Studies on calcium ion-induced conformation changes in the actin-tropomyosin-troponin system by fluorimetry: II. Effect of tropomyosin, troponin and calcium ion on conformation of anilinonaphtylmaleimide-labeled F-actin

Abstract

F-actin labeled with N-(1-anilinonaphthyl-4) maleimide, an SH-directed fluorescent probe, was used to analyse the transmission of the effect of Ca 2+ from troponin to F-actin. 1. 1.|The fluorescence emission maximum wavenumber of anilinonaphthyl-maleimide-labeled F-actin (2.29 · 10 4 · cm −1-2.27·10 4 cm −1) indicated that hydrophobicity of the microenvironments around the thiol(s) bound with the dye is 72–76 in terms of the Z value. The emission maximum did not change by combination of the actin with tropomyosin or tropomyosin-troponin complex in the presence and absence of Ca 2+, but the quantum yield and fluorescence lifetime significantly changed. The lifetime of the labeled F-actin-tropomyosin complex was restored to the level for F-actin-tropomyosin complex by adding 10 −5 M CaCl 2, but recovery of the quantum yield was incomplete. 2. 2.|The apparent Ca 2+-binding constant obtained from the change in fluorescence lifetime, 1.3·10 6 M, was in good agreement with the reported values obtained from different methods. 3. 3.|In isothermal experiments, no viscosity dependence of the fluorescence polarization, P, was detected for F-actin and all its complexes. 4. 4.|Thermal dependence of P revealed that a thermally activated portion involving the fluorescently labeled thiol(s), the rotational relaxation time of which was about 40 ns, appeared on addition of tropomyosin. A thermal transition in conformation around the labeled region was also observed on adding tropomyosin, with the transition temperature of 31 °C. 5. 5.|On addition of Ca 2+ to the F-actin-tropomyosin-troponin system, the thermally activated portion and the thermal transition in conformation completely disappeared. Based on the results mentioned above, the conformation changes at the labeled region are discussed in relation to the transmission of Ca 2+ effect on troponin to actin.