ADP- and K+-sensitive phosphorylated intermediate of Na,K-ATPase.

Yoda, Shizuko; Yoda, Atsunobu

doi:10.1016/s0021-9258(17)36066-0

Cited by 55 publications

(17 citation statements)

References 24 publications

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“…We have shown (Fortes & Lee, 1984; Lee & Fortes, 1985b) that AO can bind also to the ADP-sensitive, K-insensitive phosphoenzyme intermediate E[P(Na), which contains occluded Na (Glynn et al, 1984), and to a third phosphoenzyme form, denoted EXP, which is partially saturated with Na at low-affinity external sites (Norby et al, 1983;Lee & Fortes, 1985b;Yoda & Yoda, 1986). Energy-transfer measurements between AO and lucifer might detect possible conformational differences between the above enzyme forms and those obtained under the conditions of the experiments in Table II.…”

Section: [Ao]b = [Ao]t + [E]t + Kd-[([ao]t + [E]t + Ko)2 -4[ao]t[e]t]...mentioning

confidence: 96%

Spatial relationship and conformational changes between the cardiac glycoside site and .beta.-subunit oligosaccharides in sodium plus potassium activated adenosinetriphosphatase

Lee¹,

Fortes²

1986

Biochemistry

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(Na,K)-ATPase, the enzyme responsible for active transport of Na and K across the plasma membranes of animal cells, consists of a catalytic subunit (alpha) and a glycoprotein subunit (beta) with unknown function. We have determined the distance between fluorescent probes directed to specific sites on the alpha- and beta-subunits and ligand-induced changes in the fluorescence of a probe specifically attached to the beta-subunit. The cardiac glycoside site on the alpha-subunit was labeled with anthroylouabain [Fortes, P. A. G. (1977) Biochemistry 16, 531-540]. The oligosaccharides on the beta-subunit were labeled with lucifer yellow carbohydrazide [Lee, J. A., & Fortes, P. A. G. (1985) Biochemistry 24, 322-330]. Resonance energy transfer from anthroylouabain to lucifer yellow was measured by steady-state and time-resolved fluorescence spectroscopy. The distance between these probes was determined from the efficiency of energy transfer. The average distance between anthroylouabain and lucifer yellow was 47 A and was independent of the number of acceptor molecules attached to the beta-subunit. The measured distance corresponds to the distance between the cardiac glycoside site and the center of the labeled oligosaccharides on the beta-subunit within one alpha beta dimer. The distance was the same (47 A) when anthroylouabain was bound with ATP or Pi as phosphorylating ligands but increased to 49 A in the presence of vanadate. The change in average distance provides quantitative evidence of a conformational difference between the complexes of cardiac glycosides with (Na,K)-ATPase induced by phosphorylating ligands or by vanadate.(ABSTRACT TRUNCATED AT 250 WORDS)

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Section: [Ao]b = [Ao]t + [E]t + Kd-[([ao]t + [E]t + Ko)2 -4[ao]t[e]t]...mentioning

confidence: 96%

Spatial relationship and conformational changes between the cardiac glycoside site and .beta.-subunit oligosaccharides in sodium plus potassium activated adenosinetriphosphatase

Lee¹,

Fortes²

1986

Biochemistry

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“…The transition between E1P and E2P has several intermediate steps. Historically, E1P and E2P have been defined as ADP-sensitive and K þ -sensitive EP, respectively (52). In addition, a transition state, E*P, was defined as both ADPand K þ -sensitive, as the sum of ADP-sensitive (E1P) and K þ -sensitive (E2P) was routinely found to be greater than 100% of the total EP measured (53).…”

Section: Eda 2d Blocks Transition To the Occluded E1pmentioning

confidence: 99%

“…12), where EDA 2þ promoted a metastable state in dephosphorylated pumps that was not stabilized by TPA þ , K þ , or Na þ . Thus, in electrophysiological experiments, EDA 2þ binding might retard the enzyme in an E*P state and prevent conversion to E1P, analogous to oligomycin reacting with E1P and E*P and inhibiting conversion to E2P (52). In this case, the EDA 2þ form (possibly the dephosphorylated form E*EDA 2þ ) might be stable enough to enable crystal formation for structural analysis of this elusive transition state.…”

Section: Eda 2d Blocks Transition To the Occluded E1pmentioning

confidence: 99%

External Ion Access in the Na/K Pump: Kinetics of Na+, K+, and Quaternary Amine Interaction

Stanley

Young

Gatto

et al. 2018

Biophysical Journal

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Na/K pumps build essential ion gradients across the plasmalemma of animal cells by coupling the extrusion of three Na, with the import of two K and the hydrolysis of one ATP molecule. The mechanisms of selectivity and competition between Na, K, and inhibitory amines remain unclear. We measured the effects of external tetrapropylammonium (TPA) and ethylenediamine (EDA) on three different Na/K pump transport modes in voltage-clamped Xenopus oocytes: 1) outward pump current (I), 2) passive inward H current at negative voltages without Na or K (I), and 3) transient charge movement reporting the voltage-dependent extracellular binding/release of Na (Q). Both amines competed with K to inhibit I. TPA inhibited I without competing with H, whereas EDA did not alter I at pH 7.6. TPA competed with Na in Q measurements, reducing Na-apparent affinity, evidenced by a ∼-75 mV shift in the charge-voltage curve (at 20 mM TPA) without reduction of the total charge moved (Q). In contrast, EDA and K did not compete with Na to inhibit Q; both reduced Q without decreasing Na-apparent affinity. EDA (15 mM) right-shifted the charge-voltage curve by ∼+50 mV. Simultaneous occlusion of EDA and Na by an E2P conformation unable to reach E1P was demonstrated by voltage-clamp fluorometry. Trypsinolysis experiments showed that EDA-bound pumps are much more proteolysis-resistant than Na-, K-, or TPA-bound pumps, therefore uncovering unique EDA-bound conformations. K effects on Q and I were also evaluated in pumps inhibited with beryllium fluoride, a phosphate mimic. K reduced Q without shifting the charge-voltage curve, indicating noncompetitive effects, and partially inhibited I to the same extent as TPA in non-beryllium-fluorinated pumps. These results demonstrate that K interacts with beryllium-fluorinated pumps inducing conformational changes that alter Q and I, suggesting that there are two external access pathways for proton transport by I.

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“…However, this hydrophobic cluster is not observed in our simulations. There are several other studies which have investigated the possibility of metastable intermediates in the E1P-E2P transition of different P-type ATPases, such as Na + ,K + -ATPase (49)(50)(51)(52) and Listeria monocytogenes Ca 2+ -ATPase (48). Although P-type ATPases share a common structural architecture and reaction cycles, they have diverse functions in biological membranes.…”

Section: Discussionmentioning

confidence: 99%

Structural and energetic analysis of metastable intermediate states in the E1P–E2P transition of Ca ²⁺ -ATPase

Kobayashi

Matsunaga

Jung

et al. 2021

Proc. Natl. Acad. Sci. U.S.A.

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Sarcoplasmic reticulum (SR) Ca2+-ATPase transports two Ca2+ ions from the cytoplasm to the SR lumen against a large concentration gradient. X-ray crystallography has revealed the atomic structures of the protein before and after the dissociation of Ca2+, while biochemical studies have suggested the existence of intermediate states in the transition between E1P⋅ADP⋅2Ca2+ and E2P. Here, we explore the pathway and free energy profile of the transition using atomistic molecular dynamics simulations with the mean-force string method and umbrella sampling. The simulations suggest that a series of structural changes accompany the ordered dissociation of ADP, the A-domain rotation, and the rearrangement of the transmembrane (TM) helices. The luminal gate then opens to release Ca2+ ions toward the SR lumen. Intermediate structures on the pathway are stabilized by transient sidechain interactions between the A- and P-domains. Lipid molecules between TM helices play a key role in the stabilization. Free energy profiles of the transition assuming different protonation states suggest rapid exchanges between Ca2+ ions and protons when the Ca2+ ions are released toward the SR lumen.

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ADP- and K+-sensitive phosphorylated intermediate of Na,K-ATPase.

Cited by 55 publications

References 24 publications

Spatial relationship and conformational changes between the cardiac glycoside site and .beta.-subunit oligosaccharides in sodium plus potassium activated adenosinetriphosphatase

Spatial relationship and conformational changes between the cardiac glycoside site and .beta.-subunit oligosaccharides in sodium plus potassium activated adenosinetriphosphatase

External Ion Access in the Na/K Pump: Kinetics of Na+, K+, and Quaternary Amine Interaction

Structural and energetic analysis of metastable intermediate states in the E1P–E2P transition of Ca ²⁺ -ATPase

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ADP- and K+-sensitive phosphorylated intermediate of Na,K-ATPase.

Cited by 55 publications

References 24 publications

Spatial relationship and conformational changes between the cardiac glycoside site and .beta.-subunit oligosaccharides in sodium plus potassium activated adenosinetriphosphatase

Spatial relationship and conformational changes between the cardiac glycoside site and .beta.-subunit oligosaccharides in sodium plus potassium activated adenosinetriphosphatase

External Ion Access in the Na/K Pump: Kinetics of Na+, K+, and Quaternary Amine Interaction

Structural and energetic analysis of metastable intermediate states in the E1P–E2P transition of Ca 2+ -ATPase

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Structural and energetic analysis of metastable intermediate states in the E1P–E2P transition of Ca ²⁺ -ATPase