Oligomycin interaction with Na,K-ATPase: oligomycin binding and dissociation are slow processes

Area of Science:

• Biochemistry
• Enzyme kinetics
• Membrane transport

Background:

• The Na,K-ATPase (sodium-potassium adenosine triphosphatase) is a crucial ion pump in cell membranes.
• Oligomycin is known to affect ATPases, but its precise interaction mechanism with Na,K-ATPase requires elucidation.

Purpose of the Study:

• To investigate the interaction kinetics between oligomycin and Na,K-ATPase.
• To determine the rate constants for oligomycin binding and dissociation from Na,K-ATPase.

Main Methods:

• Utilized stop-flow fluorimetry with eosin as a fluorescent marker for the E1 state (Na+ bound) of Na,K-ATPase.
• Measured changes in eosin fluorescence to indirectly determine rate constants for oligomycin interaction.

Main Results:

• Oligomycin binding to Na,K-ATPase increased apparent Na+ affinity in the non-phosphorylated state.
• Second-order rate constants for oligomycin binding were 6-12 x 10^4 M^-1 s^-1 at 6°C.
• Dissociation rate constants were approximately 0.05 s^-1 at 6°C.
• Calculated dissociation constants were ~1 µM (shark) and ~2 µM (pig kidney) at 6°C, pH 7.0.

Conclusions:

• The slow binding and dissociation kinetics suggest oligomycin interacts with Na,K-ATPase from the membrane lipid phase.
• Oligomycin's interaction influences the Na+ binding properties of Na,K-ATPase.