Abstract
Scallop striated adductor muscle myosin is a regulatory myosin, its activity being controlled directly through calcium binding. Here, we show that millimolar concentrations of trifluoperazine were effective at removal of all regulatory light chains from scallop myosin or myofibrils. More important, 200 microM trifluoperazine, a concentration 10-fold less than that required for light-chain removal, resulted in the reversible elimination of actin-activated and intrinsic ATPase activities. Unlike desensitization induced by metal ion chelation, which leads to an elevation of activity in the absence of calcium concurrent with regulatory light-chain removal, trifluoperazine caused a decline in actin-activated MgATPase activity both in the presence and absence of calcium. Procedures were equally effective with respect to scallop myosin, myofibrils, subfragment-1, or desensitized myofibrils. Increased alpha-helicity could be induced in the isolated essential light chain through addition of 100-200 microM trifluoperazine. We propose that micromolar concentrations of trifluoperazine disrupt regulation by binding to a single high-affinity site located in the C-terminal domain of the essential light chain, which locks scallop myosin in a conformation resembling the off-state. At millimolar trifluoperazine concentrations, additional binding sites on both light chains would be filled, leading to regulatory light-chain displacement.
Highlights
Trifluoperazine (TFP),1 a member of the phenothiazine class of drugs, is one of the strongest antagonists of calmodulin action known, capable of binding to calmodulin in the presence of calcium and preventing its stimulatory effects [1,2,3]
The structure of the TFP1⁄7calmodulin complex in the presence of calcium has been determined at 2.45-Å resolution [4, 5], where it was shown that TFP induces a profound conformational change in calmodulin, converting the elongated dumbbell to a compact globular structure, analogous to the form obtained through the binding of calmodulin to a target peptide [6, 7]
Actin-activated Ca1⁄7Mg1⁄7S-1 MgATPase rates (Ϯcalcium) were measured in a buffer similar to that described for the colorimetric determination of myosin rates, except that F-actin was present at a 20ϫ molar ratio to S-1, the latter being present at a concentration of 50 g/ml
Summary
Protein Preparation and EDTA Desensitization—Myofibrils and myosin from scallop striated adductor muscles were prepared as described earlier [22]. Desensitization of scallop myofibrils and myosin through metal ion chelation was accomplished by standard procedures [17, 23]. Ca1⁄7Mg1⁄7S-1 was prepared by papain digestion [24], except that the reaction was terminated by the combined addition of N␣-p-tosyl-L-lysine chloromethyl ketone (to 10 mM from a 0.5 M stock in 50% ethanol) and leupeptin (to 10 mg/liter from a 0.5 mg/ml stock in buffer), so as to avoid covalent modification of S-1 as compared with the original procedure.
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