Regulation of Binding of Myosin Subfragments with Regulated Actin by Calcium Ions in the Presence of Magnesium ATP1

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Abstract
Previously, several workers reported that at very low ionic strength and in the presence of ATP, the extent of binding of S-1 with the F-actin-tropomyosin-tro-ponin complex or regulated actin (FA-TM-TN) is unaffected by removal of Ca2+. However, in this study we found that during the ATPase reaction at physiological ionic strength, the extent of binding of HMM with FA-TM-TN decreased markedly upon removal of Ca2+. Therefore, the effects of Ca2+ were studied on the intermediate steps in the acto-HMM or acto-S-1 ATPase reaction. 1. The nucleotide-induced dissociation of acto-S-1 was studied using AMPPNP as substrate. The extent of binding of S-1 with regulated actin in the presence of Mg2+-AMPPNP increased in a sigmoidal manner as the S-1 concentration increased. When the molar ratio of actin monomer to S-1 was higher than 5-10, the removal of Ca2+ shifted the equilibrium of the dissociation reaction, FA-S-1-AMPPNP⇌FA+ S-1 -AMPPNP, to the right. 2. The recombination rate of HMMpADP or S-1pADP with regulated actin in the absence of free Mg2+-ATP was estimated by measuring the time course of recovery in light-scattering intensity after addition of ATP. The rate decreased upon removal of Ca2+, when the molar ratio of actin monomer to S-1 was higher than 5-10. 3. The decomposition rate of HMMpADP was measured in the presence of Mg2+-ATP. In the absence of Ca2+, regulated actin did not affect this rate, whereas in its presence, regulated actin markedly accelerated the rate. These findings clearly indicated that at physiological ionic strength, removal of Ca2+ affects various elementary steps in the ATPase reaction to promote the dissociation of myosin heads from FA-TM-TN.