Topology of the 32-kd liver gap junction protein determined by site-directed antibody localizations.
Synthetic peptides corresponding to sequences in the human liver gap junction protein were chemically synthesized and used for generation of peptide antisera to defined sequences in the protein. The antibodies were affinity purified and characterized by demonstrating that they specifically recognized both their corresponding synthetic peptide (as indicated by dot blot analysis) and the native 32-kd gap junction protein (by immunoblotting). The specificity of a subset of the different site-specific antibodies was subsequently confirmed by demonstration of their binding to specific gap junction fragments produced by treatment with a lysine-specific endoproteinase. Immunoelectron microscopy was used to localize the specific peptide antibody epitopes to either the cytoplasmic or extracellular surfaces of the gap junction. Results indicate a transmembrane orientation for the protein with the amino and carboxyl termini located on the cytoplasmic side of the membrane. Based on these data, a model is proposed for the transmembrane folding of the gap junction protein.
Milks
L C
LC
Department of Molecular Biology, Research Institute of Scripps Clinic, La Jolla, CA 92037.
Kumar
N M
NM
Houghten
R
R
Unwin
N
N
Gilula
N B
NB
D000595:Amino Acid Sequence; D015153:Blotting, Western; D002462:Cell Membrane / Q000648:ultrastructure; D017630:Connexins; D000939:Epitopes; D007150:Immunohistochemistry; D008565:Membrane Proteins / Q000276:immunology / Q000648:ultrastructure*; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000276:immunology; D010447:Peptide Hydrolases / Q000378:metabolism; D010455:Peptides / Q000138:chemical synthesis / Q000276:immunology; D011487:Protein Conformation
D017630:Connexins; D000939:Epitopes; D008565:Membrane Proteins; D010446:Peptide Fragments; D010455:Peptides; D010447:Peptide Hydrolases