title: ATP binding and cross-bridge detachment steps during full Ca²⁺ activation: comparison of myofibril and muscle fibre mechanics by sinusoidal analysis.
abstract: Single myofibrils 50–60 μm length and 2–3 μm diameter were isolated from rabbit psoas muscle fibres, and cross-bridge kinetics were studied by small perturbations of the length (∼0.2%) over a range of 15 frequencies (1–250 Hz). The experiments were performed at 15◦C in the presence of 0.05–10 mM MgATP, 8mM phosphate (Pi), 200 mM ionic strength with KAc (acetate), pCa 4.35–4.65, and pH 7.0. Two exponential processes, B and C, were resolved in tension transients. Their apparent rate constants (2πb and 2πc) increased as the [MgATP] was raised from 0.05 mM to 1mM, and then reached saturation at [MgATP] ≥ 1. Given that these rate constants were similar (c/b ∼1.7) at [Pi] ≥ 4 mM, they were combined to achieve an accurate estimate of the kinetic constants: their sum and product were analysed as functions of [MgATP]. These analyses yielded K1 =2.91 ± 0.31 mM −1, k2 =288 ± 36 s−1, and k−2 =10 ± 21 s−1 (±95% confidence limit, n =13 preparations), based on the cross-bridge model: AM+ATP ↔ (step 1) AM.ATP ↔ (step 2) A+M.ATP, where K1 is the ATP association constant (step 1), k2 is the rate constant of the cross-bridge detachment (step 2), and k−2 is the rate constant of its reversal step. These kinetic constants are respectively comparable to those observed in single fibres from rabbit psoas (K1 =2.35 ± 0.31 mM −1, k2 =243 ± 22 s−1, and k−2 =6 ± 14 s−1; n =8 preparations) when analysed by the same methods and under the same experimental conditions. These values are respectively not significantly different from those obtained in myofibrils, indicating that the same kinetic constants can be deduced from myofibril and muscle fibre studies, in terms of ATP binding and cross-bridge detachments steps. The fact that K1 in myofibrils is 1.2 times that in fibres (P≈0.05) may be explained by a small concentration gradient of ATP, ADP and/or Pi in single fibres.
authors.affiliation: Institute of Vegetative Physiology, University of Cologne, 50931 Cologne, Germany.
authors.forename: Bogdan
authors.initials: B
authors.lastname: Iorga
authors.forename: Li
authors.initials: L
authors.lastname: Wang
authors.forename: Robert
authors.initials: R
authors.lastname: Stehle
authors.forename: Gabriele
authors.initials: G
authors.lastname: Pfitzer
authors.forename: Masataka
authors.initials: M
authors.lastname: Kawai
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism*; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D007700:Kinetics; D009119:Muscle Contraction; D018485:Muscle Fibers, Skeletal / Q000378:metabolism*; D009129:Muscle Tonus; D009210:Myofibrils / Q000378:metabolism*; D016658:Psoas Muscles / Q000502:physiology; D011817:Rabbits
chemical_list: D000255:Adenosine Triphosphate; D002118:Calcium