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Inhibition of actomyosin ATPase by vanadate
Abstract:
Actin-myosin subfragment-1 (SF-1) or actin-heavy meromyosin is dissociated by the binding of ADP and vanadate (Vi) under conditions such that ADP alone does not dissociate the complex. The association constant of the stable complex M.ADP.Vi, in which M indicates myosin [Goodno, C. C. (1979) Proc. Natl. Acad. Sci. USA 76, 2620-2624] with actin is smaller than the average association constant of the intermediate states of the actin-SF-1 ATPase cycle. Actin-SF-1 ATPase activity is 90% inhibited by ADP plus vanadate. The reaction of actin with M.ADP.Vi produces a slow release of ADP and vanadate and quantitative recovery of ATPase activity. The rate of dissociation of ligands was almost linear in actin concentration; consequently, the rate constant of dissociation could only be roughly estimated as 0.5-1 sec-1. The rate of dissociation of ADP and vanadate is thus increased by a factor of 10(5) compared to M.ADP.Vi. The rate of release of ligands by regulated actin (actin-tropomyosin-troponin) was reduced to 1/10th to 1/20th by removal of calcium ion. Therefore the M.ADP.Vi complex has the properties of a more stable analogue of the myosin-ADP-phosphate complex that is generated in the normal ATPase cycle. The activation of ligand release (ratio of rate of dissociation of ADP and vanadate from actomyosin relative to myosin) is much larger than the activation of myosin ATPase by actin, whereas the actual rates of the reactions are much slower.
Insights
ADP and vanadate binding to actin-myosin subfragment-1 (SF-1) creates a stable complex that inhibits ATPase activity. This complex mimics myosin-ADP-phosphate, with ligand release accelerated by actin.
Area of Science:
- Biochemistry
- Molecular Biology
- Muscle Physiology
Background:
- Actin and myosin interaction is central to muscle contraction and ATP hydrolysis.
- Myosin subfragment-1 (SF-1) is a key component in studying actin-myosin dynamics.
- The role of ADP and vanadate (Vi) in stabilizing myosin complexes is not fully understood.
Purpose of the Study:
- To investigate the properties of the stable actin-myosin subfragment-1 complex formed with ADP and vanadate (M.ADP.Vi).
- To determine if the M.ADP.Vi complex serves as an analogue for the myosin-ADP-phosphate intermediate in the actin-myosin ATPase cycle.
- To elucidate the effect of actin on the dissociation rates of ADP and vanadate from myosin.
Main Methods:
- Formation and characterization of the stable actin-myosin subfragment-1 complex with ADP and vanadate.
- Measurement of actin-SF-1 ATPase activity in the presence and absence of ADP and vanadate.
- Analysis of the dissociation kinetics of ADP and vanadate from the actin-myosin complex.
- Comparison of ligand release rates in regulated actin (with tropomyosin-troponin) versus unregulated actin.
Main Results:
- ADP and vanadate binding stabilizes actin-myosin subfragment-1, inhibiting ATPase activity by 90%.
- The M.ADP.Vi complex formation is reversible, leading to recovery of ATPase activity upon slow ligand release.
- Actin significantly accelerates the dissociation of ADP and vanadate from myosin, by a factor of 10^5 compared to M.ADP.Vi alone.
- Ligand release from regulated actin is slower, dependent on calcium ions.
Conclusions:
- The M.ADP.Vi complex acts as a stable analogue of the myosin-ADP-phosphate state in the actin-myosin ATPase cycle.
- Actin binding dramatically enhances the rate of ligand release from myosin, a key step in force generation.
- The regulation of ligand release by calcium in the actin-tropomyosin-troponin system highlights the complexity of muscle contraction control.