Abstract
The mechanism whereby oligomycin occludes Na+ within Na/K-ATPase was investigated to study Na+ and K+ transport mechanisms. Oligomycin stimulated Na+ binding to Na/K-ATPase but inhibited Na-K and Na-Na exchange. The oligomycin concentration required to stimulate Na+ binding to half-maximal was 4.5 microM, which was close to the concentration that reduced Na-Na and Na-K exchange and ATPase activity to half-maximal, suggesting that Na/K-ATPase possesses an oligomycin binding site responsible for stimulating Na+ binding and reducing ion exchange and ATPase activity. In contrast, neither K+ binding nor K+ transport was affected by oligomycin. Limited tryptic digestion of Na/K-ATPase showed that, unlike Na+, K+, and ouabain, oligomycin treatment did not result in a specific digestion pattern. Oligomycin appeared to inhibit ouabain binding in a noncompetitive manner, whereas it did not affect ATP binding. Na/K-ATPase isoforms with low and high sensitivities to ouabain were equally sensitive to oligomycin. These results suggest that the oligomycin binding site is located on the extracellular side of Na/K-ATPase, at a different position from the ouabain binding site, and this antibiotic did not induce a conformational change of Na/K-ATPase. We propose that oligomycin interacts with the Na+ occlusion site from the extracellular side of Na/K-ATPase, which delays Na+ release to the extracellular side without inducing a conformational change, suggesting that the pathways responsible for Na+ and K+ transport differ.
Highlights
Na/K-ATPase is an integral membrane protein responsible for the active transport of Naϩ and Kϩ across the cell membrane using ATP as a driving force [1,2,3]
Characterization of the Oligomycin Binding Site—The oligomycin inhibition curves for Na-Na exchange, Na-K exchange, and ATPase activity and its stimulation curve for Naϩ binding were monophasic (Figs. 1, 2, and 8)
These results suggest strongly that Na/K-ATPase possesses an oligomycin binding site responsible for enhancing Naϩ binding and inhibiting Naϩ transport and ATPase activity and that its affinity for oligomycin binding was essentially unaffected by the phosphorylation of the enzyme
Summary
Microsomes were prepared from canine kidney outer medulla and treated with sodium deoxycholate, as described by Hayashi et al [24]. The ouabain-sensitive ATPase activity of the purified ATPase obtained was usually within the range of 10 –15 mol of Pi/min/mg protein. Na/K-ATPase from rat whole kidney, which was prepared using the same method, exhibited a specific activity of 2 mol of Pi/min/mg. Canine Na/K-ATPase was used for the experiments in this study unless stated otherwise. The protein contents of the ATPase preparations were determined by the method of Lowry et al [25] using bovine serum albumin as a standard
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