The Na+/K(+)-ATPase couples the hydrolysis of ATP to the transport of Na+ and K+ via a phosphorylated intermediate and conformational changes. In order to identify these conformational changes, we have probed the sequence of steps from EP(3Na+ in) to EP + 3Na+ out with three fluorescent probes (IAF: 5-iodoacetamidofluorescein; BIPM: N-[p-(2-benzimidazolyl)phenyl]maleimide; and RH421) and the sensitivity of their fluorescence change to oligomycin and divalent cations (Ca2+ and Mn2+). The magnitude (% delta F) and rate constant (k(obs)) of ATP-induced fluorescence changes were measured on a fluorescence stopped-flow apparatus, and yielded the following results. (a) With RH421, k(obs) and % delta F varied with [Na+] (maximal k(obs) = 100 s-1, K1/2 = 6 mM; % delta Fmax = 6%, K1/2 = 1 mM); these values are comparable to those previously reported using IAF-labeled enzyme (Pratap, P.R., Robinson, J.D. and Steinberg, M.I. (1991) Biochim. Biophys. Acta 1069, 288-298). (b) With BIPM-labeled enzyme k(obs) did not vary with [Na+] over the range tested, and was twice as high as the maximum k(obs) for RH421. (c) Treatment with oligomycin reduced k(obs) for all three probes to about the same level (approximately 1-2 s-1) while % delta Fmax was largely unaffected. (d) Replacing Mg2+ with Ca2+ had similar effects to treatment with oligomycin. (e) RH421 fluorescence change was completely abolished in the presence of oligomycin and Ca2+. (f) Replacing Mg2+ with Mn2+ decreased IAF fluorescence, i.e., put the enzyme in an E2-like form, reduced k(obs), and rendered oligomycin less effective in reducing k(obs). From these results, we conclude: (a) the release of the second/third Na+ is the rate-limiting step for the conformational change measured by IAF and charge transfer measured with RH421; (b) BIPM indicates an earlier step, either the deocclusion of Na+ and/or the release of the first Na+; (c) oligomycin blocks Na+ deocclusion, and this step is sensitive to the divalent cation used to activate enzyme phosphorylation; and (d) Ca2+ slows the step reported by IAF as well. These experiments indicate that a simple model with two conformations (E1 and E2) is insufficient to explain transient kinetic data.
Calcium Kinetics