Differential effects of substrates on three transport modes of the Na+/K(+)-ATPase.

With a purified Na+/K(+)-ATPase preparation reconstituted into phospholipid vesicles, Na+/K+, Na+/Na+, and uncoupled Na+ transport were studied using three nucleotides and five substrates of the K(+)-phosphatase reaction that this enzyme also catalyzes. For Na+/K+ exchange, CTP was half as effective as ATP and GTP one-twentieth; of the phosphatase substrates only carbamyl phosphate and 3-O-methylfluorescein phosphate produced significant transport and at merely 1% of the rate with ATP. For Na+/Na+ exchange, comparable rates of transport were produced by ATP, CTP, carbamyl phosphate and acetyl phosphate, although the actual rate of transport with ATP was only 2.4% of that for Na+/K+ exchange; slower rates occurred with GTP (69%), 3-O-methylfluorescein phosphate (51%), and nitrophenyl phosphate (33%). Only umbelliferone phosphate was ineffective. For uncoupled Na+ transport results similar to those for Na+/Na+ exchange were obtained, but the actual rate of transport was still slower, 1.4% of that for Na+/K+ exchange. Thus, not only nucleotides but a variety of phosphatase substrates (which are phosphoric acid mixed anhydrides) can phosphorylate the enzyme at the high-affinity substrate site to form the E1P intermediate of the reaction sequence. Oligomycin inhibited Na+/K+ exchange with ATP by half, but with carbamyl phosphate not at all; with CTP the inhibition was intermediate, one-fourth. By contrast, oligomycin inhibited Na+/Na+ exchange by one-fifth with all three substrates. A quantitative, steady-state kinetic model accounts for the relative magnitudes of Na+/K+ and Na+/Na+ exchanges with ATP, CTP, and carbamyl phosphate as substrates, as well as the extents of inhibition by oligomycin. The model requires that even when Na+ substitutes for K+ a slow step in the reaction sequence is the E2 to E1 conformational transition.