Effects of Ca2+, Mg2+, and myosin phosphorylation on skinned smooth muscle fibers

Isometric tension, unloaded shortening velocity (Vus), and myosin light chain phosphorylation were measured with skinned chicken gizzard fibers at various Ca2+ concentrations and at two concentrations of free Mg2+, 0.7 and 2.2 mM. At low free Mg2+, an increase in Ca2+ from pCa 8.0 to 6.4 resulted in an increase of all three parameters. Between pCa 6.4 and 5.0, isometric tension and phosphorylation remained constant but Vus continued to increase. At low free Mg2+, therefore, Vus showed a dependence both on phosphorylation and on Ca2+. At high free Mg2+, tension and Vus increased as phosphorylation increased and both were maximum at pCa 6.4, where phosphorylation became constant. Therefore, at high free Mg2+, Vus was dependent only on phosphorylation and did not show an additional Ca2+ dependence. Incubation of the Ca2+-independent kinase (approximately 3 microM) with skinned fibers under various conditions resulted in a constant level of phosphorylation (49-58%). At high free Mg2+ plus the Ca2+-independent kinase Vus was independent of Ca2+, whereas at low free Mg2+ Vus increased from pCa 6.4 to 5.0. These data are consistent with the hypothesis that Ca2+ binding to the Ca2+-Mg2+ sites of myosin increase Vus and that this occurs at Ca2+ concentrations higher than those necessary to saturate calmodulin.