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ADP release from myosin in permeabilized smooth muscle
T M Butler1, D S Pacifico, M J Siegman
1Department of Physiology, Jefferson Medical College, Philadelphia, Pennsylvania 19107.
Abstract:
The purpose of this study was to determine the nucleotide bound to myosin and its rate of release under relaxed and activated conditions in permeabilized rabbit portal veins. Incubation of the muscles in a relaxing solution containing [3H]-ATP resulted in the formation of 60-70 microM radiolabeled ADP in the muscle whether or not the myosin light chains had been thiophosphorylated. This value was similar to the estimate of the concentration of myosin subfragment 1. Upon transfer of the muscles to a chase solution containing no labeled ATP, there was a very slow loss of labeled ADP when the light chains were unphosphorylated, but a much faster release occurred when the light chains were thiophosphorylated. The results suggest that smooth muscle myosin exists primarily in a complex with ADP under both relaxed and phosphorylated conditions and that phosphorylation of all of the light chains results in a large increase in the rate of release of the products of ATP splitting from all of the myosin. Interestingly, the exponential release of ADP in relaxed muscle shows two components, one of which contains about two-thirds of the total ADP and is 5- to 10-fold faster than the other. If the difference in rates of ADP release observed in relaxed muscle persists when the myosin is phosphorylated, then it is possible that there is a 5- to 10-fold difference in rates of cycling for different phosphorylated cross bridges in smooth muscle.
Insights
Smooth muscle myosin binds ADP. Phosphorylation of myosin light chains significantly accelerates ADP release, impacting ATP hydrolysis rates and cross-bridge cycling dynamics.
Area of Science:
- Biochemistry
- Muscle Physiology
- Molecular Biology
Background:
- Smooth muscle contraction is regulated by myosin phosphorylation.
- The nucleotide state of myosin is crucial for its enzymatic activity and force generation.
- Understanding myosin-ADP interactions provides insights into muscle function.
Purpose of the Study:
- To determine the nucleotide bound to myosin in rabbit portal veins.
- To measure the rate of nucleotide release under relaxed and activated conditions.
- To investigate the effect of myosin light chain thiophosphorylation on nucleotide release.
Main Methods:
- Permeabilized rabbit portal vein smooth muscle preparation.
- Incubation with radiolabeled ATP ([3H]-ATP) to label bound nucleotides.
- Transfer to a chase solution to measure the rate of radiolabeled ADP release.
- Measurement of ADP concentration and comparison to myosin subfragment 1 concentration.
Main Results:
- Radiolabeled ADP formed in the muscle, indicating myosin-ADP complex formation.
- ADP release was slow for unphosphorylated myosin light chains but significantly faster for thiophosphorylated light chains.
- ADP release in relaxed muscle exhibited two components with different rates.
- Phosphorylation of all light chains greatly increased the rate of ADP release.
Conclusions:
- Smooth muscle myosin exists mainly as a myosin-ADP complex in both relaxed and activated states.
- Myosin light chain phosphorylation dramatically enhances the release of ATP hydrolysis products.
- Potential for differential cross-bridge cycling rates based on phosphorylation status and ADP release kinetics.