Functional demonstration of connexin-protein binding using surface plasmon resonance.
Surface plasmon resonance (SPR) allows examination of protein-protein interactions in real time, from which both binding affinities and kinetics can be directly determined. We have used the SPR technique to search for proteins in heart tissue that would be candidate binding partners for the cardiac gap junction protein, connexin43 (Cx43). Heart lysate showed a strong, pH-dependent binding to the carboxyl terminus (CT) of Cx43 (amino acids 254-382) covalently linked to an SPR cuvette. Binding was inhibited by the presence of v-src transfected 3T3 cell lysate, suggesting that binding partners in these two lysates may compete for overlapping epitopes on Cx43CT. The combined application of proteomic and functional studies is expected to identify which proteins within heart tissue interact with Cx43 and what roles they may play in gap junction function.
Duffy
H S
HS
Albert Einstein College of Medicine, Bronx, New York 10464, USA.
Delmar
M
M
Coombs
W
W
Tafftet
S M
SM
Hertzberg
E L
EL
Spray
D C
DC
D016475:3T3 Cells; D000818:Animals; D018031:Connexin 43 / Q000235:genetics / Q000378:metabolism*; D017629:Gap Junctions / Q000737:chemistry / Q000378:metabolism; D016391:Genes, src; D006863:Hydrogen-Ion Concentration; D051379:Mice; D009206:Myocardium / Q000737:chemistry*; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012756:Sheep; D020349:Surface Plasmon Resonance / Q000295:instrumentation / Q000379:methods; D014020:Tissue Extracts / Q000737:chemistry / Q000378:metabolism
D018031:Connexin 43; D011993:Recombinant Fusion Proteins; D014020:Tissue Extracts