Kinetics of the conformational change of skeletal troponin C induced by Ca2+-Mg2+ exchange at the high-affinity Ca2+-binding sites.
The rate constant of the conformational change of skeletal troponin C (TnC) induced by the Ca2+ binding reaction with the high-affinity Ca2+-binding sites was determined in the presence of Mg2+ by the fluorescence stopped-flow method in 0.1 M KCl, 50 mM Na-cacodylate-HCl pH 7.0 at 20 degrees C. The [MgCl2] dependence of the rate constants of the observed biphasic conformational change leveled off at the high [MgCl2] region: the rate constants were 60 +/- 9 s-1 and 8 +/- 2 s-1, respectively. These values are larger than the rate constants of the biphasic fluorescence intensity change of TnC induced by Mg2+ removal reaction at the high-affinity Ca2+-binding sites (37 +/- 7 s-1 and 3.0 +/- 0.6 s-1) under the same experimental conditions. These results suggest that the Ca2+-Mg2+ exchange reaction at the high-affinity Ca2+-binding sites is faster than the resultant conformational change accompanying the fluorescence intensity change. Based on these results, we also reexamine the molecular kinetic mechanism of the conformational change of the protein induced by the Mg2+ binding or removal reaction with the high affinity Ca2+-binding sites of skeletal TnC.
Department of Physics, Faculty of Science, Nagoya University, Aichi.
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D000818:Animals; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism*; D007700:Kinetics; D008274:Magnesium / Q000378:metabolism*; D009132:Muscles / Q000378:metabolism; D011189:Potassium Chloride / Q000494:pharmacology; D011487:Protein Conformation; D011817:Rabbits; D013050:Spectrometry, Fluorescence; D014336:Troponin / Q000032:analysis*; D019209:Troponin C
D014336:Troponin; D019209:Troponin C; D011189:Potassium Chloride; D008274:Magnesium; D002118:Calcium