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Kinetics of oligomycin inhibition and activation of Na+/K(+)-ATPase
1Danish Biomembrane Research Center, Aarhus.
Abstract:
Oligomycin inhibition of the maximal hydrolysis activity of ox brain Na+/K(+)-ATPase was studied at varying NaCl concentrations and it was found that for a given amount of live enzyme, the observed inhibition of a particular total oligomycin concentration decreased as the amount of added, (heat-) denatured enzyme increased. In the present article we derive a scale factor for the oligomycin concentration, i.e., the fraction of the total concentration of oligomycin which is free in solution, as a function of the enzyme concentration used. This fraction decreased linearly with the protein concentration and may attain quite small values. We also study the Na(+)-dependence of the hydrolysis rate at saturating substrate concentrations ([Mg2+] = [ATP] = 3 mM), in the presence as well as the absence of KCl, at various concentrations of oligomycin. These data may be explained if it is assumed that the sole effect of oligomycin is to confer upon the enzyme an increased affinity for Na+, i.e., oligomycin merely enhances the inhibitory effect of Na+ on the (maximal) activity seen at high Na(+)-concentrations. The increased Na(+)-affinity in the presence of oligomycin should result in activation of the hydrolysis rate measured under conditions where Na(+)-activation is predominant, i.e., at low Na(+)-concentration and sub-saturating substrate concentrations. This prediction is verified for both Na(+)-ATPase and for Na+/K(+)-ATPase. This proposed action of oligomycin seems to be corroborated also by other evidence discussed in the text.
Insights
Oligomycin enhances the sodium (Na+) affinity of Na+/K(+)-ATPase, increasing its activity at low Na+ concentrations. This effect was observed in both Na+-ATPase and Na+/K(+)-ATPase, suggesting a consistent mechanism of action for oligomycin.
Area of Science:
- Biochemistry
- Enzymology
- Membrane Transport
Background:
- Na+/K(+)-ATPase is a crucial enzyme for maintaining ion gradients across cell membranes.
- Oligomycin is known to inhibit ATPases, but its precise mechanism of action on Na+/K(+)-ATPase requires further elucidation.
- Understanding oligomycin's interaction with Na+/K(+)-ATPase is vital for comprehending cellular energy metabolism and ion transport regulation.
Purpose of the Study:
- To investigate the mechanism by which oligomycin inhibits the maximal hydrolysis activity of ox brain Na+/K(+)-ATPase.
- To determine the relationship between oligomycin concentration, enzyme concentration, and free oligomycin in solution.
- To elucidate the effect of oligomycin on the Na+-dependence of Na+/K(+)-ATPase activity.
Main Methods:
- Enzyme kinetic studies were performed on ox brain Na+/K(+)-ATPase at varying NaCl concentrations.
- The study involved measuring hydrolysis activity in the presence and absence of KCl and varying oligomycin concentrations.
- A scale factor was derived to quantify the fraction of free oligomycin as a function of enzyme concentration.
Main Results:
- The observed inhibition by oligomycin decreased as the concentration of denatured enzyme increased, indicating oligomycin binding to the active enzyme.
- The fraction of free oligomycin decreased linearly with increasing protein concentration.
- Oligomycin was found to increase the enzyme's affinity for Na+, enhancing the inhibitory effect of Na+ at high concentrations and activating the enzyme at low Na+ concentrations.
Conclusions:
- Oligomycin's primary effect is to increase the Na+ affinity of Na+/K(+)-ATPase, rather than directly inhibiting its maximal activity.
- This increased Na+ affinity explains the observed activation of Na+/K(+)-ATPase at low Na+ concentrations.
- The findings provide a new perspective on oligomycin's role in modulating ATPase activity and cellular ion transport.