A radioimmunoblotting method for measuring myosin light chain phosphorylation levels in smooth muscle.
A method for measuring the molar stoichiometry of myosin light chain phosphorylation in intact smooth muscle has been developed. Antiserum to the 20,000-Da light chains of bovine aortic smooth muscle was harvested from rabbits and used to label light chains by a radioimmunoblotting procedure. In the initial characterization it was found that the 20,000-Da light chains could be transferred by electroblotting from polyacrylamide gels to nitrocellulose paper with an efficiency of approximately 80% over a protein range of 0.1-5.0 micrograms. At a dilution of 1:500, the unpurified light chain antiserum required approximately 10-12 h at 22 degrees C to reach equilibrium binding to the transferred light chains. Moreover, equilibrium labeling of the light chain-antibody complex with 125I-protein A required 4-6 h of incubation at 22 degrees C. By using these conditions, a radioimmunoassay for the 20,000-Da light chains was developed that was linear over a protein range of 0.1-5.0 micrograms (5-250 pmol). As little as 20 ng of light chains could be measured if a second antibody procedure (goat anti-rabbit immunoglobulin G Fab fragments) was used. Phosphorylated and unphosphorylated myosin light chains were separated by glycerol-urea polyacrylamide gel electrophoresis. This procedure, combined with radioimmunoblot, gave similar estimates of phosphorylation levels when compared with direct assay for phosphate or scanning of Coomassie blue-stained gels. Moreover, when applied to intact uterine smooth muscle, the glycerol-urea gel radioimmunoblot gave values of myosin light chain phosphorylation for relaxed and contracted muscles that were not statistically different from those obtained with a two-dimensional electrophoretic method.(ABSTRACT TRUNCATED AT 250 WORDS)
D000818:Animals; D001011:Aorta / Q000378:metabolism; D002217:Carbachol / Q000494:pharmacology; D002417:Cattle; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D005260:Female; D007106:Immune Sera; D007700:Kinetics; D009130:Muscle, Smooth / Q000378:metabolism*; D009131:Muscle, Smooth, Vascular / Q000378:metabolism*; D009218:Myosins / Q000302:isolation & purification / Q000378:metabolism*; D010121:Oxytocin / Q000494:pharmacology; D010766:Phosphorylation; D011863:Radioimmunoassay / Q000379:methods; D051381:Rats; D014590:Uterine Contraction / Q000187:drug effects; D014599:Uterus / Q000378:metabolism
D007106:Immune Sera; D010121:Oxytocin; D002217:Carbachol; D009218:Myosins