Quantitation of myosin light chain phosphorylation in intact smooth muscle.
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 fraction 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 (KI, 0.6 M) was added to the crude supernatant fraction, myosin migrated into the gels during electrophoresis. Without adding KI, 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 KI. After the PPi-PAGE, myosin was subjected to isoelectric focusing (IEF) on polyacrylamide slab gels to separate the phosphorylated from the nonphosphorylated 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 exhibited 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+.calmodulin-induced activation of myosin light chain kinase and phosphorylation of smooth muscle myosin.
Department of Pharmacology, Nagoya City University Medical School, Japan.
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Asano
D000818:Animals; D002217:Carbachol / Q000494:pharmacology; D002417:Cattle; D011756:Diphosphates / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D006168:Guinea Pigs; D066298:In Vitro Techniques; D009130:Muscle, Smooth / Q000378:metabolism*; D009218:Myosins / Q000378:metabolism*; D010766:Phosphorylation; D011193:Potassium Iodide / Q000494:pharmacology; D011817:Rabbits; D012834:Silver; D013194:Staining and Labeling; D014132:Trachea / Q000187:drug effects
D011756:Diphosphates; D011193:Potassium Iodide; D012834:Silver; D002217:Carbachol; D009218:Myosins