OLIGOMYCIN INTERACTION WITH NA,K-ATPASE - OLIGOMYCIN BINDING AND DISSOCIATION ARE SLOW PROCESSES

Oligomycin interacts with the Na,K-ATPase by increasing the apparent Na+ affinity in the non-phosphorylated state of the enzyme. This property is used to estimate rate constants attributed to oligomycin binding and dissociation reactions with Na,K-ATPase. The rate constants are determined indirectly, employing stop-flow fluorimetry of eosin, the fluorescence of which is a marker for the E1 state of the enzyme, i.e. for Na+ binding. The second-order rate constants derived for oligomycin binding are in the range (6-12) . 10(4) M-1 s-1 at 6-degrees-C for both shark rectal gland and pig kidney enzyme. Rate constants for dissociation of the enzyme-oligomycin complex are about 0.05 s-1 at 6-degrees-C. The slow rates of binding and dissociation suggest that oligomycin acts from within the membrane lipid phase rather than from the aqueous phase. The dissociation constant at 6-degrees-C for the enzyme-oligomycin complex can be calculated to be about 1-mu-M for shark enzyme and about 2-mu-M for kidney enzyme, at pH 7.0 in 2 mM NaCl.