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Kinetic analysis of amyloid fibril polymerization in vitro
H Naiki1, K Higuchi, K Nakakuki
1Department of Pathology, Fukui Medical School, Matsuoka, Japan.
Abstract:
We investigated the polymerization kinetics of murine senile amyloid fibrils (fASSAM) in vitro. When sonicated murine senile amyloid fibrils was incubated with its constituent monomer protein, the extension of amyloid fibrils was observed in an electron microscopic analysis. Quantitative fluorometric analysis with thioflavine T (Naiki H, Higuchi K, Hosokawa M, Takeda T: Anal Biochem 177:244, 1989) revealed that (a) extension of amyloid fibrils occurred by a pseudo-first-order exponential increase in the fluorescence of thioflavine T; (b) the rate of extension was maximal around pH 7.5, and was inhibited with the increase in KCl or NaCl concentration in the reaction mixture; (c) the rate of polymerization was proportional to the product of the murine senile amyloid fibrils number concentration and the constituent monomer protein concentration; (d) the net rate of extension was the sum of the rates of polymerization and depolymerization with the equilibrium association constant K of 5 x 10(7) M-1. These results show that amyloid fibril formation can apparently be explained by a first-order kinetic model: that is, extension of amyloid fibrils proceeds by consecutive association of precursor proteins onto the ends of existing fibrils.
Insights
This study reveals that amyloid fibril extension follows a first-order kinetic model. Amyloid fibril formation involves monomer proteins adding to existing fibrils, a process influenced by pH and salt concentration.
Area of Science:
- Biochemistry
- Biophysics
- Molecular Biology
Background:
- Amyloid fibrils are implicated in various neurodegenerative diseases.
- Understanding the kinetics of amyloid fibril formation is crucial for developing therapeutic strategies.
Purpose of the Study:
- To investigate the in vitro polymerization kinetics of murine senile amyloid fibrils (fASSAM).
- To elucidate the mechanism of amyloid fibril extension at the molecular level.
Main Methods:
- Electron microscopy was used to observe fibril extension.
- Quantitative fluorometric analysis with thioflavine T was employed to measure polymerization rates.
Main Results:
- Amyloid fibril extension followed a pseudo-first-order kinetic model.
- Optimal extension rates were observed around pH 7.5.
- Polymerization rates were dependent on fibril and monomer concentrations and inhibited by increased salt concentrations (KCl, NaCl).
- The equilibrium association constant (K) was determined to be 5 x 10^7 M^-1.
Conclusions:
- Amyloid fibril formation can be explained by a first-order kinetic model.
- Fibril extension occurs through the sequential addition of monomer proteins to the ends of existing fibrils.