ATP, uncomplexed by divalent cations, and the LC2 light chain are interdependent modifiers of the skeletal actomyosin MgATPase activity

Abstract

In the absence of troponin and tropomyosin, skeletal actomyosin MgATPase activity can be altered by 2-3-fold by divalent cations. The 'sign' of this effect (i.e. inhibition or activation) varies with ionic strength. To investigate the mechanism, P(i) liberation was analysed at both low and high ionic strength with three concentrations of MgATP and over a wide range of Mg2+ concentrations. This procedure separated the effects of two dependent variables, Mg2+ and ATP4-/3- (ATPfree), to provide the following observations. (1) ATPfree, not Mg2+ (nor Ca2+), was the modifier. (2) ATPfree was an activator at low ionic strength and an inhibitor at high ionic strength, with half-maximal activation/inhibition occurring between 0.75 and 0.8 mM-ATPfree. (3) The rate constants controlling Vmax. with respect to actin were increased up to 3-fold by ATPfree at low ionic strength, and decreased up to 3-fold by ATPfree at high ionic strength. (4) The effect of ATPfree required near-native levels of the LC2 light chain bound to myosin (i.e. 2 mol of LC2/mol of myosin). (5) Sensitivity of P(i) liberation to a 50% decrease in the LC2 content of myosin required high ATPfree concentrations. It is concluded that LC2 and ATPfree are interdependent, non-additive, modifiers of MgATPase. These results are consistent with thin filament regulation of skeletal muscle contraction, and begin to explain why both positive and negative effects on MgATPase have been attributed to LC2.

Slowing effects of Mg 2+ on contractile kinetics of skinned preparations of rat hearts depending on myosin heavy chain isoform content

“…The concentration of free ATP (=ATP 4− +HATP 3− ) was 1.3 and 0.8 mM in our 0.5 and 1 mM free Mg 2+ solutions, respectively. Although unlikely, it cannot be excluded that the observed deceleration of contractile kinetics is based on a decrease of free ATP concentration [39]. In living cells changes of free Mg 2+ probably occur under the same concomitant circumstances as in our artificial solutions, i.e.…”