Kinetic studies of calcium binding to regulatory complexes from skeletal muscle.
The kinetic mechanism of the binding and release of calcium by troponin and by the complexes troponin: tropomyosin, troponin:tropomyosin:actin, and troponin (TN)-tropomyosin (TM)-actin:myosin subfraction 1 (SF-1) was investigated using troponin labeled on the TN-I subunit with the fluorophore 4-(N-iodoac etoxyethyl-N-methyl)-7-nitrobenz-2-oxa-1,3-diazole. The apparent association constant is five to 10 times smaller for TN:TM:actin compared to TN:TM or TN and saturation of actin sites with SF-1 increased the binding constant approximately to the value for TN:TM. Kinetic measurements on TN or TN:TM fitted a single rate process for association or dissociation which is consistent with a model in which the calcium sites are equivalent and independent and each calcium induces a change in structure of the complex. TN:TM:actin gave biphasic transients for association and dissociation of calcium. The two binding sites are no longer equivalent and independent. The TN:TM:actin:SF-1 complex gave kinetic behavior essentially equivalent to TN:TM. The kinetics of calcium dissociation from the various complexes was also measured by the fluorescent calcium indicator quin 2, which gave the same values for the rate constants as for the labeled protein. The evidence is interpreted in terms of a model in which regulated actin can exist in two states and the binding of each calcium and SF-1 displaces the equilibrium between states. Formation of the complex of TN:TM with actin yielded an enhancement of the fluorescence of the labeled TN-I moiety of approximately 30%. The rate of constant for association of the complex decreased 6-fold in the presence of calcium while the rate constant for dissociation of the protein complex was essentially unchanged. Saturation of actin sites with SF-1 had no effect on the rate constant for association with TN:TM in the presence of calcium.
D000199:Actins / Q000378:metabolism; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D003285:Contractile Proteins / Q000378:metabolism*; D005456:Fluorescent Dyes; D007700:Kinetics; D008433:Mathematics; D008954:Models, Biological; D009132:Muscles / Q000378:metabolism*; D009218:Myosins / Q000378:metabolism; D010069:Oxadiazoles; D011817:Rabbits; D014335:Tropomyosin / Q000378:metabolism; D014336:Troponin / Q000378:metabolism
D000199:Actins; D003285:Contractile Proteins; D005456:Fluorescent Dyes; D010069:Oxadiazoles; D014335:Tropomyosin; D014336:Troponin; C035573:4-(N-(iodoacetoxy)ethyl-N-methyl)amino-7-nitrobenz-2-oxa-1,3-diazole; D009218:Myosins; D002118:Calcium