An in vitro assay reveals essential protein components for the 'catch' state of invertebrate smooth muscle
| Autor: | Kazuhiro Oiwa, Maki Yoshio, Hiroaki Kojima, Akira Yamada |
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| Rok vydání: | 2001 |
| Předmět: |
Multidisciplinary
Phosphatase Muscle Proteins Skeletal muscle Muscle Smooth macromolecular substances Myosins Biological Sciences Biology Bivalvia medicine.anatomical_structure Biochemistry ATP hydrolysis Myosin Cyclic AMP medicine Biophysics Animals Phosphorylation Calcium medicine.symptom Protein kinase A Actin Muscle Contraction Muscle contraction |
| Zdroj: | Proceedings of the National Academy of Sciences. 98:6635-6640 |
| ISSN: | 1091-6490 0027-8424 |
| DOI: | 10.1073/pnas.111585098 |
| Popis: | “Catch,” a state where some invertebrate muscles sustain high tension over long periods of time with little energy expenditure (low ATP hydrolysis rate) is similar to the “latch” state of vertebrate smooth muscles. Its induction and release involve Ca 2+ -dependent phosphatase and cAMP-dependent protein kinase, respectively. Molecular mechanisms for catch remain obscure. Here, we describe a quantitative microscopic in vitro assay reconstituting the catch state with proteins isolated from catch muscles. Thick filaments attached to glass coverslips and pretreated with ≈10 −4 M free Ca 2+ and soluble muscle proteins bound fluorescently labeled native thin filaments tightly in catch at ≈10 −8 M free Ca 2+ in the presence of MgATP. At ≈10 −4 M free Ca 2+ , the thin filaments moved at ≈4 μm/s. Addition of cAMP and cAMP-dependent protein kinase at ≈10 −8 M free Ca 2+ caused their release. Rabbit skeletal muscle F-actin filaments completely reproduced the results obtained with native thin filaments. Binding forces >500 pN/μm between thick and F-actin filaments were measured by glass microneedles, and were sufficient to explain catch tension in vivo . Synthetic filaments of purified myosin and twitchin bound F-actin in catch, showing that other components of native thick filaments such as paramyosin and catchin are not essential. The binding between synthetic thick filaments and F-actin filaments depended on phosphorylation of twitchin but not of myosin. Cosedimentation experiments showed that twitchin did not bind directly to F-actin in catch. These results show that catch is a direct actomyosin interaction regulated by twitchin phosphorylation. |
| Databáze: | OpenAIRE |
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