Calcium-binding properties of troponin C in detergent-skinned heart muscle fibers.
In order to obtain information with regard to behavior of the Ca2+ receptor, troponin C (TnC), in intact myofilament lattice of cardiac muscle, we investigated Ca2+-binding properties of canine ventricular muscle fibers skinned with Triton X-100. Analysis of equilibrium Ca2+-binding data of the skinned fibers in ATP-free solutions suggested that there were two distinct classes of binding sites which were saturated over the physiological range of negative logarithm of free calcium concentration (pCa): class I (KCa = 7.4 X 10(7) M-1, KMg = 0.9 X 10(3) M-1) and class II (KCa = 1.2 X 10(6) M-1, KMg = 1.1 X 10(2) M-1). The class I and II were considered equivalent, respectively, to the Ca2+-Mg2+ and Ca2+-specific sites of TnC. The assignments were supported by TnC content of the skinned fibers determined by electrophoresis and 45Ca autoradiograph of electroblotted fiber proteins. Dissociation of rigor complexes by ATP caused a downward shift of the binding curve between pCa 7 and 5, an effect which could be largely accounted for by lowering of KCa of the class II sites. When Ca2+ binding and isometric force were measured simultaneously, it was found that the threshold pCa for activation corresponds to the range of pCa where class II sites started to bind Ca2+ significantly. We concluded that the low affinity site of cardiac TnC plays a key role in Ca2+ regulation of contraction under physiological conditions, just as it does in the regulation of actomyosin ATPase. Study of kinetics of 45Ca washout from skinned fibers and myofibrils revealed that cardiac TnC in myofibrils contains Ca2+-binding sites whose off-rate constant for Ca2+ is significantly lower than the Ca2+ off-rate constant hitherto documented for the divalent ion-binding sites of either cardiac/slow muscle TnC or fast skeletal TnC.
D000818:Animals; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism*; D002132:Calcium Radioisotopes; D003902:Detergents; D004285:Dogs; D006352:Heart Ventricles / Q000378:metabolism; D066298:In Vitro Techniques; D007700:Kinetics; D008433:Mathematics; D009206:Myocardium / Q000378:metabolism*; D009210:Myofibrils / Q000378:metabolism; D011485:Protein Binding; D014336:Troponin / Q000378:metabolism*; D019209:Troponin C
D002132:Calcium Radioisotopes; D003902:Detergents; D014336:Troponin; D019209:Troponin C; D002118:Calcium