Weak static magnetic field modulation of myosin phosphorylation in a cell-free preparation: Calcium dependence

Ca²⁺-calmodulin-dependent myosin phosphorylation is studied here to assess magnetic field effects on Ca²⁺ binding. The cell-free reaction mixture was chosen for the phosphorylation rate to be linear in time for several minutes, and to be rate limited by Ca²⁺. To this end the ratio of substrate to enzyme was <100:1 in order to slow the overall reaction rate, and the Ca²⁺ concentration was well below saturation for calmodulin. The Ca²⁺ concentration was varied from 1 to 7 μM with appropriate EGTA buffering. Phosphorylation was allowed to proceed for 5 min at 44 μT (ambient) and 200 μT (vertical). The results showed that phosphorylation increased up to 2× at 200 μT (vs. 44 μT) dependent upon [Ca²⁺]. In addition, as [Ca²⁺] approaches saturation for calmodulin, the magnetic field effect disappears. Effects between 1 and 5 μM Ca²⁺ were analyzable using a Lineweaver-Burk plot, showing a negligible change in affinity, K_D, and a relatively large increase in V_MAX (1.8× at 200 μT vs. ambient) suggesting Ca²⁺ binding kinetics are affected.