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Magnesium adenosine 5'-diphosphate influences proteolytic susceptibility of myosin in myofibrils
Abstract:
The proteolytic susceptibility of the subfragment 2/light meromyosin junction [heavy meromyosin (HMM) junction] of myosin was employed as a probe of the cross-bridge conformation. The proteolysis was carried out in the myofibrils where myosin assembled in arrays typical of the in vivo organization. When subfragment I formation was inhibited by saturating the Nbs2 [5,5'-dithiobis(2-nitrobenzoic acid)] light chains with Mg2+ ions, chymotrypsin attacked exclusively the HMM junction. The rate of this attack was assessed by measuring the rate of HMM formation by quantitative polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and by following absorbance changes associated with the solubilization of myofibrillar suspensions. Under rigor conditions, the myofibrils were relatively resistant to the chymotryptic attack. The presence of MgAMP-PNP or MgPPi did not affect the rate of proteolytic attack. On the other hand, binding of MgADP had a powerful stimulating influence on the HMM site digestibility. The dissociation constant for the effect of MgADP was 10 microM less than Kd less than 50 microM. MgADP did not exercise its unique effect through destabilization of myosin filaments or through dissociation of the actomyosin complex. These results are explained in terms of a change in the myosin cross-bridge conformation brought about by the binding of MgADP to the active site.
Insights
Myosin cross-bridge conformation changes are revealed by studying how enzymes break down myosin. MgADP binding significantly increases the breakdown rate at a specific junction, indicating a conformational shift.
Area of Science:
- Muscle physiology
- Protein biochemistry
- Biophysics
Background:
- Myosin cross-bridge conformation is crucial for muscle contraction.
- Understanding these conformational changes requires specific molecular probes.
- Myofibrils provide an in vivo-like environment for studying myosin organization.
Purpose of the Study:
- To investigate myosin cross-bridge conformation using proteolytic susceptibility.
- To probe the subfragment 2/light meromyosin (HMM) junction of myosin.
- To determine the influence of nucleotide binding on myosin structure.
Main Methods:
- Proteolysis of myosin in myofibrils using chymotrypsin.
- Inhibition of subfragment I formation using Mg2+ saturated Nbs2 light chains.
- Quantification of heavy meromyosin (HMM) formation via SDS-PAGE and absorbance changes.
- Assessment of proteolytic susceptibility under different nucleotide conditions (rigor, MgAMP-PNP, MgPPi, MgADP).
Main Results:
- Chymotrypsin selectively attacked the HMM junction when subfragment I formation was inhibited.
- Myofibrils were resistant to proteolysis under rigor conditions.
- MgAMP-PNP and MgPPi did not alter the proteolytic attack rate.
- MgADP binding strongly stimulated HMM site digestibility, with a dissociation constant between 10-50 microM.
- MgADP's effect was independent of myosin filament destabilization or actomyosin dissociation.
Conclusions:
- MgADP binding induces a significant conformational change in the myosin cross-bridge.
- This conformational change enhances the susceptibility of the HMM junction to proteolysis.
- The study provides insights into the molecular mechanisms of muscle contraction regulation by nucleotides.