title: Enzyme kinetic characterization of the smooth muscle myosin phosphorylating system: activation by calcium and calmodulin and possible inhibitory mechanisms of antagonists.
abstract: A native-like smooth muscle filamentous myosin system was characterized from an enzyme kinetic point of view. The system contains endogenous myosin light 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 phosphorylation 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 (vi/vo) and total inhibitor concentration (It) which cover most of the inhibitory mechanisms applicable to the myosin system or to other CM-dependent enzymes. The three binding constants were subsequently evaluated from experimental data for filamentous myosin and for its isolated regulatory light chain (ReLC) using a non-linear regression software. They indicated differences in the interaction of myosin filament with the active CM-MLCKase complex in comparison to that of the isolated ReLC. The derived vi/vo versus It relationships, together with the software, make it possible to evaluate the inhibition constants and binding stoichiometries of CM-antagonists and other compounds inhibiting myosin phosphorylation. This approach was successfully applied to experimental data on inhibition of MLCKase by amiloride, cadmium, or CM-binding peptide (M-12) for simple mechanisms. For more complex mechanisms, inhibition by calmidozolium, trifluoperazine or melittin, the analysis showed that only calmidozolium acted specifically at the CM level in a multiple-site activator-depletion mechanism. Melittin and trifluoperazine inhibited the phosphorylation rate by a novel substrate-and-activator depletion mechanism, in which additional inhibition of the substrate resulted in the removal of the inhibition at lower range of the antagonists' concentration.
authors.affiliation: Institute of Molecular Biology, Austrian Academy of Sciences, Billrothstrasse 11, A-5020, Salzburg, Austria. asobieszek@compuserve.com
authors.forename: A
authors.initials: A
authors.lastname: Sobieszek
mesh_terms: D000818:Animals; D002104:Cadmium / Q000494:pharmacology; D002118:Calcium / Q000494:pharmacology; D002147:Calmodulin / Q000494:pharmacology; D003201:Computers; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005895:Gizzard, Avian; D007700:Kinetics; D008555:Melitten / Q000494:pharmacology; D009130:Muscle, Smooth / Q000201:enzymology*; D009219:Myosin-Light-Chain Kinase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D010766:Phosphorylation; D013379:Substrate Specificity; D014268:Trifluoperazine / Q000494:pharmacology; D014422:Turkeys
chemical_list: D002147:Calmodulin; D004791:Enzyme Inhibitors; D002104:Cadmium; D008555:Melitten; D014268:Trifluoperazine; D009219:Myosin-Light-Chain Kinase; D002118:Calcium