ISOLATION OF MOUSE HEPATOCYTE GAP JUNCTIONS - PRELIMINARY CHEMICAL CHARACTERIZATION AND X-RAY-DIFFRACTION
ISOLATION OF MOUSE HEPATOCYTE GAP JUNCTIONS - PRELIMINARY CHEMICAL CHARACTERIZATION AND X-RAY-DIFFRACTION
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DOI：
10.1083/jcb.54.3.646
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发表时间：
1972-01-01
影响因子：
7.8
通讯作者：
STOECKENIUS, W
中科院分区：
文献类型：
--
作者：
GOODENOUGH, DA;STOECKENIUS, W
本文报道了一种从小鼠肝细胞中分离肝间隙连接的方法。该方法包括胶原酶消化，用洗涤剂Sarkosyl NL-97处理，超声处理，然后用蔗糖梯度洗脱。36只动物的运行产生0.1-0.5 mg蛋白质。电子显微镜与薄切片和负染色技术表明，最终的小球是一个非常纯的制备间隙连接，伴随着少量的无定形污染。十二烷基硫酸钠（SDS）增溶材料的聚丙烯酰胺凝胶电泳显示，连接处有一种主要蛋白质，表观工具wt为20，000，还有两种次要组分。薄层色谱显示一种主要磷脂和一种次要磷脂，以及一些中性脂质。低角X-射线衍射的湿和干标本显示的反射，其指数上的86 A中心到中心的六方晶格，密切对应的电子显微镜数据。干燥的样本也显示出片状衍射，对应于结的总轮廓厚度（150埃）。
A method is reported for isolating a preparation of hepatic gap junctions from the mouse. The method involves a collagenase digestion, treatment with the detergent Sarkosyl NL-97, and ultrasonication, followed by sucrose gradient nltracentrifugadon. A run with 36 animals yields 0.1-0.5 mg protein. Electron microscopy with thin-sectioning and negative staining techniques reveals that the final pellet is a very pure preparation of gap junctions, accompanied by a small amount of amorphous contamination. Polyacrylamide-gel electrophoresis of sodium dodecyl sulfate (SDS)-solubilized material shows one major protein in the junction, with an apparent tool wt of 20,000, and two minor components. Thin-layer chromatography demonstrates one major and one minor phospholipid, and some neutral lipid. Lowangle X-ray diffraction of wet and dried specimens show reflections which index on an 86 A center-to-center hexagonal lattice, corresponding closely to electron microscope data. Dried specimens also show a lamellar diffraction, corresponding to the total profile thickness of the junction (150 A).