ARTICLE

THE ISOLATION OF MOUSE HEPATOCYTE GAP JUNCTIONS : Preliminary Chemical Characterization and X-Ray Diffraction

Daniel A. Goodenough ¹ and Walther Stoeckenius ¹

¹ From the Cardiovascular Research Institute, University of California, San Francisco Medical Center, San Francisco, California 94122.

Dr. Goodenough's present address is the Department of Anatomy, Harvard Medical School, Boston, Massachusetts 02115.

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).

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