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 ultracentrifugation. 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 mol wt of 20,000, and two minor components. Thin-layer chromatography demonstrates one major and one minor phospholipid, and some neutral lipid. Low-angle 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).
THE ISOLATION OF MOUSE HEPATOCYTE GAP JUNCTIONS : Preliminary Chemical Characterization and X-Ray Diffraction
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Daniel A. Goodenough, Walther Stoeckenius; THE ISOLATION OF MOUSE HEPATOCYTE GAP JUNCTIONS : Preliminary Chemical Characterization and X-Ray Diffraction . J Cell Biol 1 September 1972; 54 (3): 646–656. doi: https://doi.org/10.1083/jcb.54.3.646
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