Formation of Acto-H-meromyosin and Acto-subfragment-1 Complexes and Their Dissociation by Adenosine Triphosphate*

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Abstract
1. The binding ratios of heavy meromyosin (HMM) and subfragment-1 (S-l) of myosin to F-actin were measured by light-scattering and ultracentrifugal methods. The ratios were one mole of HMM per mole of actin dimer with a dissociation constant of 1.6′ 2.6×10−7M and one mole of S-l per mole of actin monomer. They were not affected by the method of preparation of actin or the KC1 and MgCl2 concentrations. The distribution of HMM on the F-actin filament was analyzed by electron microscopy. Some acto-H-meromyosin preparations showed a random distribution of HMM on F-actin, while others showed a distribution suggesting cooperative binding of HMM to F-actin. 2. The amounts of the initial burst of P1-liberation from the HMM-ATP and the S-l-ATP systems were one mole and 0.5 mole per mole of protein, respectively. The values were not affected by ionic strength. 3. The decrease in light-scattering intensity after adding ATP to acto-HMM or acto-S-l complexes was measured by a stopped flow method. The minimum amounts of ATP required for the maximum decrease in light-scattering intensity were 2.2 moles and 1.0mole per mole of HMM and S-l, respectively. 4. From these results it was concluded that each of the two head parts of HMM has one binding site for actin, and that the binding of one site with actin is dissociated by formation of the HMM-phosphate-ADP complex, while the binding of the other site is broken by simple binding of ATP.

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