Abstract
Actin and tropomyosin were labeled with fluorescent thiol reagents, N- (1-anilinonaphthy 1-4) maleimide (ANM) or N- (p- (2-benzimidazolyl) phenyl) maleimide (BIPM), and the transmission of the effect of Ca2+ from troponin to F-actin on the thin filament was studied fluorometrically. 1. The chemical modification of actin and tropomyosin did not essentially alter their physiological functions. 2. The fluorescence characteristics of labeled actin were altered by combination with H-meromyosin and by G-F transformation. On the other hand, those of labeled tropomyosin were altered by combination with actin or troponin. 3. The microenvironment around the thiol (s) bound with the dye in actin was appresiably hydrophobic. However, its polarity did not change by combination with tropomyosin or tropomyosin-troponin complex in the presence and absence of Ca2+. Under the same conditions, on the other hand, quantum yield and fluorescence lifetime significantly changed. The latter of the ANM-F-actin-tropomyosin-troponin complex was restored to the level for F-actin-tropomyosin complex by adding 10-5 M CaCl2, but the former was incompletely recovered. 4. The apparent Ca2+ -binding constant obtained from the change in lifetime, 1.3×106 M, was in good agreement with the value reported on troponin alone. 5. Thermal dependence studies on the fluorescence polarization of ANM-F-actin revealed that a thermally activated portion and thermal transition of structure at 31°C appeared on addition of tropomyosin and/or troponin. On addition of Ca2+ to the complete system, the above phenomena completely disappeared. 6. Quantum yield of ANM-tropomyosin decreased by complex formation with either skeletal or cardiac troponin. Those of ANM-tropomyosin-troponin complexes further decreased by the same degree on addition of 10-4M CaCl2 or SrCl2 in the presence of 1mM MgCl2. From the above results it can be concluded that a conformation change in troponin first triggered by Ca2+-binding is transmitted to the actin molecule via tropomyosin without any leakage,.
