Isolation and protein composition of gap junctions from rabbit hearts.
We have modified a method for isolating gap-junctional membrane from mouse hearts [Kensler & Goodenough (1980) J. Cell Biol. 86, 755-764] to isolate gap junctions of comparable purity from rabbit hearts more rapidly, with better yield, and without resort to non-ionic detergents. Purification was monitored by electron microscopy of thin-sectioned membrane pellets and by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. Gap junctions were obtained as vesicles whose mean surface area approximated that of junctions in intact myocardial cells. About 10-20% of the vesicles were ferritin-impermeable. Approx. 125 micrograms of membrane protein was obtained per 8 g of rabbit heart. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis of purified gap junctions showed five major protein bands of mol.wts. 46 000, 44 000, 33 000, 30 000 and 28 500 that co-purified with the junctions. This protein composition was nearly identical with that published for gap junctions of mouse hearts, and differed markedly from the protein composition of gap junctions from non-excitable cells (lens and liver). The constancy of junctional protein composition between hearts of two different species and its non-identity with that from liver and lens suggest that, although gap-junctional structure in mammalian tissues seems to be remarkably similar by electron-microscopic techniques, junctional-channel protein composition actually varies from tissue to tissue and may be adapted to the permeability requirements of the tissue.
D000818:Animals; D002458:Cell Fractionation / Q000379:methods; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D005293:Ferritins / Q000378:metabolism; D066298:In Vitro Techniques; D007365:Intercellular Junctions / Q000378:metabolism* / Q000648:ultrastructure; D008297:Male; D008565:Membrane Proteins / Q000378:metabolism*; D009206:Myocardium / Q000378:metabolism* / Q000648:ultrastructure; D011506:Proteins / Q000032:analysis; D011817:Rabbits
D008565:Membrane Proteins; D011506:Proteins; D005293:Ferritins