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Acylphosphate formation by the Menkes copper ATPase
1Department of Clinical Pharmacology, University of Berne, Switzerland. solioz@ikp.unibe.ch
Abstract:
The Menkes ATPase is the product of the MNK gene, defective in some inherited human disorders of copper metabolism. We here show the formation of an acylphosphate intermediate by the murine MNK homologue in membranes from normal and copper resistant Chinese hamster ovary cells. In the latter, fivefold higher levels of acylphosphate were formed. Challenging these cells with copper, which induces relocation of the MNK ATPase from the trans-Golgi network to the plasma membrane, did not influence acylphosphate formation. The kinetics of phosphorylation, metal dependence, and sensitivity to inhibitors were investigated. The results show that the MNK ATPase is an active P-type ATPase and provide a direct functional test for this enzyme.
Insights
The Menkes ATPase enzyme, crucial for copper metabolism, forms an acylphosphate intermediate. This finding provides a direct functional test for this P-type ATPase, important in inherited copper disorders.
Area of Science:
- Biochemistry
- Molecular Biology
- Cell Biology
Background:
- The Menkes ATPase, encoded by the MNK gene, is implicated in inherited human copper metabolism disorders.
- Understanding the enzymatic activity of the Menkes ATPase is critical for elucidating these conditions.
Purpose of the Study:
- To investigate the enzymatic activity of the murine Menkes ATPase homologue.
- To characterize the formation of an acylphosphate intermediate by the Menkes ATPase.
- To establish a functional assay for the Menkes ATPase.
Main Methods:
- Enzyme assays were performed on membranes from normal and copper-resistant Chinese hamster ovary cells.
- Acylphosphate formation was measured under varying conditions.
- Kinetics of phosphorylation, metal dependence, and inhibitor sensitivity were analyzed.
Main Results:
- The murine Menkes ATPase homologue formed an acylphosphate intermediate in cell membranes.
- Copper-resistant cells exhibited fivefold higher acylphosphate formation compared to normal cells.
- Copper-induced relocation of the Menkes ATPase did not affect acylphosphate formation.
- Detailed kinetic and inhibitor sensitivity data were obtained.
Conclusions:
- The Menkes ATPase functions as an active P-type ATPase.
- Acylphosphate intermediate formation serves as a direct functional test for Menkes ATPase activity.
- This research offers insights into copper metabolism and related genetic disorders.