Immunocytochemical localization of the gap junction 26 K protein in mouse liver plasma membranes.
Specific binding sites for anti-26 K antibodies directed against the liver gap junction protein (26 K) were localized by immunoelectron microscopy in gap junction plaques purified from hepatic plasma membranes. Using immunofluorescence microscopy we found discrete fluorescent spots on plasma membranes in cross sections of liver tissues after incubation with anti-26 K antibodies. This is consistent with the notion of specific binding to gap junction plaques. Quantitative binding of anti-26 K antibodies was indirectly measured by the protein A-gold technique. We found that urea/detergent-treated, purified gap junction plaques bind 30-fold more anti-26 K antibodies than preimmune serum. Anti-26 K antibodies also bind specifically to native gap junction plaques within hepatic plasma membranes although only about one fifth as efficiently as to purified plaques. Possibly the anti-26 K antibodies raised after injection of SDS-denatured 26 K protein into rabbits recognize the cytoplasmic face of urea/detergent-treated plaques better than that of native plaques. Some, if not most, of the vesicular structures in preparations of purified plaques appear to be derived from split gap junction plaques and are probably sheets of gap junction hemichannels. In some vesicles the former cytoplasmic face of the hemichannels is turned outside, other vesicles have the former cell surface turned outside. The anti-26 K antibodies do not recognize any 26 K protein on the sheets of partially split gap junction plaques, on the heterogeneous vesicular structures, or on non-junctional areas of hepatic plasma membranes. These results suggest that the conformation of the 26 K protein in plaques must be different from that of the 26 K protein in earlier biosynthetic steps of plaque assembly.
Janssen-Timmen
U
U
Institut für Zellbiologie, Universität Essen, FRG.
Dermietzel
R
R
Frixen
U
U
Leibstein
A
A
Traub
O
O
Willecke
K
K
D000818:Animals; D000906:Antibodies / Q000276:immunology; D002462:Cell Membrane / Q000737:chemistry; D000072259:Connexin 26; D017630:Connexins / Q000032:analysis* / Q000276:immunology; D007150:Immunohistochemistry; D008099:Liver / Q000737:chemistry*; D051379:Mice; D008807:Mice, Inbred BALB C; D008856:Microscopy, Fluorescence / Q000379:methods; D008867:Microtomy; D011817:Rabbits
D000906:Antibodies; D017630:Connexins; D000072259:Connexin 26