A Preparative Method for the Isolation of Calponin from Molluscan Catch Muscle
We describe the development of a preparative method to isolate molluscan catch muscle, calponin. This method is based on the ability of calponin to interact with actin in a temperature-dependent manner. After extracting thin filaments, as previously described, the extract was ultracentrifuged at 2 °...
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MDPI AG
2022-07-01
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| Series: | International Journal of Molecular Sciences |
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| Online Access: | https://www.mdpi.com/1422-0067/23/14/7993 |
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| author | Stanislav S. Lazarev Ulyana V. Shevchenko Vyacheslav A. Dyachuk Ilya G. Vyatchin |
| author_facet | Stanislav S. Lazarev Ulyana V. Shevchenko Vyacheslav A. Dyachuk Ilya G. Vyatchin |
| author_sort | Stanislav S. Lazarev |
| collection | DOAJ |
| description | We describe the development of a preparative method to isolate molluscan catch muscle, calponin. This method is based on the ability of calponin to interact with actin in a temperature-dependent manner. After extracting thin filaments, as previously described, the extract was ultracentrifuged at 2 °C. While other surface proteins of thin filaments co-precipitated with actin, calponin, along with some minor contaminants, remained in the supernatant. Calponin was purified through cation-exchange chromatography. The yield of pure protein was four-fold higher than that achieved through high-temperature extraction. To evaluate functionally isolated proteins, we determined the effect of calponin on Mg<sup>2+</sup>-ATPase activity of hybrid and non-hybrid actomyosin. The degree of ATPase inhibition was consistent with previously published data but strongly dependent on the environmental conditions and source of actin and myosin used. Furthermore, at low concentrations, calponin could induce the ATPase activity of hybrid actomyosin. This result was consistent with data indicating that calponin can modulate actin conformation to increase the relative content of “switched on” actin monomers in thin filaments. We assume that calponin obtained by the isolation method proposed herein is a fully functional protein that can both inhibit and induce the ATPase activity. |
| first_indexed | 2024-03-09T03:20:29Z |
| format | Article |
| id | doaj.art-92fccadeeca94ddb9d91f5e070f7b942 |
| institution | Directory Open Access Journal |
| issn | 1661-6596 1422-0067 |
| language | English |
| last_indexed | 2024-03-09T03:20:29Z |
| publishDate | 2022-07-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | International Journal of Molecular Sciences |
| spelling | doaj.art-92fccadeeca94ddb9d91f5e070f7b9422023-12-03T15:10:42ZengMDPI AGInternational Journal of Molecular Sciences1661-65961422-00672022-07-012314799310.3390/ijms23147993A Preparative Method for the Isolation of Calponin from Molluscan Catch MuscleStanislav S. Lazarev0Ulyana V. Shevchenko1Vyacheslav A. Dyachuk2Ilya G. Vyatchin3Laboratory of Cell Biophysics, A.V. Zhirmunsky National Scientific Center of Marine Biology, Far Eastern Branch, Russian Academy of Sciences, 17 Palchevsky Str., 690041 Vladivostok, RussiaLaboratory of Cell Biophysics, A.V. Zhirmunsky National Scientific Center of Marine Biology, Far Eastern Branch, Russian Academy of Sciences, 17 Palchevsky Str., 690041 Vladivostok, RussiaLaboratory of Cell Biophysics, A.V. Zhirmunsky National Scientific Center of Marine Biology, Far Eastern Branch, Russian Academy of Sciences, 17 Palchevsky Str., 690041 Vladivostok, RussiaLaboratory of Cell Biophysics, A.V. Zhirmunsky National Scientific Center of Marine Biology, Far Eastern Branch, Russian Academy of Sciences, 17 Palchevsky Str., 690041 Vladivostok, RussiaWe describe the development of a preparative method to isolate molluscan catch muscle, calponin. This method is based on the ability of calponin to interact with actin in a temperature-dependent manner. After extracting thin filaments, as previously described, the extract was ultracentrifuged at 2 °C. While other surface proteins of thin filaments co-precipitated with actin, calponin, along with some minor contaminants, remained in the supernatant. Calponin was purified through cation-exchange chromatography. The yield of pure protein was four-fold higher than that achieved through high-temperature extraction. To evaluate functionally isolated proteins, we determined the effect of calponin on Mg<sup>2+</sup>-ATPase activity of hybrid and non-hybrid actomyosin. The degree of ATPase inhibition was consistent with previously published data but strongly dependent on the environmental conditions and source of actin and myosin used. Furthermore, at low concentrations, calponin could induce the ATPase activity of hybrid actomyosin. This result was consistent with data indicating that calponin can modulate actin conformation to increase the relative content of “switched on” actin monomers in thin filaments. We assume that calponin obtained by the isolation method proposed herein is a fully functional protein that can both inhibit and induce the ATPase activity.https://www.mdpi.com/1422-0067/23/14/7993molluscan catch musclesmolluscan thin filamentscatch muscle calponin extractionactin-activated myosin Mg<sup>2+</sup>-ATPase activity |
| spellingShingle | Stanislav S. Lazarev Ulyana V. Shevchenko Vyacheslav A. Dyachuk Ilya G. Vyatchin A Preparative Method for the Isolation of Calponin from Molluscan Catch Muscle International Journal of Molecular Sciences molluscan catch muscles molluscan thin filaments catch muscle calponin extraction actin-activated myosin Mg<sup>2+</sup>-ATPase activity |
| title | A Preparative Method for the Isolation of Calponin from Molluscan Catch Muscle |
| title_full | A Preparative Method for the Isolation of Calponin from Molluscan Catch Muscle |
| title_fullStr | A Preparative Method for the Isolation of Calponin from Molluscan Catch Muscle |
| title_full_unstemmed | A Preparative Method for the Isolation of Calponin from Molluscan Catch Muscle |
| title_short | A Preparative Method for the Isolation of Calponin from Molluscan Catch Muscle |
| title_sort | preparative method for the isolation of calponin from molluscan catch muscle |
| topic | molluscan catch muscles molluscan thin filaments catch muscle calponin extraction actin-activated myosin Mg<sup>2+</sup>-ATPase activity |
| url | https://www.mdpi.com/1422-0067/23/14/7993 |
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