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. © 1979, by the Japanese Biochemical Society.
