Abstract
"Catch" is the state where some invertebrate muscles sustain high tension for long periods at low ATP hydrolysis rates. Physiological studies using muscle fibers have not yet fully provided the details of the initiation process of the catch state. The process was extensively studied by using an in vitro reconstitution assay with several phosphatase inhibitors. Actin filaments bound to thick filaments pretreated with the soluble protein fraction of muscle homogenate and Ca2+ (catch treatment) in the presence of MgATP at a low free Ca2+ concentration (the catch state). Catch treatment with > 50 microm okadaic acid, > 1 microm microcystin LR, 1 microm cyclosporin A, 1 microm FK506, or 0.2 mm calcineurin autoinhibitory peptide fragment produced almost no binding of the actin filaments, indicating protein phosphatase 2B (PP2B) was involved. Use of bovine calcineurin (PP2B) and its activator calmodulin instead of the soluble protein fraction initiated the catch state, indicating that only PP2B and calmodulin in the soluble protein fraction are essential for the initiation process. The initiation was reproduced with purified actin, myosin, twitchin, PP2B, and calmodulin. 32P autoradiography showed that only twitchin was dephosphorylated during the catch treatment with either the soluble protein fraction or bovine calcineurin and calmodulin. These results indicate that PP2B directly dephosphorylates twitchin and initiates the catch state and that no other component is required for the initiation process of the catch state.
Highlights
Invertebrate muscles such as the smooth adductor and byssus retractor muscles of bivalves maintain high tension with little energy expenditure after active contractions [1, 2]
By using thick and thin filaments isolated from catch muscles of the mussel Mytilus galloprovincialis, our group devised a powerful assay based on direct visualization of the binding of thin filaments to thick filaments in vitro under conditions corresponding to the catch state [15]
Reconstitution of Catch State Using Synthetic Thick Filaments—In our previous study, the catch state was reconstituted by using native thick filaments, and nearly half of them did not bind to fluorescence-labeled actin filaments tightly in the catch state [15]
Summary
Invertebrate muscles such as the smooth adductor and byssus retractor muscles of bivalves maintain high tension with little energy expenditure after active contractions [1, 2]. Actin filaments bound to thick filaments pretreated with the soluble protein fraction of muscle homogenate and Ca2؉ (catch treatment) in the presence of MgATP at a low free Ca2؉ concentration (the catch state). Catch treatment with >50 M okadaic acid, >1 M microcystin LR, 1 M cyclosporin A, 1 M FK506, or 0.2 mM calcineurin autoinhibitory peptide fragment produced almost no binding of the actin filaments, indicating protein phosphatase 2B (PP2B) was involved.
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