Affinity purification of a rat-brain junctional protein, connexin 43.
Immunocytochemical investigations have previously shown that antibodies specific for mammal connexins labeled in situ rat and mouse brain gap junctions. However brain gap-junction proteins have neither been identified with certainty, nor purified. By immunoblotting, anti-peptide antibodies directed against rat heart connexin 43 (CX43) detect a major protein of 41 kDa in rat brain homogenates. The specificity of these antibodies made it possible to establish an affinity-chromatography purification procedure of the 41-kDa protein. Purified antibodies specific for the sequence SAEQNRMGQ (residues 314-322) of rat heart CX43 were covalently bound to a protein-A-Sepharose-CL-4B matrix. Rat brain homogenates were recycled through the immunomatrix and the material specifically bound to the matrix was then competitively eluted with the peptide SAEQNRMGQY. Analysis by SDS/PAGE of eluates demonstrated that they contain a 41-kDa protein associated with low amounts of high-molecular-mass proteins. By immunoblotting, these proteins were shown to be specifically recognized by antibodies directed against residues 5-17, 55-56, and 314-322 of rat heart CX43. The NH2-terminal partial sequence for the 41-kDa protein was determined by microsequencing and shown to be similar to alpha 1 connexins. This is the first successful purification of a junctional protein from brain tissue and provides direct evidence that the 41-kDa protein is a CX43 gene product.
Dupont
E
E
Laboratoire de Biologie de la Différenciation Cellulaire, Faculté des Sciences de Luminy, Université d'Aix-Marseille II, France.
el Aoumari
A
A
Fromaget
C
C
Briand
J P
JP
Gros
D
D
D000595:Amino Acid Sequence; D000818:Animals; D000917:Antibody Formation; D015152:Blotting, Northern; D001923:Brain Chemistry; D002846:Chromatography, Affinity; D017630:Connexins; D004591:Electrophoresis, Polyacrylamide Gel; D015151:Immunoblotting; D008565:Membrane Proteins / Q000737:chemistry / Q000276:immunology / Q000302:isolation & purification*; D008969:Molecular Sequence Data; D009206:Myocardium / Q000737:chemistry; D009842:Oligopeptides / Q000138:chemical synthesis; D051381:Rats; D012689:Sequence Homology, Nucleic Acid
D017630:Connexins; D008565:Membrane Proteins; D009842:Oligopeptides