Enzyme kinetic characterization of the smooth muscle myosin phosphorylating system: activation by calcium and calmodulin and possible inhibitory mechanisms of antagonists

A native-like smooth muscle filamentous myosin system was characterized fro m an enzyme kinetic point of view. The system contains endogenous myosin li ght chain kinase (MLCKase) and calmodulin (CM) (A. Sobieszek, J. Muscle Res . Cell Motil. 11 (1990) 114-124) and is, therefore, well suited for testing the action of CM-antagonists or other inhibitory compounds. However, this has not been done due to its complexity. The characterization of the system includes: (1) derivation of a relationship for rate of myosin phosphorylat ion in terms of total CM, free Ca2+ and total MLCKase concentrations, which includes only three binding constants; and (2) derivation of relationships between fractional inhibition rate (nu(i)/nu(o)) and total inhibitor conce ntration (I-t) which cover most of the inhibitory mechanisms applicable to the myosin system or to other CM-dependent enzymes. The three binding const ants were subsequently evaluated from experimental data for filamentous myo sin and for its isolated regulatory light chain (ReLC) using a non-linear r egression software. They indicated differences in the interaction of myosin filament with the active CM-MLCKase complex in comparison to that of the i solated ReLC. The derived nu(i)/nu(o) versus I-t relationships, together wi th the software, make it possible:to evaluate the inhibition constants and binding stoichiometries of CM-antagonists and other compounds inhibiting my osin phosphorylation. This approach was successfully applied to experimenta l data on inhibition of MLCKase by amiloride, cadmium, or CM-binding peptid e (M-12) for simple mechanisms. For more complex mechanisms, inhibition by calmidozolium, trifluoperazine or melittin, the analysis showed that only c almidozolium acted specifically at the CM level in a multiple-site activato r-depletion mechanism. Melittin and trifluoperazine inhibited the phosphory lation rate by a novel substrate-and-activator depletion mechanism, in whic h additional inhibition of the substrate resulted in the removal of the inh ibition at lower range of the antagonists' concentration. (C) 1999 Elsevier Science B.V. All rights reserved.