Removal of troponin C and desensitization of myosin B from ascidian smooth muscle by treatment with ethylene diamine tetraacetate.
On treatment with 10 mM EDTA at 30 degrees C, protein of 18,000 daltons was released from myofibrils, thin filaments and myosin B prepared from the smooth muscle of an ascidian, Halocynthia roretzi. This protein was purified from the EDTA extract of myofibrils by differential centrifugation, freeze-drying and gel-filtration. Based on its molecular weight, electrophoretic mobilities in the presence and absence of Ca2+ and other properties, it was identified as troponin C. By EDTA treatment, ascidian myosin B lost the Ca2+-sensitivity of Mg2+-ATPase, and EDTA-treated myosin B recovered the sensitivity by mixing with the EDTA extract of myosin B in the presence of Mg2+. Gel-electrophoretic patterns indicated that desensitization and resensitization of ascidian myosin B were accompanied by the removal and binding of troponin C. These results indicate that ascidian smooth muscle is regulated by a troponin-tropomyosin system, and desensitization induced by EDTA treatment is due to the removal of troponin C but not the release of the light chains of the myosin molecule. Based on these findings, we have established a simple method for the purification of troponin C from ascidian smooth muscle.
D000251:Adenosine Triphosphatases / Q000378:metabolism; D000818:Animals; D004492:Edetic Acid; D004586:Electrophoresis / Q000379:methods; D009130:Muscle, Smooth / Q000032:analysis; D009218:Myosins / Q000302:isolation & purification*; D012967:Sodium Dodecyl Sulfate; D014336:Troponin / Q000302:isolation & purification*; D019209:Troponin C; D014508:Urea; D014561:Urochordata / Q000032:analysis*
D014336:Troponin; D019209:Troponin C; D012967:Sodium Dodecyl Sulfate; D014508:Urea; D004492:Edetic Acid; D000251:Adenosine Triphosphatases; D009218:Myosins