The reaction sequence of the Na+/K(+)-ATPase: rapid kinetic measurements distinguish between alternative schemes.
Conformational changes between E1 and E2 enzyme forms of a dog kidney Na+/K(+)-ATPase preparation labeled with 5-iodoacetamidofluorescein were followed with a stopped-flow fluorimeter, in terms of the rate constant, kobs, and the steady-state magnitude, % delta F of fluorescence change. On rapid mixing of enzyme plus Mg2+ plus Na+ with saturating (0.5 mM) ATP in the absence of K+, kobs varied with Na+ concentration in the range 0-155 mM, with a K1/2 of 10 mM, while % delta F was relatively insensitive to Na+, with a K1/2 of 0.5 mM. Oligomycin reduced kobs by 98-99% for Na+ greater than or equal to 10 mM, but only by 50% for Na+ = 1 mM; % delta F was reduced at most by 20%. At 155 mM Na+, both kobs and % delta F changed if K+ was present with the enzyme. kobs decreased by 50% when K+ was increased from 0 to 0.2 mM, but increased when K+ was varied in the range 0.2-5 mM. K+ increased % delta F by a factor of 3 with a K1/2 of 0.3-0.5 mM as measured in both stopped-flow and steady-state experiments. These data are considered in terms of the derived presteady-state equations for two alternate schemes for the enzyme, with the E1P to E2P conformational change either preceding (Albers-Post) or following (Nørby-Yoda-Skou) Na+ transport and release. The analysis indicates that: (i) Na+ must be released before the conformational transition, from an E1 form; (ii) the step in which the second and/or third Na+ is released is rate-limiting, but this release is accelerated by Na+; and (iii) the release is also accelerated by K+ acting with low affinity (possibly at extracellular sites).
Pratap
P R
PR
Department of Pharmacology, SUNY Health Science Center, Syracuse 13210.
Robinson
J D
JD
Steinberg
M I
MI
D000818:Animals; D001692:Biological Transport; D004285:Dogs; D005456:Fluorescent Dyes; D007679:Kidney Medulla / Q000201:enzymology; D007700:Kinetics; D009840:Oligomycins / Q000494:pharmacology; D011188:Potassium / Q000494:pharmacology; D011487:Protein Conformation / Q000187:drug effects; D012964:Sodium / Q000494:pharmacology; D000254:Sodium-Potassium-Exchanging ATPase / Q000737:chemistry*
D005456:Fluorescent Dyes; D009840:Oligomycins; D012964:Sodium; D000254:Sodium-Potassium-Exchanging ATPase; D011188:Potassium