Related Experiment Videos
The effects of pyrophosphate on myosin heads and on fiber stiffness
1Department of Biochemistry and Biophysics, University of California, San Francisco 94143-0524.
Abstract:
We have investigated the effect of magnesium pyrophosphate (MgPPi) on the orientation and stiffness of myosin heads in glycerinated muscle fibers. MgPPi is known to bind to the nucleotide site of myosin and to weaken the bond between myosin and actin. The spectra of paramagnetic probes attached to the reactive sulfhydryl on the myosin head were used to monitor the orientation of the head. Addition of 3 mM MgPPi to rigor fibers resulted in a shift of approximately 50% of the myosin heads from a state in which the probes are highly oriented to a state in which the probes are disordered. Experiments in which MgPPi dissociated labeled subfragment-1 from unlabeled fibers support the conclusion that the disordered probes are on myosin heads that are not attached to actin. Addition of MgPPi causes only a small decrease in fiber stiffness, measured by step changes in fiber length. We conclude that up to 50% of the myosin heads can be dissociated from actin with little change in muscle stiffness. Thus stiffness is not a linear measure of the fraction of myosin heads attached to actin. We present a model in which one head of the pair of a myosin molecule binds more weakly to actin and can be dissociated preferentially by the binding of MgPPi.
Insights
Magnesium pyrophosphate (MgPPi) detaches up to 50% of myosin heads from muscle fibers with minimal impact on stiffness. This suggests muscle stiffness doesn't solely depend on myosin head attachment.
Area of Science:
- Muscle physiology
- Biochemistry
- Molecular motor function
Background:
- Myosin heads bind to actin filaments to generate muscle force.
- Magnesium pyrophosphate (MgPPi) is known to interact with the myosin nucleotide-binding site.
- Understanding myosin head detachment is crucial for muscle function research.
Purpose of the Study:
- To investigate the effect of MgPPi on myosin head orientation and muscle fiber stiffness.
- To determine the relationship between myosin head attachment and muscle stiffness.
- To elucidate the mechanism of MgPPi-induced myosin head dissociation.
Main Methods:
- Utilized glycerinated muscle fibers.
- Employed paramagnetic probes attached to myosin heads to assess orientation via spectral analysis.
- Measured muscle fiber stiffness through step changes in fiber length.
- Performed experiments dissociating labeled subfragment-1 from fibers.
Main Results:
- Addition of 3 mM MgPPi caused approximately 50% of myosin heads to shift from an oriented to a disordered state.
- Disordered probes indicated myosin heads detached from actin.
- MgPPi addition resulted in only a minor decrease in muscle fiber stiffness.
- Dissociation of labeled subfragment-1 confirmed MgPPi's effect on myosin-actin binding.
Conclusions:
- Up to 50% of myosin heads can detach from actin with minimal change in muscle stiffness.
- Muscle stiffness is not a direct linear indicator of the fraction of attached myosin heads.
- A model is proposed where one myosin head in a pair binds more weakly and is preferentially dissociated by MgPPi.