DP
用硝酸纤维素贴壁法从牛蛙囊状毛细胞中分离出静纤毛。经显微镜观察，证明该方法纯度高，收率高。静纤毛蛋白SDS/PAGE分离出12-15条主带。肌动蛋白，以前确定为一个组成部分的静纤毛核心，被确定在纯化的静纤毛作为一个带comigrating与真实的肌动蛋白和鬼笔环肽标记的完整的孤立静纤毛。在纯化的静纤毛的免疫印迹中鉴定了Fimmunoglobulin。最丰富的其他蛋白质迁移在11，14，16-19，27和36 KDa。脱膜的静纤毛核心主要由对应于肌动蛋白和fimalgins的蛋白带和几种蛋白质组成，分子量从43到63 kDa。由于毛细胞的适应机制是钙敏感的，似乎定位于静纤毛，我们寻找证据的钙结合蛋白在静纤毛。钙调素和钙结合素抗体标记的静纤毛在完整的细胞。纯化静纤毛中的蛋白条带表现出与真实钙调素相同的电泳迁移率的钙离子依赖性移位，并且27-kDa条带可能代表钙结合蛋白。这些生化数据表明，静纤毛由一组相对较小的蛋白质。其中大多数，包括那些参与转导和适应，是尚未定性。纯化的静纤毛的可用性应被证明是有用的，在这些机械敏感的细胞器的结构-功能关系的进一步研究。
Stereocilia were isolated from bullfrog (Rana catesbeiana) saccular hair cells by nitrocellulose adhesion. The high purity and high yield of the preparation were demonstrated by microscopy. SDS/PAGE of stereociliary proteins resolved 12-15 major bands. Actin, previously identified as a component of the stereociliary core, was identified in purified stereocilia as a band comigrating with authentic actin and by phalloidin labeling of intact isolated stereocilia. Fimbrin was identified in immunoblots of purified stereocilia. The most abundant other proteins migrated at 11, 14, 16-19, 27, and 36 KDa. Demembranated stereociliary cores consisted primarily of protein bands corresponding to actin and fimbrin and several proteins ranging from 43 to 63 kDa. Because the adaptation mechanism in hair cells is calcium-sensitive and seems localized to stereocilia, we sought evidence for calcium-binding proteins in stereocilia. Calmodulin and calbindin antibodies labeled stereocilia in intact cells. A protein band in purified stereocilia exhibited a Ca2+-dependent shift in electrophoretic mobility identical to that of authentic calmodulin, and the 27-kDa band may represent calbindin. These biochemical data demonstrate that stereocilia consist of a relatively small set of proteins. Most of these, including those involved in transduction and adaptation, are as yet uncharacterized. The availability of purified stereocilia should prove useful in further studies of structure-function relationships in these mechanically sensitive organelles.