Effect of myosin cross-bridge interaction with actin on the Ca2(+)-binding properties of troponin C in fast skeletal myofibrils.
Ca2+ binding to fast skeletal muscle troponin C reincorporated into troponin C-depleted (CDTA-treated) myofibrils has been measured directly by using 45Ca and indirectly by using a fluorescent probe. Direct Ca2(+)-binding measurements have shown that the Ca2+ affinity of the low-affinity sites is enhanced in the absence of ATP and conversely reduced when myosin is selectively extracted from myofibrils, compared to the Ca2+ affinity in the presence of ATP. Fluorescence intensity changes of a dansylaziridine label at the Met-25 residue of troponin C have shown the same Ca2(+)-sensitivity whether or not ATP is present, while much lower Ca2(+)-sensitivity is seen in the myosin-extracted myofibrils. Since the Met-25 residue is in the amino terminal side alpha-helix of Ca2(+)-binding site I and far from Ca2(+)-binding site II in the primary structure, Ca2+ binding to site II has been evaluated by assuming that the fluorescence change monitors Ca2+ binding to site I alone. Ca2+ binding to site II thus estimated has shown high positive cooperativity only in the presence of ATP and has been found to be nearly proportional to the activation of myofibrillar ATPase, suggesting that Ca2(+)-binding site II is directly involved in the activation of myofibrillar ATPase activity. On the other hand, Ca2(+)-binding site I has been suggested to regulate the interaction of weakly binding cross-bridges with the thin filament, since the fluorescence change in the presence of ATP is saturated at the free Ca2+ concentration required for the activation of myofibrillar ATPase.
Morimoto
S
S
Department of Pharmacology, Faculty of Medicine, Kyushu University, Fukuoka.
D000199:Actins / Q000378:metabolism*; D000251:Adenosine Triphosphatases / Q000378:metabolism; D000818:Animals; D001388:Aziridines; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism*; D003619:Dansyl Compounds; D005456:Fluorescent Dyes; D066298:In Vitro Techniques; D007700:Kinetics; D008297:Male; D009210:Myofibrils / Q000378:metabolism; D009218:Myosins / Q000378:metabolism*; D011817:Rabbits; D014336:Troponin / Q000378:metabolism*; D019209:Troponin C
D000199:Actins; D001388:Aziridines; D003619:Dansyl Compounds; D005456:Fluorescent Dyes; D014336:Troponin; D019209:Troponin C; C017173:dansylaziridine; D000251:Adenosine Triphosphatases; D009218:Myosins; D002118:Calcium