Abstract
The rate and equilibrium constants for the formation and dissociation of the bovine ventricular (BV) actomyosin-S1-ADP have been measured by stopped flow light scattering. A comparison of the rate constants obtained here with those for rabbit skeletal (RS) actomyosin-S1 indicates that there are large differences in several of the rate and equilibrium constants. 1) The rate constant of ADP dissociation from BV actomyosin-S1 is 65 +/- 10 s-1 at 15 degrees C compared to a lower limit of 500 s-1 previously observed for RS actomyosin-S1. 2) The association constant for ADP binding to actomyosin-S1 is increased from 6 X 10(3) M-1 for RS to 1.5 X 10(5) M-1 for BV at 15 degrees C. The following rate and equilibrium constants differ by less than a factor of 2 between RS and BV actomyosin-S1: 1) the second order rate constant for the dissociation of actomyosin-S1 by MgATP; 2) the second order rate constant of myosin-S1 and myosin-S1-ADP binding to actin; and 3) the association constant of myosin-S1 to actin. The rate constant for ADP dissociation from BV actomyosin-S1 is at least 10-fold greater than the Vmax for the steady state ATPase and therefore cannot be the rate-limiting step of ATP hydrolysis. However, at physiological temperature, 38 degrees C, and ATP concentration, greater than 3 mM, ADP dissociation is sufficiently slow to limit the rate of myosin-S1 dissociation from actin by ATP and is likely to be the rate-limiting step of cross-bridge dissociation in muscle. Moreover, the rate constant of ADP dissociation is sufficiently slow to be the molecular step which limits the unloaded shortening velocity in cardiac muscle.
Highlights
The rate and equilibrium constants for the formatiionnEquation 1 havebeenmeasuredfor RS myosin-S1 and and dissociation of the bovine ventricular (BV) acto- actomyosin-Sl,butthere is considerablyless information myosin-S1-ADP have been measured by stopped flow available regarding the kineticsof the mechanismfor cardiac light scattering
The following rate andequilibrium constants differ previously measured for BV myosin-Sl (6,7).I n general, they by less than a factor of 2 between RS and BV acto- are equalin magnitude to within a factor of 2 of those myosin-S1: 1)the second order rate constant for the measured for RS myosin-Sl
At physiological temperature, 38 “C, and ATP concentration, >3 mM, ADP dissociation is sufficiently slow to ATPase of actomyosin-S1 in solution is that the rate and equilibrium constants obtainedmay berelated to the contractile properties of muscle
Summary
At standard low ionic strength conditions (I = 0.02), the second order rate constant kA is observed to be 2 X lo[7] M". There is good agreement between the values obtained by methods which depend upon measurement of the concentration of the actomyosin-S1complex (light scatteringand kinetic) whereas methods measuring the concentration of free myosin-Sl (centrifugation and fluorescence depolarization) tend to give somewhat lower values. Both the apparent second order rate constant k A and association constant Ka observed for various actomyosin-SI species display essentially the same ionic. Myosin-S1 type and experimental method are: BV, stopped flow and steady state light scattering, this paper (0,O)R;S, stopped flow light scattering and fluorescence (21) (0,a);RS, stopped flow light scattering (14) (A, A);RS, stopped flow light scattering, K A values determined according to detailed balance from values for K A N , K N A , and KN ( N is ADP or AMP-PNP)
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