Differences in the Mechanisms of Calcium Regulation of the Acceleration of ADP Dissociation from Myosin-ADP and Myosin-ADP-Pi by Native Cardiac Thin Filaments

Abstract

We have used double mixing stopped-flow fluorescence to measure the kinetics of the dissociation of the hydrolysis products deoxymantADP (mdADP) from cardiac myosin-mdADP and cardiac myosin-mdADP-Pi by native cardiac thin filaments. Increasing the calcium concentration increases the rate of dissociation of mdADP from cardiac myosin-S1-ADP-Pi ∼100 fold from 0.5 s−1 at pCa > 7 to 50 s−1 at pCa < 4. Increasing the calcium concentration increases the rate of dissociation of mdADP from cardiac myosin-S1-ADP-Pi only 10 fold from 15 s−1 at pCa > 7 to 150 s−1 at pCa < 4. These results indicate that slow dissociation of phosphate limits the rate of ADP dissociation from acto(thinfilaments)myosin-ADP-Pi and that there are different mechanisms for the calcium regulation of dissociation of the two products of myosin ATP hydrolysis, ADP and phosphate. These results support a mechanism in which the step of the hydrolysis cycle that is principally regulated by calcium is phosphate dissociation from actomyosin-ADP-Pi and do not support a mechanism such as the three state mechanism in which the regulation is a result different distributions of thin filament states in presence and absence of bound calcium that occur prior to myosin binding. This work is supported by a NIH HL84604.