1. Gap (communicating) junctions are plasma-membrane specializations of characteristic morphology that form transmembrane channels allowing direct communication between cells. Their preparation is described starting from mouse liver plasma membranes and the constituent polypeptides are deduced. 2. Gap junctions co-purify with collagen fibres when the plasma-membrane residues insoluble in N-dodecyl sarcosinate are fractionated on sucrose gradients. Sucrose-density perturbation by relipidation of isolated gap junctions or the use of urea to remove non-junctional membranes both failed to diminish the collagen content of fractions. 3. Removal of collagen by treatment with purified collagenase preparations yielded morphologically satisfactory gap-junction fractions. Analysis by polyacrylamide-gel electrophoresis of the polypeptides present in gap junctions prepared by procedures omitting or using collagenases indicated two non-glycosylated polypeptides, a major component of apparent mol.wt. 38000 and a minor 40000-mol.wt. component. These two polypeptides were also present in plasma membranes and the intermediate fractions. 4. Proteolysis of the gap-junction polypeptides yielding components of mol.wt. 34000, 25000 and below 20000 occurred when iodinated gap junctions were subject to prolonged collagenase treatment, thus explaining the variable polypeptide composition of gap junctions reported by others. 5. The morphological properties of the isolated gap junctions prepared by the various procedures are described
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