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Effect of pH on the mechanism of actin polymerization
1Department of Biological Chemistry, Washington University School of Medicine, St. Louis, Missouri 63110.
Abstract:
The effect of pH on the Mg2+-induced polymerization of rabbit skeletal muscle G-actin at 20 degrees C was examined. Polymerization data were obtained at various initial concentrations of Mg2+, Ca2+, and G-actin between pH 6 and 7.5. The data were found to fit a kinetic mechanism for actin polymerization previously proposed at pH 8 in which Mg2+ binding at a moderate-affinity site on actin induces an isomerization of the protein enabling more favorable nucleation [Frieden, C. (1982) J. Biol. Chem. 257, 2882-2886]. The data also suggest the formation of actin dimers induced by Mg2+ binding is over 2 orders of magnitude more favorable at pH 6 than at pH 8. Little effect on trimer formation is found over this pH range. In addition, the conformation induced by nonspecific binding of metal to low-affinity sites becomes more favorable as the pH is lowered. The critical concentration for filament formation is also decreased at lower pH. The kinetic data do not support fragmentation occurring under any of the conditions examined. Furthermore, as Mg2+ exchange for Ca2+ at a high-affinity site (Kd less than 10(-9) M) fails to alter significantly the polymerization kinetics, Ca2+ release from this site appears unnecessary for either the nucleation or the elongation of actin filaments.
Insights
Lowering pH significantly enhances magnesium-induced actin polymerization by promoting dimer formation and favorable nucleation. This study clarifies the pH-dependent kinetics of G-actin polymerization, crucial for understanding muscle function.
Area of Science:
- Biochemistry
- Molecular Biology
- Protein Polymerization
Background:
- Actin polymerization is essential for muscle contraction and cellular structure.
- Magnesium ions (Mg2+) are known to induce G-actin polymerization.
- The influence of pH on these polymerization kinetics remains incompletely understood.
Purpose of the Study:
- To investigate the effect of pH on Mg2+-induced G-actin polymerization.
- To elucidate the kinetic mechanisms governing actin polymerization across a pH range.
- To determine the role of specific Mg2+ binding sites and Ca2+ in polymerization.
Main Methods:
- Kinetic analysis of rabbit skeletal muscle G-actin polymerization.
- Experiments conducted at 20°C across a pH range of 6 to 7.5.
- Varying initial concentrations of Mg2+, Ca2+, and G-actin were employed.
Main Results:
- Mg2+-induced actin polymerization kinetics fit a previously proposed mechanism involving nucleation.
- Actin dimer formation is over 100 times more favorable at pH 6 compared to pH 8.
- Lowering pH decreases the critical concentration for filament formation and favors conformations from nonspecific metal binding.
Conclusions:
- pH significantly impacts the kinetics of Mg2+-induced actin polymerization, primarily by enhancing dimer formation and nucleation.
- Ca2+ release from a high-affinity site is not essential for actin filament nucleation or elongation.
- The findings provide a detailed kinetic model for actin polymerization influenced by pH and divalent cations.