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Purification of active Na+-K+-ATPase using a new ouabain-affinity column
D R Yingst1, S Y Yang, R Schiebinger
1Department of Physiology, Division of Endocrinology, Wayne State University School of Medicine, Detroit, Michigan 48201, USA.
The American Journal of Physiology
|October 1, 1998
Summary
Researchers developed an ouabain-affinity column to purify Na+-K+-ATPase (sodium-potassium adenosine triphosphatase). This method successfully isolated active enzyme from rat kidney and adrenal cells, demonstrating its utility in biochemical studies.
Area of Science:
- Biochemistry
- Molecular Biology
- Enzymology
Background:
- Na+-K+-ATPase (sodium-potassium adenosine triphosphatase) is a crucial ion pump involved in maintaining cellular homeostasis.
- Specific inhibitors like ouabain are essential tools for studying Na+-K+-ATPase activity and function.
- Developing affinity chromatography methods can enhance the purification and characterization of membrane-bound enzymes.
Purpose of the Study:
- To create and validate an affinity column for the specific isolation of Na+-K+-ATPase using ouabain.
- To investigate the binding and elution characteristics of Na+-K+-ATPase based on enzyme conformation and ligand interactions.
- To purify active Na+-K+-ATPase from complex biological sources like kidney microsomes and adrenal cells.
Main Methods:
- Coupling ouabain to epoxy agarose via a spacer to create an affinity chromatography column.
- Binding and eluting Na+-K+-ATPase from rat and dog kidney using specific ligand combinations (Na+, ATP, Mg2+, K+).
- Assessing enzyme activity and phosphorylation status post-purification using specific activity measurements and [32P]orthophosphate labeling.
Main Results:
- The ouabain-affinity column successfully bound and eluted Na+-K+-ATPase under specific ionic and ligand conditions, mirroring untethered ouabain binding.
- Competitive elution with Na+/ouabain or Na+/ATP allowed for the recovery of Na+-K+-ATPase, with Na+/ATP eluting active enzyme.
- Purification from rat kidney microsomes increased enzyme specific activity significantly, and phosphorylation of the alpha-subunit was observed in purified adrenal enzyme.
Conclusions:
- The developed ouabain-affinity column is an effective tool for purifying active Na+-K+-ATPase.
- Enzyme conformation, influenced by ligands, dictates binding affinity to the ouabain-affinity matrix.
- This purification method facilitates further studies on Na+-K+-ATPase structure, function, and regulation in various cell types.
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