Multimeric species in equilibrium in detergent-solubilized Na,K-ATPase

Area of Science:

• Biochemistry
• Biophysics
• Membrane protein biochemistry

Background:

• Na,K-ATPase (NKA) is a crucial ion pump involved in maintaining cellular homeostasis.
• Understanding the oligomeric state of NKA is essential for elucidating its function and regulation.
• Solubilization in detergents can alter the native structure and oligomeric state of membrane proteins.

Purpose of the Study:

• To investigate the oligomeric equilibrium of NKA solubilized in C12E8 detergent.
• To correlate NKA oligomeric states with its enzymatic activity.
• To understand the impact of protein concentration and detergent concentration on NKA aggregation.

Main Methods:

• Dynamic Light Scattering (DLS)
• Analytical Ultracentrifugation (AUC)
• Small Angle X-ray Scattering (SAXS)
• Spectrophotometry
• Enzymatic activity assays

Main Results:

• AUC identified seven populations of NKA, with monomers and tetramers initially comprising ~55% of the mass.
• Increasing NKA concentration shifted the equilibrium, reducing monomers and tetramers to <40%.
• Filtering aggregates increased specific enzymatic activity, but NKA reformed aggregates over time.
• High detergent concentrations led to NKA dissociation into inactive smaller aggregates.

Conclusions:

• C12E8-solubilized NKA exists in a dynamic equilibrium of monomers, tetramers, and inactive higher-order aggregates.
• Higher-order aggregates and dissociated forms exhibit reduced or null enzymatic activity.
• NKA aggregation is a time-dependent process that impacts its functional state.