Structural changes in (Na+ + K+)-ATPase accompanying detergent inactivation

Area of Science:

• Biochemistry
• Membrane Protein Structure
• Enzyme Kinetics

Background:

• The sodium-potassium ATPase ((Na+ + K+)-ATPase) is crucial for cellular ion transport.
• Understanding its inactivation mechanisms is key to comprehending enzyme function and stability.
• Detergent interactions can alter enzyme structure and activity.

Purpose of the Study:

• To investigate the structural changes in purified (Na+ + K+)-ATPase during detergent-induced inactivation.
• To elucidate the role of associated lipids in enzyme activity and stability.
• To differentiate between rapid and slow inactivation phases.

Main Methods:

• Monitoring light scattering and intrinsic protein fluorescence.
• Utilizing tryptophan to beta-parinaric acid fluorescence resonance energy transfer (FRET).
• Employing non-ionic detergents: digitonin, Lubrol WX, and Triton X-100.

Main Results:

• Two distinct inactivation phases were observed with different detergents.
• The rapid phase involved detergent insertion with minimal structural changes.
• The slow phase led to a 400,000-dalton inactive particle with 20 mol/mol associated phospholipid, indicating lipid dissociation and conformational changes.

Conclusions:

• At least 20 mol/mol of tightly associated lipid are essential for (Na+ + K+)-ATPase and p-nitrophenylphosphatase activity.
• The rate-limiting step in slow inactivation involves the dissociation of essential lipids.
• Digitonin's non-parallel inhibition does not require a change in the enzyme's oligomeric state.