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Monomeric and trimeric structures of active Na,K-ATPase in C12E8 solution.
Biochemical and Biophysical Research Communications
|May 31, 1983
Summary
Horse kidney Na,K-ATPase exists as both a trimer and monomer. These distinct molecular forms were identified using advanced chromatography and light scattering techniques, revealing their oligomeric states.
Area of Science:
- Biochemistry
- Molecular Biology
- Enzymology
Background:
- Na,K-ATPase is a crucial ion pump in kidney cells.
- Understanding its oligomeric state is key to its function.
- Detergent solubilization is often necessary to study membrane proteins like Na,K-ATPase.
Purpose of the Study:
- To determine the molecular weight and oligomeric state of purified horse kidney Na,K-ATPase.
- To characterize different active forms of Na,K-ATPase present after purification.
Main Methods:
- High-performance gel chromatography (HPLC) coupled with on-line low angle laser light scattering (LS) and refractive index (RI) detection.
- Measurement of refractive index increment (dn/dc) for molecular weight determination.
- Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and glycerol density gradient centrifugation.
Main Results:
- Two peaks with Na,K-ATPase activity were detected by HPLC, with estimated molecular weights of approximately 535K (peak-2) and 175K (peak-3).
- Both peaks contained alpha and beta subunits but not gamma subunits, as shown by SDS-PAGE.
- Analysis of LS/RI and LS/UV ratios indicated peak-2 represents a trimer ((alpha beta)3) and peak-3 represents a monomer (alpha beta).
Conclusions:
- Horse kidney Na,K-ATPase exists in both monomeric and trimeric forms in solution.
- These distinct oligomeric states likely reflect different functional or regulatory states of the enzyme.