A Further Study on the Phosphorylation and Dephosphorylation of a Light-Chain Subunit of Chicken Gizzard Myosin

Abstract

Ikebe et al,. ((1977) J. Biochem. 82, 299–302, and (1978) J. Biochem. 83,1643-1655) proposed that calcium is required only for the activation of myosin light-chain kinase, and that phos-phorylation and dephosphorylation of a specific light-chain component of gizzard myosin are required for superprecipitation (increase in turbidity) and for its reversal (decrease in turbidity), respectively. The following results support their proposals: 1. Gizzard myosin was freed from myosin light-chain kinase by chromatography on a DEAE-Sephadex A-50 column or by washing with 10 mM EDTA solution. 2. Phosphorylated myosin was chromatographed on a DEAE-Sephadex A-50 column, and combined with actin to obtain acto-phosphorylated myosin. The Mg-ATPase activity of the acto-phosphorylated myosin was always insensitive to calcium. On the other hand, the superprecipitation activity of acto-phosphorylated myosin in the absence of calcium (i.e., in the presence of EGTA) was lower than that in the presence of 0.1 mM CaCl² (showing an apparent calcium sensitivity). However, some results were obtained suggesting that a trace amount of heavy-metal contaminant in the buffer used may have been responsible for the apparent calcium sensitivity. For example, the superprecipitation activity in the presence of 0.2 mM CaCl² plus; 0.1 mM EGTA was equal to that in the presence of 1 mM EGTA. 3. With a combined system of acto-phosphorylated myosin and gizzard native tropomyosin, both the ATPase reaction and superprecipitation proceeded in a manner independent of calcium in the early stages of the reactions, but both reactions in the absence of calcium some times slowed down in the later stages. Urea-gel electrophoresis revealed that myosin light-chain phosphatase, which is contained in native tropomyosin, caused dephosphorylation of phosphorylated myosin in the absence of calcium.