ANALYSIS OF VERTEBRATE GAP JUNCTION PROTEIN
ANALYSIS OF VERTEBRATE GAP JUNCTION PROTEIN
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DOI：
10.1002/j.1460-2075.1983.tb01611.x
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发表时间：
1983-01-01
期刊：
影响因子：
11.4
通讯作者：
PITTS, JD
中科院分区：
文献类型：
--
作者：
FINBOW, ME;SHUTTLEWORTH, J;PITTS, JD
描述了一种纯化间隙连接的新方法，该方法依赖于使用 Triton X-100 提取细胞单层或组织匀浆。来自多种脊椎动物来源的连接制剂的 SDS[十二烷基硫酸钠]-聚丙烯酰胺凝胶电泳 (PAGE) 上的主条带的表观分子量为 16,000 (16 K)。 4 种不同实验方法的结果提供了 16 K 蛋白连接起源的进一步证据。连接处在 1.195 g/cm3 的碘化钾密度梯度中形成一条尖锐的带，并且 16 K 蛋白是该浮力密度分数中唯一可检测到的带。通过增加 SDS 浓度（范围为 0.1-0.5%），连接逐渐溶解，并且连接结构的溶解（通过 EM 观察）与 16 K 蛋白的释放平行。完整连接处的戊二醛固定可交联 16 K 蛋白。 16K蛋白的可回收量与部分肝切除后再生的断奶大鼠肝脏和暴露于4.β.-佛波醇12-肉豆蔻酸酯13-乙酸酯的[仓鼠肺]V79细胞培养物中间隙连接区域的已知变化相关。
A new method for the purification of gap junctions is described which depends on the extraction of cell monolayers or tissue homogenates with Triton X-100. The major band on SDS[sodium dodecyl sulfate]-polyacrylamide gel electrophoresis (PAGE) of junctional preparations from a variety of vertebrate sources has an apparent MW of 16,000 (16 K). Further evidence for the junctional origin of the 16 K protein is provided by the results of 4 different experimental approaches. The junctions form a sharp band in potassium iodide density gradients at 1.195 g/cm3 and the 16 K protein is the only detectable band in fractions of this bouyant density. The junctions are progressively solubilized by increasing concentrations of SDS (in the range 0.1-0.5%) and the dissolution of the junctional structure (observed by EM) parallels the release of the 16 K protein. Glutaraldehyde fixation of intact junctions cross-links the 16 K protein. The recoverable amount of the 16 K protein correlates with known changes in gap junctional area in the regenerating weanling rat liver after partial hepatectomy and in [hamster lung] V79 cell cultures exposed to 4.beta.-phorbol 12-myristate 13-acetate.