title: Reaction intermediates formed by myofibrils during the ATPase reaction under relaxed conditions.
abstract: The species and amounts of intermediates formed by myosin in myofibrils during the ATPase reaction under relaxed conditions were examined. The amount of total nucleotides (ADP + ATP) bound to myofibrils, determined by a centrifugation method or a rapid filtration method, was 0.86 mol/mol myosin head. The amount of bound ADP, determined as the ADP remaining in the mixture after free ADP had been rapidly converted into ATP by an ATP-regenerating system, was found to be 0.67 mol/mol myosin head. We examined the time courses of free-Pi and total-Pi (TCA-Pi) formation after adding ATP to the myofibrils. The amount of Pi bound to myofibrils, calculated by subtracting the burst size of free Pi (0.23 mol/mol myosin head) from that of TCA-Pi (0.60 mol/mol myosin head), was found to be 0.37 mol/mol myosin head. The amount of tightly bound ATP determined by an ATP-quenching method was very low (0.03 mol/mol myosin head). If there is no myosin-phosphate complex, then the amounts of the myosin-phosphate-ADP complex, MADPP, and the tightly bound myosin-ATP complex, M*ATP, are 0.37 and 0.03 mol/mol myosin head, respectively, whereas the amounts of myosin-ADP and loosely bound myosin-ATP complexes are 0.30 and 0.16 mol/mol myosin head, respectively. Thus, half of the myosin heads forms MADPP or M*ATP, and the equilibrium between MADPP and M*ATP shifts to the MADPP side. These results agree with those obtained for myosin in solution (Inoue, A., Takenaka, H., Arata, T., & Tonomura, Y. (1979) Adv. Biophys. 13, 1-194). Therefore, in relaxed myofibrils the active site of myosin does not interact with actin.
authors.affiliation: Department of Biology, Faculty of Science, Osaka University.
authors.forename: M
authors.initials: M
authors.lastname: Miyata
authors.forename: T
authors.initials: T
authors.lastname: Arata
authors.forename: A
authors.initials: A
authors.lastname: Inoue
mesh_terms: D000244:Adenosine Diphosphate / Q000378:metabolism; D000251:Adenosine Triphosphatases / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D066298:In Vitro Techniques; D009126:Muscle Relaxation; D009210:Myofibrils / Q000201:enzymology*; D010761:Phosphorus Radioisotopes; D011817:Rabbits; D013997:Time Factors
chemical_list: D010761:Phosphorus Radioisotopes; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases