Magnetic resonance and kinetic studies of the mechanism of membrane‐bound sodium and potassium ION‐activated adenosine triphosphatase

Abstract

EPR and water proton relaxation rate (1/T1) studies of partially (40%) and "fully" (90%) purified preparations of membrane-bound (Na+ + K+) activated ATPase from sheep kidney indicate one tight binding site for Mn2+ per enzyme dimer, with a dissociation constant (KD = 0.88 muM) in agreement with the kinetically determined activator constant, identifying this Mn2+-binding site as the active site of the ATPase. Competition studies indicate that Mg2+ binds at this site with a dissociation constant of 1 mM in agreement with its activator constant. Inorganic phosphate and methylphosphonate bind to the enzyme-Mn2+ complex with similar high affinities and decrease 1/T1 of water protons due to a decrease from four to three in the number of rapidly exchanging water protons in the coordination sphere of enzyme-bound Mn2+. The relative effectiveness of Na+ and K+ in facilitating ternary complex formation with HPO2-4 and CH3PO2-3 as a function of pH indicates that Na+ induces the phosphate monoanion to interact with enzyme-bound Mn2+. Thus protonation of an enzyme-bound phosphoryl group would convert a K+-binding site to a Na+-binding site. Dissociation constants for K+ and Na+, estimated from NMR titrations, agreed with kinetically determined activator constants of these ions consistent with binding to the active site. Parallel 32Pi-binding studies show negligible formation (less than 7%) of a covalent E-P complex under these conditions, indicating that the NMR method has detected an additional noncovalent intermediate in ion transport. Ouabain, which increases the extent of phosphorylation of the enzyme to 24% at pH 7.8 and to 106% at pH 6.1, produced further decreases in 1/T1 of water protons. Preliminary 31P- relaxation studies of CH3PO2-3 in the presence of ATPase and Mn2+ yield an Mn to P distance (6.9 +/- 0.5 A) suggesting a second sphere enzyme-Mn-ligand-CH3PO2-3 complex. Previous kinetic studies have shown that T1+ substitutes for K+ in the activation of the enzyme but competes with Na+ at higher levels. From the paramagnetic effect of Mn2+ at the active site on the enzyme on I/T1 of 205T1 bound at the Na+ site, a Mn2+ to T1+ distance of 4.0 +/- 0.1 A is calculated, suggesting the sharing of a common ligand atomy by Mn2+ and T1+ on the ATPase. Addition of Pi increases this distance to 5.4 A consistent with the insertion of P between Mn2+ and T1+. These results are consistent with a mechanism for the (Na+ + K+)-ATPase and for ion transport in which the ionization state of Pi at a single enzyme active site controls the binding and transport of Na+ and K+, and indicate that the transport site for monovalent cations is very near the catalytic site of the ATPase. Our mechanism also accounts for the order of magnitude weaker binding of Na+ compared to K+.

Kinetics of thallium exchange in cultured rat myocardial cells.

“…If all of the ouabain-inhibitable Tl + influx represents Na + ,K + -ATPase activity, then the results of this study would give an apparent K m for Tl + on the enzyme activity of 2-7 HM. This is considerably different than the K m of Tl + on renal Na + ,K + -ATPase of 0.17 mM found by Britten and Blank (1968) and Grisham et al (1974). The latter authors, however, noted K m of Tl + on Na + ,K + -AT-Pase of 29 ^M in the absence of Mn ++ , a value not greatly different from that of the present study.…”

Distinct Structural Identities of Catalytic and Ca²⁺ Binding Domains in the Sarcoplasmic Reticulum ATPase

“…An example of shortrange transmission is the phosphate-cation symport hypothesis, assuming a direct inter action of the phosphoryl group undergoing transfer and the cation undergoing transloca tion [78]. This hypothesis is supported by magnetic resonance studies demonstrating the presence of cation binding sites at the catalytic site, in close proximity of the y-phosphoryl group of bound ATP [79][80][81]. How ever, the current information derived from mutagenesis, chemical derivatization and spectroscopy (as outlined above) indicates that catalytic and Ca2+ binding domains are separated by a distance of approximately 50 A, thereby requiring a long-range linkage [59], In fact, a peptide segment (residues 308-355 in the SR ATPase) connecting the catalytic and Ca2+ binding domains is highly homolo gous in several cations' ATPases aligned with reference to the phosphorylation site [39] and may be instrumental in the linkage mecha nism.…”

“…(2) Enzyme activation by Ca2+ occurs with rather slow kinetics [67,86] and is accompa nied by fluorescence changes involving tryptophanyl residues near the membrane-bound re gion [79,[87][88][89] and extended to a Lys515 label at the upper end of the cytosolic region [90]. The fluorescence changes are reversed as the catalytic cycle proceeds through the phos phorylation and Ca2+ translocation steps [91].…”

Wholebody autoradiographic study after a single dose of204thallium in young rats

“…Il is interesting to note ihat the kidney medullary uptake in the 10-d-o!d animals reached a very high value. Aiso in the medulla thallium may interfere with the sodium/ potas.sium transport system (2,4,7,19,22).…”

“…This inhibiting effect is restricted to potassium and does not involve the sodium pumping directly (2,4,7,19,22). At low concentrations thallium may substitute for potassium whereas it becomes toxically inhibitory at higher doses.…”