Cross-linking of cardiac gap junction connexons by thiol/disulfide exchanges

Summary SDS-polyacrylamide gel electrophoresis and immunoblotting were used to investigate inter- and intramolecular disulfide bonds to connexin 43 (the cardiac gap junctional protein) in isolated rat heart gap junctions and in whole heart fractions. In gap junctions isolated in the absence of alkylating agent, connexin 43 molecules are cross-linked by disulfide bonds. The use of iodoacetamide (100mm) for the first steps of isolation procedure prevents the formation of these artifactual linkages. Investigation of connexin 43 in whole heart fractions by means of antibodies confirms the results obtained with isolated gap junctions; that is, connexin 43 molecules are not interconnected with disulfide bridges. In whole heart fractions treated with alkylating agents, a 38 kD protein, immunologically related to connexin 43, and containing intramolecular disulfide bonds is detected. It is hypothesized that this protein might be a folded form of connexin 43, a precursory form of the molecules embedded in the gap junctions.The abbreviations used are BSA bovine serum albumin - EDTA ethylene diamine tetra-acetic acid - IAA iodoacetamide - NEM N-ethylmaleimide - PAGE polyacrylamide gel electrophoresis - PMSF phenylmethylsfonyl fluoride - SDS sodium dodecyl sulfate - Tris trishydroxymethyl-aminomethane