Binary interactions of troponin subunits.
The association constants for the formation of the binary complexes of rabbit fast skeletal muscle troponin subunits have been determined for three solution conditions: (a) 1 mM CaCl2, (b) 3 mM MgCl2 and 1 mM EGTA, and (c) 2 mM EDTA. The subunits were labeled with extrinsic fluorescence probes, either 5-(iodoacetamido)eosin (IAE) or dansylaziridine (DANZ), and the binding was detected by enhancement or quenching of the probe fluorescence. The association constant for the TnI X TnT (where TnI and TnT are the inhibitory subunit and the tropomyosin-binding subunit, respectively, of troponin) complex was measured with two different probes, IAE-TnI and IAE-TnT. The measured values were not affected by the presence of Ca2+ or Mg2+, and the mean values for the three buffer conditions are, respectively, 8.0 X 10(6) and 9.0 X 10(6) M-1 for the two probes. The association constant for TnC-TnI (where TnC is the Ca2+-binding subunit of troponin) interaction was measured with three probes, IAE-TnC, DANZ-TnC, and IAE-TnI. Values of 1.7 X 10(9), 1.2 X 10(8), and 1.0 X 10(6) M-1 were obtained, respectively, in the presence of calcium ion, in the presence of magnesium ion (no calcium), and in the absence of divalent metal ions. A mean value of 4.0 X 10(7) M-1 was obtained for the association constant of TnC X TnT using DANZ-TnC and IAE-TnC as probes in the presence of calcium or magnesium ions. A value of 4.5 X 10(6) M-1 was obtained in the absence of divalent metal ions. The results show that the presence of magnesium ion in the Ca2+-Mg2+ sites strengthens the TnC-TnI and the TnC-TnT interactions and suggest that the troponin structure would be stabilized. This likely results from the effect of magnesium ion on the Ca2+-Mg2+ domains of TnC. The presence of calcium ion in the Ca2+-specific sites provides an additional binding free energy for the TnC-TnI interaction which presumably reflects the changes in the subunit interactions required for the calcium regulatory switch.
D000818:Animals; D001388:Aziridines / Q000378:metabolism; D002122:Calcium Chloride / Q000378:metabolism; D003619:Dansyl Compounds / Q000378:metabolism; D004492:Edetic Acid / Q000378:metabolism; D004533:Egtazic Acid / Q000378:metabolism; D004801:Eosine Yellowish-(YS) / Q000031:analogs & derivatives / Q000378:metabolism; D005456:Fluorescent Dyes / Q000378:metabolism; D046911:Macromolecular Substances; D008274:Magnesium / Q000378:metabolism; D015636:Magnesium Chloride; D009132:Muscles / Q000032:analysis; D011817:Rabbits; D014336:Troponin / Q000378:metabolism*; D019209:Troponin C; D019210:Troponin I; D020107:Troponin T
D001388:Aziridines; D003619:Dansyl Compounds; D005456:Fluorescent Dyes; D046911:Macromolecular Substances; D014336:Troponin; D019209:Troponin C; D019210:Troponin I; D020107:Troponin T; D015636:Magnesium Chloride; C017173:dansylaziridine; D004533:Egtazic Acid; C018572:iodoacetamidoeosin; D004492:Edetic Acid; D008274:Magnesium; D002122:Calcium Chloride; D004801:Eosine Yellowish-(YS)