Heat Inactivation of the Relaxing Site of Actomyosin: Prevention and Reversal with Dithiothreitol

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Abstract
Adenosine triphosphate and magnesium (MgATP) inhibit contraction by binding to a specific relaxing site on natural actomyosin gel. This inhibitory control site is distinct from the active sites where MgATP causes contraction. In high concentrations of MgATP, calcium triggers contraction by releasing the protein from substrate inhibition, allowing the contractile reactions to occur. Heating the protein for 5 minutes at 43°C selectively inactivates the relaxing site. After this treatment, actomyosin with MgATP contracts as well without calcium as with it. That this effect of heat is prevented and reversed by dithiothreitol (an agent that reduces disulfide bonds) indicates that the structure of the relaxing site depends on certain labile sulfhydryl groups, which may be those of tropomyosin. When these are oxidized to disulfide bonds, the site loses its activity; when the disulfide bonds are reduced, the site regains its activity.