CROSSBRIDGE SCHEME AND THE KINETIC CONSTANTS OF ELEMENTARY STEPS DEDUCED FROM CHEMICALLY SKINNED PAPILLARY AND TRABECULAR MUSCLES OF THE FERRET

Elementary steps of the crossbridge cycle in chemically skinned ferret myocardium were investigated with sinusoidal analysis. The muscle preparations were activated at pCa 4.82 and an ionic strength of 200 mM, and the effects of the change in the MgATP (S) and phosphate (Pi) concentrations on three exponential processes were studied at 20-degrees-C. Results are consistent with the following crossbridge scheme: [GRAPHICS] where A is actin, M is myosin, D is MgADP, and Det includes all detached states (MS and MDP) and weakly attached states (AMS and AMDP). From our studies, we obtained K1a=0.99 mM-1 (MgATP association), k1b=270 s-1 (ATP isomerization), k-1b=280 s-1 (reverse isomerization), K1b=k1b/k-1b=0.95, k2=48 s-1 (crossbridge detachment), k-2=14 s-1 (reverse detachment), K2=3.5, k4=11 s-1 (crossbridge attachment), k-4=107 s-1 (reverse attachment), K4=0.11, and K5=0.06 mM-1 (Pi association). k6 is the rate-limiting step, and it is the slowest forward reaction in the cycle, which results in the rigorlike AM state. K1a (MgATP binding) is four times that of rabbit psoas, and K5 (Pi binding) is 0.3 times that of psoas, demonstrating that crossbridges in myocardium bind MgATP more and Pi less than psoas. The rate constants of ATP isomerization (k1b, k-1b), Crossbridge detachment (k2, k-2), and crossbridge attachment (k4) steps are generally an order of magnitude slower than rabbit psoas. The reverse attachment step (k-4) is similar to that in psoas, indicating that this step may occur irrespective of the myosin type and possibly spontaneously. The above scheme with the deduced kinetic constants predicts the following crossbridge distributions at 5 mM MgATP2- and 8 mM Pi: AM (3%), AM dagger S (15%0, AM*S (14%), Det (50%), AM*DP (6%), and AM*D (12%). The actual number of attached crossbridges was measured to be 51+/-4% by the stiffness ratio during activation and after rigor induction, and a strong correlation was seen with the prediction. Our results are consistent with the hypothesis that force generation occurs at the Det-->AM*DPi transition, and the same force is maintained after the release of Pi.