Quantitation of myosin light chain phosphorylation in intact smooth muscle.

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Abstract
An improved method for quantitating the extent of myosin light chain (P-LC) phosphorylation in small smooth muscle samples is described. Native myosin was isolated from other cellular proteins in a crude supernatant frction prepared from a few milligrams of bovine tracheal smooth muscle by polyacrylamide gel electrophoresis (PAGE) in the presence of sodium pyrophosphate (PPi). When potassium iodide (Kl, 0.6 M) was added to the crude supernatant fraction, myosin migrated into the gels during electrophoresis. Without adding Kl, myosin remained at the top of the gel so that the myosin content in the gel was 20 times less than in the presence of Kl. After the PPi-PAGE, myosin was subjected to isoelectric focusing (IEF) on polyacrylamide slab gels to separate the phosphorylated from the nonphoshorylated forms of the P-LC. The extent of P-LC phosphorylation was quantitated after densitometric scanning of silver-stained IEF gels. Examination of the temporal changes in carbachol-induced contraction and P-LC phosphorylation in tracheal smooth muscle strips exhibied a relatively transient change in the P-LC phosphorylation as shown in other smooth muscle preparations. This procedure is applicable to investigations on the role of Ca2+.cntdot.calmodulin-induced activation of myosin light chain kinase and phosphorylation of smooth muscle myosin.