[19] Interaction of cardiac glycosides with Na+,K+-ATPase

Wallick, Earl T.; Schwartz, Arnold

doi:10.1016/0076-6879(88)56022-6

Cited by 66 publications

(43 citation statements)

References 11 publications

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“…The interaction of ouabain with the H5-H6 and the H7-H8 transmembrane domains of the Na,K-ATPase may inhibit the protein by sterically blocking the cation access channel (49 -50) or by stabilizing an intermediate conformation of the protein, effectively locking the movement of the H5-H6 transmembrane domains. Both of these mechanisms are supported by the allosteric effects of cations on ouabain binding (25)(26)(27). In the wild type sheep ␣1 protein the hydrophobic nature of the residues at positions 793, 786, and 863 allows cations to bind to the carboxyl-and hydroxyl-containing residues located in the cytoplasmic half of the membrane (Asp-804, Asp-808, Glu-779, and Ser-775).…”

Section: Fig 3 Dose-response Curves For Inhibition Of Nak-atpase Amentioning

confidence: 88%

“…It is known that the phosphorylated form of Na,K-ATPase binds ouabain with higher affinity (1 ϫ 10 Ϫ9 M, with Mg 2ϩ and P i ) compared with the unphosphorylated form (2 ϫ 10 Ϫ8 M, with Mg 2ϩ alone) (25,26). This change in affinity indicates that the binding site for ouabain changes upon phosphorylation.…”

Section: Fig 3 Dose-response Curves For Inhibition Of Nak-atpase Amentioning

confidence: 92%

“…The complex nature of the protein-drug interaction is also suggested by binding kinetics that indicate that the interaction of the glycoside with the Na,K-ATPase is dependent on the conformation of the protein (25)(26)(27). For example, when the enzyme is phosphorylated in the catalytic cycle (E 2 P) it binds ouabain with high affinity (K D Ϸ1 ϫ 10 Ϫ9 M).…”

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confidence: 99%

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Ouabain Interactions with the H5-H6 Hairpin of the Na,K-ATPase Reveal a Possible Inhibition Mechanism via the Cation Binding Domain

Palasis

Kuntzweiler

Argüello

et al. 1996

Journal of Biological Chemistry

101

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Section: Fig 3 Dose-response Curves For Inhibition Of Nak-atpase Amentioning

confidence: 88%

Section: Fig 3 Dose-response Curves For Inhibition Of Nak-atpase Amentioning

confidence: 92%

See 1 more Smart Citation

Ouabain Interactions with the H5-H6 Hairpin of the Na,K-ATPase Reveal a Possible Inhibition Mechanism via the Cation Binding Domain

Palasis

Kuntzweiler

Argüello

et al. 1996

Journal of Biological Chemistry

101

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“…As in the case of vanadate, the inhibition of the signal is consistent with it arising from a reaction beginning in the E 2 conformation. The absence of complete inhibition may be due to the lack of Mg 2+ ions in the buffer, which are known to significantly enhance ouabain binding (44).…”

Section: Namentioning

confidence: 99%

Mechanism of the Rate-Determining Step of the Na⁺,K⁺-ATPase Pump Cycle

et al. 2002

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The kinetics of the E 2 f E 1 conformational change of unphosphorylated Na + ,K + -ATPase from rabbit kidney and shark rectal gland were investigated via the stopped-flow technique using the fluorescent label RH421 (pH 7.4, 24°C). The enzyme was pre-equilibrated in a solution containing 25 mM histidine and 0.1 mM EDTA to stabilize initially the E 2 conformation. When rabbit kidney enzyme was mixed with NaCl alone, tris ATP alone or NaCl, and tris ATP simultaneously, a fluorescence decrease was observed. The reciprocal relaxation time, 1/τ, of the fluorescent transient was found to increase with increasing NaCl concentration and reached a saturating value in the presence of 1 mM tris ATP of 54 ( 3 s -1 in the case of rabbit kidney enzyme. The experimental behavior could be described by a binding of Na + to the enzyme in the E 2 state with a dissociation constant of 31 ( 7 mM, which induces a subsequent rate-limiting conformational change to the E 1 state. Similar behavior, but with a decreased saturating value of 1/τ, was found when NaCl was replaced by choline chloride. Analogous experiments performed with enzyme from shark rectal gland showed similar effects, but with a significantly lower amplitude of the fluorescence change and a higher saturating value of 1/τ for both the NaCl and choline chloride titrations. The results suggest that Na + ions or salt in general play a regulatory role, similar to that of ATP, in enhancing the rate of the rate-limiting E 2 f E 1 conformational transition by interaction with the E 2 state.The Na + ,K + -ATPase is known to play a fundamental role in numerous physiological processes, e.g., nerve, kidney, and heart function. Its activity in the cell must, therefore, be under tight metabolic control. A major site of regulation of the enzyme at the molecular level must be at its rate-determining steps, since only changes in the rates of these steps will result in any significant change in the overall turnover number of the enzyme.The kinetics of the Na + ,K + -ATPase are generally described in terms of the Albers-Post model (1, 2), a simplified version of which is shown in Figure 1. This simple model considers two conformations of the enzyme, E 1 and E 2 , which can be either in a phosphorylated or an unphosphorylated state. The model, furthermore, describes a consecutive mechanism of Na + and K + ion transport across the membrane, whereby Na + ions normally bind from the cytoplasm and K + ions from the extracellular fluid. The location of the rate-determining steps within this cycle and the determination of rate constants for the various steps have been the subject of an intense research effort by many groups over the past thirty years. Although several different reaction steps have been discussed as possible candidates for the rate-determining step of the enzyme, there now appears to be conclusive evidence and an overall consensus that under physiological conditions it is in fact the E 2 f E 1 transition of unphosphorylated enzyme (3).It is known that the enzyme can be ...

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“…The (H++K+)-ATPase was not inhibited by ouabain and oligomycin even at concen trations that inhibited (Na++K+)-ATPase (31) and mitochondrial ATPase (32), respectively. It was inhibited by DCCD, pCMBS and omeprazole.…”

Section: Methodsmentioning

confidence: 99%

Purification and characterization of (H++K+)-ATPase from hog gastric mucosa.

1990

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Abstract-A (H++K+)-ATPase-enriched membrane fraction derived from the fundic portion of hog gastric mucosa was obtained by a combination of differential and repeated 7% Ficoll gradient centrifugation.The microsomal membrane fraction isolated by repeated 7% Ficoll gradient centrifugation was free of ouabain-sensitive (Na++K+)-ATPase, 5'-nucleotidase and succinate dehydrogenase; and it was highly enriched in (H++K+)-ATPase and K+-stimulated p-nitrophenylphosphatase (p-NPPase).The (H++K+)-ATPase had a pH optimum of 7.4 and was stimulated by TI', K+, Rb+ and NH4+ with Ka values of 0.0667, 0.526, 0.667 and 3.03 mM , respectively, at this pH. On the other hand, monovalent cations such as Na+ , Li+ and (CH3)4N+ as well as divalent cations such as Cue', Ca t+, Bat+, Sr2+ and Cd2+ inhibited this enzyme activity concentration-dependently.Ouabain and oligomycin had no effect, whereas omeprazole, a specific (H++K+)-ATPase inhibitor, inhibited this enzyme activity in a pH-dependent manner. Sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis showed a major band (>90% of protein) at 97,400 daltons, which was phosphorylated in the presence of Mg2+ and [r-32P] ATP and dephosphorylated in the presence of K+. The present method was very simple, and the (H++K+)-ATPase activity of the microsomal fraction obtained by this method was much higher compared with those obtained by other methods such as free-flow electrophoresis.

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[19] Interaction of cardiac glycosides with Na+,K+-ATPase

Cited by 66 publications

References 11 publications

Ouabain Interactions with the H5-H6 Hairpin of the Na,K-ATPase Reveal a Possible Inhibition Mechanism via the Cation Binding Domain

Ouabain Interactions with the H5-H6 Hairpin of the Na,K-ATPase Reveal a Possible Inhibition Mechanism via the Cation Binding Domain

Mechanism of the Rate-Determining Step of the Na⁺,K⁺-ATPase Pump Cycle

Purification and characterization of (H++K+)-ATPase from hog gastric mucosa.

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[19] Interaction of cardiac glycosides with Na+,K+-ATPase

Cited by 66 publications

References 11 publications

Ouabain Interactions with the H5-H6 Hairpin of the Na,K-ATPase Reveal a Possible Inhibition Mechanism via the Cation Binding Domain

Ouabain Interactions with the H5-H6 Hairpin of the Na,K-ATPase Reveal a Possible Inhibition Mechanism via the Cation Binding Domain

Mechanism of the Rate-Determining Step of the Na+,K+-ATPase Pump Cycle

Purification and characterization of (H++K+)-ATPase from hog gastric mucosa.

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Mechanism of the Rate-Determining Step of the Na⁺,K⁺-ATPase Pump Cycle