Use of antibodies in the analysis of connexin 43 turnover and phosphorylation.
A series of antipeptide antibodies designed to recognize specific sequences of the gap junction protein connexin 43 (Cx43) were developed and characterized immunochemically and immunohistologically. These antibodies bound to gap junctions and, on Western blots, to 43-kDa (often resolved as a doublet) and 41-kDa proteins in samples from heart, leptomeningeal cells, and brain. Relatively little of the 41-kDa protein was detectable in heart homogenates. Cultured rat leptomeningeal cells expressed high levels of the gap junction protein Cx43 and were used to analyze its turnover and phosphorylation. Pulse-chase experiments in leptomeningeal cells with [(35)S]methionine indicated that the 41-kDa form of connexin 43 was the first immunoprecipitable translation product. Radiolabel subsequently appeared in the lower band of the doublet at 43 kDa, followed by a shift into the higher band and turnover of the protein with a t(1/2) of 2.7 h. Pulse-chase labeling with [(32)P]P(i) indicated that phosphorylation of connexin 43 was limited to the 43-kDa protein, with a t(1/2) of 1.7 h. Treatment with alkaline phosphatase shifted the apparent molecular mass of the 43-kDa protein doublet such that it comigrated with the 41-kDa form. Hence, the 43-kDa protein observed on Western blots of both leptomeningeal cells and heart arises by phosphorylation of the 41 kDa precursor. Phosphorylation of serine residues accounts for most, if not all, of Cx43 phosphorylation in this system.
Hertzberg
E L
EL
Department of Neuroscience, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, New York 10461, USA. ehertz@aecom.yu.edu
Sáez
J C
JC
Corpina
R A
RA
Roy
C
C
Kessler
J A
JA
D000595:Amino Acid Sequence; D000818:Animals; D000831:Animals, Newborn; D000906:Antibodies; D001921:Brain / Q000166:cytology / Q000378:metabolism*; D018031:Connexin 43 / Q000737:chemistry / Q000378:metabolism*; D017629:Gap Junctions / Q000378:metabolism* / Q000648:ultrastructure; D008578:Meninges / Q000166:cytology / Q000378:metabolism*; D016253:Microscopy, Immunoelectron; D008969:Molecular Sequence Data; D009206:Myocardium / Q000166:cytology / Q000378:metabolism*; D010446:Peptide Fragments / Q000138:chemical synthesis / Q000276:immunology; D010766:Phosphorylation; D051381:Rats
D000906:Antibodies; D018031:Connexin 43; D010446:Peptide Fragments