title: Two step mechanism of phosphate release and the mechanism of force generation in chemically skinned fibers of rabbit psoas muscle.
abstract: The elementary steps of contraction in rabbit fast twitch muscle fibers were investigated with particular emphasis on the mechanism of phosphate (Pi) binding/release, the mechanism of force generation, and the relation between them. We monitor the rate constant 2 pi b of a macroscopic exponential process (B) by imposing sinusoidal length oscillations. We find that the plot of 2 pi b vs. Pi concentration is curved. From this observation we infer that Pi released is a two step phenomenon: an isomerization followed by the actual Pi release. Our results fit well to the kinetic scheme: [formula: see text] where A = actin, M = myosin, S = MgATP (substrate), D = MgADP, P = phosphate, and Det is a composite of all the detached and weakly attached states. For our data to be consistent with this scheme, it is also necessary that step 4 (isomerization) is observed in process (B). By fitting this scheme to our data, we obtained the following kinetic constants: k4 = 56 s-1, k-4 = 129 s-1, and K5 = 0.069 mM-1, assuming that K2 = 4.9. Experiments were performed at pCa 4.82, pH 7.00, MgATP 5 mM, free ATP 5 mM, ionic strength 200 mM in K propionate medium, and at 20 degrees C. Based on these kinetic constants, we calculated the probability of each cross-bridge state as a function of Pi, and correlated this with the isometric tension. Our results indicate that all attached cross-bridges support equal amount of tension. From this, we infer that the force is generated at step 4. Detailed balance indicates that 50-65% of the free energy available from ATP hydrolysis is transformed to work at this step. For our data to be consistent with the above scheme, step 6 must be the slowest step of the cross-bridge cycle (the rate limiting step). Further, AM*D is a distinctly different state from the AMD state that is formed by adding D to the bathing solution. From our earlier ATP hydrolysis data, we estimated k6 to be 9 s-1.
authors.affiliation: Department of Anatomy, University of Iowa, Iowa City 52242.
authors.forename: M
authors.initials: M
authors.lastname: Kawai
authors.forename: H R
authors.initials: HR
authors.lastname: Halvorson
mesh_terms: D000199:Actins / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism*; D000818:Animals; D004548:Elasticity; D066298:In Vitro Techniques; D007537:Isometric Contraction / Q000187:drug effects; D007700:Kinetics; D008433:Mathematics; D008962:Models, Theoretical; D009119:Muscle Contraction; D009132:Muscles / Q000502:physiology*; D009218:Myosins / Q000378:metabolism; D010710:Phosphates / Q000378:metabolism* / Q000494:pharmacology; D011817:Rabbits
chemical_list: D000199:Actins; D010710:Phosphates; D000255:Adenosine Triphosphate; D009218:Myosins