Reversible Changes in the State of Phosphorylation of Gizzard Myosin, in That of Gizzard Myosin Assembly, in the ATPase Activity of Gizzard Myosin, in That of Actomyosin and in the Superprecipitation Activity1

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Abstract
Previously, we (Onishi et al. 1978, Suzuki et al. 1978, and Suzuki et al. 1981) reported studies on gizzard unphosphorylated myosin (UM) in MgCl2 concentrations lower than 10 mM. In the present report, we describe some findings of a study of UM in MgCl2 concentrations higher than 10 mM. 1. The turbidity of UM suspensions in the presence of 1 ms ATP increased as the MgCI2 concentration increased to higher than 10 mM, suggesting that UM can form “ATP-resistant” filaments in such extremely high concentrations of MgCl2. This suggestion was supported by electron microscopic observation. 2. The ATPase activity of UM alone also increased as the MgCI2 concentration increased, indicating that the activity is much higher with UM in a filamentous form than with UM in a disassembled form. 3. As the MgCl2 concentration increased to higher than 10 nm, both actin-activation of UM-ATPase and ATP-induced superprecipitation occurred with increasing magnitude. Summarizing these results, we observed that the requirement of MgCI2 in a concentration higher than 10 mM was common for formation of ATP-resistant filaments from UM, for increase in the ATPase activity of UM, and for superprecipitation of acto-UM. Moreover, since these findings for UM are very similar to those obtained previously (Suzuki et al. 1978, and 1981) with gizzard phosphorylated myosin (PM), except that MgCI, in a concentration higher than 2 m was the common requirement for PM, we propose a) that formation of ATP-resistant filaments from myosin is the cause of the increase in the ATPase activity of myosin alone and for activation of ATP-induced superprecipitation, and b) that the primary effect of phosphorylation of gizzard myosin light chains is on the MgCl2 concentration required for formation of ATP-resistant fflaments of myosin, reducing it (>10 mM) to the physiological concentration (>2 mM). 4. In connection with the latter proposal, we observed, by measuring the turbidity of myosin suspensions, that Ca2+, Sr2+, and Mn2+ can be used in place of Mg2+ to form ATP-resistant filaments of UM or PM, and that the concentration of these divalent cations required for formation of ATP-resistant filaments was again lower with PM than with UM. 5. We also measured the sedimentation rate and the ATPase activity of both UM and PM in the presence of MgCl2 where neither UM nor PM formed ATP-resistant filaments. We thus observed (in the presence of 1 mi.t ATP) a) that the ATPase activities of UM alone and of PM alone were both very low, and b) that both UM and PM sedimented at the same rate of approximately lOS on ultracentrifugation. These observations are in support of our electron microscopic observation that high concentrations of MgCl2 were required to observe ATP-resistant filaments of gizzard myosin, either UM or PM. From these studies, it seems that the various differences so far reported between skeletal and gizzard contractile proteins arise from one basic difference, that high concentrations of magnesium are required for formation of ATP-resistant filaments from gizzard myosin whereas they are not required at all for that from skeletal myosin. 6. The turbidity of gizzard myosin suspensions underwent a reversible change on addition of myosin light-chain kinase (MLCK) with C2+ and on subsequent addition of myosin light-chain phosphatase (MLCP) with or without Ca2+. The reversible change in the turbidity (of myosin suspensions) was associated with that in the state of phosphorylation of myosin light chains, as well as with that in the assembly of myosin molecules which was examined by electron microscopy and by ultracentrifugation. These results provide further support for and are an extension of a previous proposal (Suzuki et al. 1978) for the mechanism of contraction and relaxation in smooth muscle. Moreover, it seems that not just in smooth muscle but in muscle in general, the filamentous structure of myosin assembly is the structure essential for conversion of chemical energy into mechanical energy.