Eosin, a fluorescent probe of ATP binding to the (Na+ + K+)-ATPase

Skou, J.C.; Esmann, Mikael

doi:10.1016/0005-2736(81)90251-0

Cited by 143 publications

(82 citation statements)

References 32 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…From their data, however, it is not possible to conclude anything concerning the relative effectiveness of Na + and choline + in stimulating the transition. Similar to the case of the experiments of Karlish (6), however, the observed rate constants found by Skou and Esmann (7) are very low, due to a blockage of the ATP binding site by eosin (24)(25)(26)(27).…”

supporting

confidence: 86%

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

et al. 2002

View full text Add to dashboard Cite

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 ...

show abstract

supporting

confidence: 86%

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

et al. 2002

View full text Add to dashboard Cite

show abstract

“…This dye binds to the ATP binding site noncovalently. Its fluorescence can be related to the Na+-and K+-induced conformations of the enzyme (14). Because the concentration of Na+,K+-ATPase in the bovine lens was too low for this investigation, bovine kidney Na+,K+-ATPase was modified with H202 under conditions in which the resultant modified enzyme resembled the enzyme of H202-modified lenses on the basis of ATP and p-nitrophenyl'phosphate hydrolysis.…”

Section: Resultsmentioning

confidence: 99%

H2O2-induced uncoupling of bovine lens Na+,K+-ATPase.

Garner¹,

Garner²,

Spector³

1983

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

View full text Add to dashboard Cite

A 1-hr exposure of bovine lenses in organ culture to H202 concentrations in the range found in the aqueous fluid of patients with cataracts inhibits 86Rb+ influx. At 1 mM H202, complete inhibition was observed and further investigated. Membrane permeability is slightly decreased. Although lactate concentrations increase 2-fold, lens ATP concentrations decrease -10%, suggesting that glycolysis may be stimulated but ATP production is not able to keep up with the demand for energy. Examination of epithelial cell Mg-+-stimulated Na+,K+-ATPase isolated from the cultured lenses indicates H202-induced modification. At 5 mM MgATP, ATP hydrolysis is accelerated 30%; at 3 mM MgATP, hydrolysis is normal; and at 0.75 mM MgATP, it is inhibited 75%. p-Nitrophenyl phosphate hydrolysis and eosin maleimide binding indicate that K+ control of the enzyme is modified. Thus, a very early effect of H202 upon the lens, well before the formation of opacity, appears to be the uncoupling of Na+ and K+ transport from ATP hydrolysis.In cataracts Na', K+, and Ca2" concentrations appear to be altered (1). The change in cation concentrations may reflect either increased membrane permeability or altered function of the major lenticular cation pumps, Mg2+-stimulated Na+/ K+ ATPase and Ca2 ATPase. Recent studies indicate that with cataract development Na+,K+-ATPase appears to be increasingly inhibited (2).With cataract development there also is increased oxidation of certain lens protein amino-acid side chains (3). This fundamental oxidation process, which appears to start at the cell membrane, leads to conformational changes and crosslinking of the lens proteins with plasma membrane polypeptides acting as nucleation points. Eventually the fiber cell membrane ruptures and large aggregates containing membrane fragments, extrinsic membrane protein, and cytosol protein are formed (4).Because the oxidation of the membrane protein appeared to precede the oxidation of the cytoplasmic protein (3), these studies suggested that possibly deleterious oxidizing agents may arise outside the lens. Examination of the aqueous fluid surrounding the lenses indicated that in patients with cataracts, there are dramatically elevated concentrations of H202, ranging from 45 to 663 uM (5). A patient with Marfan syndrome had a H202 concentration >3 mM. These observations suggest that elevated exogenous H202 may be an important oxidizing agent involved in the formation of certain cataracts. This viewpoint is supported by the finding that in organ culture, H202 concentrations comparable to concentrations found in the aqueous fluid of patients with cataracts will cause the development of opacity within 48 hr (6).The lens has a number of defenses against oxidation caused by H202 and other agents (7). These defenses include catalase, glutathione peroxidase, superoxide dismutase, glutathione reductase, methionine sulfoxide reductase (8), a high concentration of glutathione, and a very active hexose monophosphate shunt. However, many of these defenses reside primarily...

show abstract

“…Eosin-Eosin, tetrabromofluorescein, has been shown to be a potent inhibitor of the Na,K-ATPase by competing with ATP (32). Thus, we performed the domain-domain interaction experiments described above in the presence and absence of 10 M eosin.…”

Section: Treatment Of Fusion Protein Abds With Atp Site Probesmentioning

confidence: 99%

“…For example, eosin is a reversible inhibitor of the Na,K-ATPase, and the binding of eosin and ATP are mutually exclusive (32). Thus, eosin should block GST-ABD and His 6 -ABD interactions, by preventing MgATP binding.…”

Section: Fig 3 His 6 -Abd Interaction With C 12 E 8 -Solubilized Domentioning

confidence: 99%

Interactions between Na,K-ATPase α-Subunit ATP-binding Domains

Costa¹,

Gatto²,

Kaplan³

2003

Journal of Biological Chemistry

View full text Add to dashboard Cite

The reaction mechanism of the Na,K-ATPase is thought to involve a number of ligand-induced conformational changes. The specific amino acid residues responsible for binding many of the important ligands have been identified; however, details of the specific conformational changes produced by ligand binding are largely undescribed. The experiments described in this paper begin to identify interactions between domains of the Na,K-ATPase ␣-subunit that depend on the presence of particular ligands. The major cytoplasmic loop (between TM4 and TM5), which we have previously shown contains the ATP-binding domain, was overexpressed in bacteria either with a His 6 tag or as a fusion protein with glutathione S-transferase. We have observed that these polypeptides associate in the presence of MgATP. Incubation with [␥-32 P]ATP under conditions that result in phosphorylation of the full-length Na,K-ATPase did not result in 32 P incorporation into either the His 6 tag or glutathione S-transferase fusion proteins. The MgATPinduced association was strongly inhibited by prior modification of the fusion proteins with fluorescein isothiocyanate or by simultaneous incubation with 10 M eosin, indicating that the effect of MgATP is due to interactions within the nucleotide-binding domain. These data are consistent with Na,K-ATPase associating within cells via interactions in the nucleotide-binding domains. Although any functional significance of these associations for ion transport remains unresolved, they may play a role in cell function and in modulating interactions between the Na,K-ATPase and other proteins.

show abstract

Eosin, a fluorescent probe of ATP binding to the (Na+ + K+)-ATPase

Cited by 143 publications

References 32 publications

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

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

H2O2-induced uncoupling of bovine lens Na+,K+-ATPase.

Interactions between Na,K-ATPase α-Subunit ATP-binding Domains

Contact Info

Product

Resources

About

Eosin, a fluorescent probe of ATP binding to the (Na+ + K+)-ATPase

Cited by 143 publications

References 32 publications

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

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

H2O2-induced uncoupling of bovine lens Na+,K+-ATPase.

Interactions between Na,K-ATPase α-Subunit ATP-binding Domains

Contact Info

Product

Resources

About

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

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