Biochemical and Biophysical Research Communications
Nucleotide dependent differences between the α-skeletal and α-cardiac actin isoforms
Section snippets
Materials and methods
Sample preparation. The cardiac actin was prepared from acetone powder of bovine heart muscle [21], [22]. The calcium saturated actin monomers were stored in a 2 mM Mops buffer (pH 8.0) containing 0.2 mM ATP, 0.1 mM CaCl2, 0.1 mM β-mercaptoethanol and 0.005% NaN3. The concentration of the actin monomers was calculated by using the extinction coefficient of 0.63 mg−1 ml cm−1 at 290 nm [23]. The actin bound calcium was changed for magnesium by incubating the samples for 5 min in the presence of 0.2 mM EGTA
Sample characterisation
Actin filaments prepared from the actin monomers of cardiac muscle cells were investigated with the method of DSC. Before the polymerisation process the ATP was replaced with ADP in the binding-pocket of the actin monomers (Fig. 1). The critical concentration of the ADP-actin is ∼ 1 μM [32], [33], therefore, the contribution of the ADP-actin monomers to the amount of the total actin was so small (<1.5%) that their effect on the calorimetric results was negligible under the applied conditions.
The comparison of the α-cardiac and α-skeletal actin isoforms
One of the characteristic features of the DSC results was the appearance of the two peaks in the heat absorption curve (Fig. 3). A possible explanation for this observation could be that the nucleotide exchange was not complete during the preparation of ADP-actin monomers, and the samples contained ATP-actin before the polymerisation. It is unlikely that the method fails to replace the bound ATP with ADP in the case of the α-cardiac actin isoform because the method of nucleotide exchange is
Conclusion
In this study the comparison of the α-actin isoforms revealed that the filaments of the α-skeletal actin were more resistant to heat denaturation than the α-cardiac filaments, when they were polymerised from ADP-actin monomers. This difference was not found in filaments polymerised from ATP-actin monomers, which indicates that the conformational differences between the isoforms were nucleotide dependent. The structure and the dynamics of proteins can be related to their biological functions [41]
Acknowledgements
We are grateful to Professor Béla Somogyi for the support he produced during the preparation of this study. This work was supported by the Hungarian Academy of Sciences and by grants from the Hungarian Scientific Research Fund (OTKA Grant No. K60186 and K60968 (Miklós Nyitrai)). The SETARAM Micro DSC-II was purchased with a Grant (CO-272) from the Hungarian Scientific Research Fund (Dénes Lőrinczy). Miklós Nyitrai holds a Wellcome Trust International Senior Research Fellowship in Biomedical
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