A radioimmunoblotting method for measuring myosin light chain phosphorylation levels in smooth muscle

Abstract

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)

Quantitation of myosin light chain phosphorylation in intact smooth muscle.

“…P-LC phosphate contents in resting and contracting smooth muscle strips were similar to those measured with other methods (6)(7)(8)(9)(10). Antagonistic effects of atropine and W-7 on the responses to car bachol demonstrated the pharmacological relevance of this method for quantitating the P-LC phosphorylation during isometric tension development in intact smooth muscles.…”

“…Recently, a method has been described that involved radioimmunoblotting with antibodies raised to P-LC after urea/glycerol-PAGE of de natured protein (9). Without using radioiso topes, a combined method of two electro phoretic procedures, PPi-PAGE and IEF, has been reported to quantitate the extent of P LC phosphorylation (10).…”

Vinculin Phosphorylation at Tyr1065 Regulates Vinculin Conformation and Tension Development in Airway Smooth Muscle Tissues

“…The proteins were extracted using an 8 M urea buffer. Phosphorylated and unphosphorylated MLCs were separated by glycerol-urea polyacrylamide gel electrophoresis, transferred to nitrocellulose, and then immunoblotted for MLC (2,4,44). MLC and phosphorylated MLC were visualized by ECL and digitally quantified using a Bio-Rad ChemiDoc XRS detection system.…”

Involvement of rho in GTPγS‐induced enhancement of phosphorylation of 20 kDa myosin light chain in vascular smooth muscle cells: inhibition of phosphatase activity

“…The inclusion of 1 pM ionomycin in both the permeabilization and the phosphorylation buffers did not alter the results. The extent of MLC20 phosphorylation was determined by glycerol-urea gel electrophoresis followed by quantitation of the relative amounts of non-phosphorylated and phosphorylated forms of MLC20 with Western blot technique [13]. Densities of bands corresponding to non-phosphorylated, monophosphorylated and diphosphorylated forms of MLC20 were quantitated by using a scanning densitometer (The Discovery Series, PDI).…”