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Ascorbate-induced high-affinity binding of copper to cytosolic proteins
1Department of Biochemistry, Wakayama Medical University, 811-1 Kimiidera, Wakayama 641-0012, Japan.
Biochemical and Biophysical Research Communications
|September 28, 2001
Summary
Cytosolic proteins, particularly protein thiols, effectively bind and sequester copper within cells. Ascorbate significantly enhances this copper-binding capacity and affinity in mouse brain and liver tissues.
Area of Science:
- Biochemistry
- Cell Biology
- Metalloprotein Chemistry
Background:
- Copper is essential for cellular function, requiring chaperones for intracellular transport.
- The cellular environment can sequester essential metals like copper.
- Understanding copper binding to cytosolic proteins is crucial for cellular homeostasis.
Purpose of the Study:
- To investigate copper binding characteristics of whole cytosolic proteins from mouse brain and liver.
- To determine the effect of ascorbate on copper binding capacity and affinity.
- To compare the copper-chelating ability of cytosolic proteins with glutathione (GSH).
Main Methods:
- Fluorometry utilizing a fluorescent Cu(II) chelator.
- Centrifugal ultrafiltration to study copper binding.
- Assessment of copper-catalyzed ascorbate oxidation to determine redox activity.
Main Results:
- Ascorbate markedly increased the binding capacity and affinity of cytosolic proteins for copper.
- Mouse brain cytosolic protein exhibited high-affinity binding (63 nmol copper/mg), with over half being redox-inactive.
- The majority of bound copper was in the Cu(I) state, coordinated to protein thiols.
- Cytosolic proteins demonstrated stronger copper competition than GSH at physiological concentrations.
Conclusions:
- Protein thiols in the cytosol possess a strong capacity to sequester copper.
- Ascorbate enhances the intracellular copper-binding potential of cytosolic proteins.
- Cytosolic proteins play a significant role in intracellular copper homeostasis.