STRUCTURE AND BIOCHEMISTRY OF MOUSE HEPATIC GAP-JUNCTIONS
STRUCTURE AND BIOCHEMISTRY OF MOUSE HEPATIC GAP-JUNCTIONS
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DOI：
10.1016/0022-2836(79)90391-7
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发表时间：
1979-01-01
影响因子：
5.6
通讯作者：
WEBER, K
中科院分区：
文献类型：
--
作者：
HENDERSON, D;EIBL, H;WEBER, K
本文报道了一种从小鼠肝脏分离缝隙连接的新方法。已特别注意将分离过程中蛋白质分解的影响降至最低。纯化的膜片段保留了在完整肝脏连接处发现的典型形态特征。在十二烷基硫酸钠存在下，聚丙烯酰胺凝胶电泳法显示连接处有两个主要的多肽。较丰富的物种的表观相对分子质量为26,000，次要成分为21,000。指纹图谱初步分析表明，这两种多肽在结构上具有亲缘关系。虽然不能排除21,000 mW物种的体内来源，但连接蛋白对体外蛋白质降解的敏感性表明，21,000 mW分子可能是主要成分的分解产物。将图像重建方法应用于负染孤立结的显微照片，结果表明，膜包含紧密堆积的六方晶格成分，具有明显的6重对称性。这些代表26,000兆瓦蛋白质的六角体。对通过不同程序分离的缝隙连接进行的脂质分析表明，在分离过程中使用的洗涤剂对脂质成分有很大影响。可以从结构中提取大量的磷脂，但不能从结构中提取胆固醇，而不会影响其大体形态。胆固醇可能与连接蛋白紧密结合，并可能在决定缝隙连接结构中发挥作用。
A new method for the isolation of gap junctions from mouse liver is described. Particular attention has been directed to minimizing the effects of proteolysis during isolation. The purified membrane fragments retain the typical morphological features found in junctions of the intact liver. The junctions show 2 major polypeptides upon polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The apparent MW are 26,000 for the more abundant species and 21,000 for the minor component. Preliminary protein chemical characterization by fingerprint analysis suggests that the 2 polypeptides are structurally related. While an in vivo origin of the 21,000 MW species cannot be excluded, the sensitivity of the junction proteins to proteolytic degradation in vitro suggests that the 21,000 MW molecule may be a breakdown product of the major component. Image reconstruction methods applied to micrographs of negatively stained isolated junctions show that the membrane contains a close-packed hexagonal lattice of components with marked 6-fold symmetry. These represent hexamers of the 26,000 MW protein. Lipid analysis performed on gap junctions isolated by different procedures shows that the lipid composition is strongly affected by the detergents employed during the isolation. A large amount of phospholipid, but not cholesterol, can be extracted from the structure without affecting its gross morphology. Cholesterol may be tightly bound to the junction protein and may play a role in determining the structure of the gap junction.