Native Pyrophosphate Gel Analysis of Smooth Muscle Myosin Phosphorylated with Protein Kinase C

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We have shown that the phosphorylation of smooth muscle regulatory myosin light chain (L₂₀) with myosin light chain kinase (MLCK) produces faster moving bands (GMP1: heterodimer myosin with 1 unphosphorylated L₂₀ and 1 mono-phospho-rylated L₂₀, GMP2: homodimer myosin with 2 mono-phosphorylated L₂₀s) on native pyrophosphate polyacrylamide gel electrophoresis (PP₁ PAGE) (J. Biochem. 100, 259-268, 1986; J. Biochem. 100, 1681-1684, 1986). However, the mobility of the myosin phosphorylated, at its L₂₀, with protein kinase C (PK-C) was the same that of the unphosphorylated myosin (GM) on PP₁ PAGE. When the myosin prephos-phorylated with MLCK was further phosphorylated with PK-C, PP₁ PAGE analysis showed only one band comigrating with GM, i.e., GMPI and GMP2 migrated to the same position as GM. Conversely, when the myosin prephosphorylated with PK-C was further phosphorylated with MLCK, GMP1 and GMP2 were not pro-duced. Thus the effect of L₂₀ phosphorylated with PK-C is quite the opposite of that with MLCK, and the former predominated over the latter. We speculate that phosphorylation of L₂₀ with PK-C “freezes” myosin in the inactive state.