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Myosin subfragment 1 activates ATP hydrolysis on Mg(2+)-G-actin
1Centre de Recherches de Biochimie Macromoléculaire, CNRS-INSERM U. 249, Université de Montpellier I, France.
Abstract:
The interaction of myosin subfragment 1 isoenzyme A2 (S1A2) with Mg(2+)-G-actin was studied. Polarization titrations of 1,5-IAEDANS-Mg(2+)-G-actin and of epsilon ATP-Mg(2+)-G-actin with S1A2 provided evidence that, similar to Ca(2+)-G-actin, the proteins form a tight binary complex. Significant amounts of oligomeric forms of actin in the presence and absence of S1 were not detected. The effect of S1A2 on the rates of nucleotide and metal dissociation and hydrolysis from Mg(2+)-actin was measured. The hydrolysis rate for [gamma-32P]ATP-actin in the G-acto-S1A2 complex (k- = 0.016 s-1) was faster than the rate of 32P liberation from the complex (k- = 0.004 s-1), obtained by measuring the liberation of [32P]orthophosphate from [alpha-32P]ATP-actin in the presence of a large excess of alkaline phosphatase. This indicates that most of actin's ATP was hydrolyzed before it was released to solution and that the dissociating nucleotide was ADP, for which the dissociation rate is higher than that for ATP. In agreement with this mechanism, S1A2 accelerated the dissociation of epsilon ATP but inhibited the dissociation of epsilon ADP from the complex. The activation of actin's ATPase is specific for Mg(2+)-G-actin and does not occur in Ca(2+)-G-actin. The effect of deoxyribonuclease I on the rates of nucleotide dissociation and hydrolysis was examined.(ABSTRACT TRUNCATED AT 250 WORDS)
Insights
Myosin subfragment 1 isoenzyme A2 (S1A2) forms a tight complex with Mg(2+)-G-actin, accelerating ATP hydrolysis. This interaction is specific for Mg(2+)-G-actin, not Ca(2+)-G-actin.
Area of Science:
- Biochemistry
- Molecular Biology
- Muscle Contraction
Background:
- Actin and myosin are key proteins in muscle contraction.
- Understanding their interaction is crucial for elucidating muscle function.
- Myosin subfragment 1 (S1) is a functional unit of myosin that binds actin.
Purpose of the Study:
- To investigate the interaction between myosin subfragment 1 isoenzyme A2 (S1A2) and Mg(2+)-G-actin.
- To determine the effect of S1A2 on nucleotide and metal ion binding and hydrolysis kinetics.
- To explore the specificity of this interaction regarding different actin-metal complexes.
Main Methods:
- Polarization titrations using fluorescently labeled actin (1,5-IAEDANS-Mg(2+)-G-actin and epsilon ATP-Mg(2+)-G-actin) with S1A2.
- Measurement of nucleotide and metal dissociation rates.
- Quantification of ATP hydrolysis rates using radiolabeled ATP and alkaline phosphatase.
Main Results:
- S1A2 forms a tight binary complex with Mg(2+)-G-actin, similar to Ca(2+)-G-actin.
- Actin's ATP hydrolysis rate within the S1A2 complex is significantly faster than nucleotide dissociation.
- S1A2 accelerates ATP dissociation but inhibits ADP dissociation, indicating hydrolysis precedes release.
- The ATPase activation by S1A2 is specific to Mg(2+)-G-actin and absent in Ca(2+)-G-actin.
Conclusions:
- Myosin subfragment 1 isoenzyme A2 (S1A2) forms a stable complex with Mg(2+)-G-actin, facilitating ATP hydrolysis.
- The hydrolysis of ATP by actin is a key step in the actin-myosin interaction cycle.
- The observed specificity for Mg(2+)-G-actin highlights the distinct roles of divalent cations in actin-myosin dynamics.