Calmodulin antagonist action in smooth-muscle myosin phosphorylation. Different mechanisms for trifluoperazine and calmidazolium inhibition.
The mechanism of inhibition of myosin phosphorylation by calmodulin antagonists [trifluoperazine (TFP) and calmidazolium (CAL)] was investigated in two enzyme-substrate systems: (1) mixtures of isolated myosin phosphorylatable light-chain (L20), myosin light-chain kinase (MCLKase) and calmodulin (CM); (2) synthetic self-assembled myosin filaments containing tightly bound endogenous MLCKase and CM. Double-reciprocal plots obtained with the first system were non-linear, indicating that the antagonists did not act exclusively on CM to inhibit MLCKase. First-order phosphorylation progress curves obtained at different CM antagonist concentrations for the more native filamentous myosin system indicated that the antagonists could also inhibit phosphorylation by interaction with the myosin phosphorylation site. Further analysis of these data in accordance with Reiner [(1969) Behavior of Enzyme Systems, 2nd edn., pp. 185-201, Van Nostrand-Reinhold, New York] showed that over a range of concentrations required to inhibit phosphorylation TFP interacted with free CM as well as with the myosin phosphorylation site: accordingly inhibition was of an activator- and substrate-depletion type. CAL inhibition was more CM-specific and operated via an activator-combination mechanism, inhibiting free CM as well as the CM-MLCKase complex. Both CM and the isolated L20 light chain antagonized the inhibitory effects of CAL and TFP, consistent with the above analysis.
Sobieszek
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Institute of Molecular Biology, Austrian Academy of Sciences, Salzburg.
D000818:Animals; D002147:Calmodulin / Q000037:antagonists & inhibitors*; D004791:Enzyme Inhibitors / Q000378:metabolism; D007093:Imidazoles / Q000494:pharmacology; D007700:Kinetics; D009130:Muscle, Smooth / Q000378:metabolism*; D015879:Myosin Subfragments / Q000378:metabolism; D009219:Myosin-Light-Chain Kinase / Q000378:metabolism; D009218:Myosins / Q000378:metabolism*; D010766:Phosphorylation; D014268:Trifluoperazine / Q000494:pharmacology
D002147:Calmodulin; D004791:Enzyme Inhibitors; D007093:Imidazoles; D015879:Myosin Subfragments; D014268:Trifluoperazine; C031938:calmidazolium; D009219:Myosin-Light-Chain Kinase; D009218:Myosins