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

Lee, J. A.; Fortes, P. A. G.

doi:10.1021/bi00373a002

Cited by 21 publications

(12 citation statements)

References 68 publications

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“…In the millimolar concentration range, magnesium (AF = -3% and -5% at 1 and 5 mM, respectively), manganese, and calcium each were found to cause modest decreases in the LY-Na+,K+-ATPase fluorescence. There decreases were very similar to that reported previously by Lee and Fortes (1986). When the lifetime(s) of LY were further analyzed by frequency-domain fluorometry (over the modulation frequency range of 7-125 MHz), either as distributions or as discrete values, they were found to be altered by 5 mM Mg2+.…”

Section: Labeling Of the Na+k+-atpase By Lysupporting

confidence: 88%

The carbohydrate moieties of the beta-subunit of Na+, K(+)-ATPase: their lateral motions and proximity to the cardiac glycoside site

Amler

Abbott

Malak

et al. 1996

Biophysical Journal

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The beta-subunit associated with the catalytic (alpha) subunit of the mammalian Na+, K(+) -ATPase is a transmembrane glycoprotein with three extracellularly located N-glycosylation sites. Although beta appears to be essential for a functional enzyme, the role of beta and its sugars remains unknown. In these studies, steady-state and dynamic fluorescence measurements of the fluorophore lucifer yellow (LY) covalently linked to the carbohydrate chains of beta have demonstrated that the bound probes are highly solvent exposed but restricted in their diffusional motions. Furthermore, the probes' environments on beta were not altered by Na+ or K+ or ouabain-induced enzyme conformational changes, but both divalent cation and oligomycin addition evoked modest changes in LY fluorescence. Frequency domain measurements reflecting the Förster fluorescence energy transfer (FET) occurring between anthroylouabain (AO) bound to the cardiac glycoside receptor site on alpha and the carbohydrate-linked LY demonstrated their close proximity (18 A). Additional FET determinations made between LY as donor and erythrosin-5-isothiocyanate, covalently bound at the enzyme's putative ATP binding site domain, indicated that a distance of about 85 A separates these two regions and that this distance is reduced upon divalent cation binding and increased upon the Na+E1-->K+E2 conformational transition. These data suggest a model for the localization of the terminal moieties of the oligosaccharides that places them, on average, about 18 A from the AO binding site and this distance or less from the extracellular membrane surface.

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Section: Labeling Of the Na+k+-atpase By Lysupporting

confidence: 88%

The carbohydrate moieties of the beta-subunit of Na+, K(+)-ATPase: their lateral motions and proximity to the cardiac glycoside site

Amler

Abbott

Malak

et al. 1996

Biophysical Journal

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“…Section VI). This agrees with estimates of distances in fluorescence studies [118]. A notable difference is a 10-20 A protrusion on the extracellular surface of the model for Na,K-ATPase while the Ca-ATPase model has a smooth extracytoplasmic surface.…”

Section: B Crystallization In the Membranesupporting

confidence: 84%

Structural basis for E1–E2 conformational transitions in Na, K-pump and Ca-pump proteins

1988

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“…It had been found previously by energy transfer measurements that ATP and cardiac glycoside binding sites are 7.2 nm apart (57) and that the cytosolic part carrying the ATP site sits 3 nm above the inner side of the plasma membrane (57,58). Additionally, energy transfer measurements between the AOcontaining ouabain binding site and the lucifer yellow-modified ␤-subunit (51,59) give an overall arrangement of distances (Fig. 7).…”

Section: Discussionmentioning

confidence: 99%

Molecular Distance Measurements Reveal an (αβ)2Dimeric Structure of Na+/K+-ATPase

Linnertz

Urbanova²,

Obšil³

et al. 1998

Journal of Biological Chemistry

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ATP hydrolysis by Na؉ /K ؉ -ATPase proceeds via the interaction of simultaneously existing and cooperating high (E 1 ATP) and low (E 2 ATP) substrate binding sites. It is unclear whether both ATP sites reside on the same or on different catalytic ␣-subunits. To answer this question, we looked for a fluorescent label for the E 2 ATP site that would be suitable for distance measurements by Fö rster energy transfer after affinity labeling of the E 1 ATP site by fluorescein 5-isothiocyanate (FITC). Naϩ /K ϩ -ATPase is an integral membrane protein that transports sodium and potassium ions against an electrochemical gradient. The transport of Na ϩ and K ϩ ions is presumably connected to an oscillation of the enzyme between two major conformational states, namely the E 1 Na ϩ and the E 2 K ϩ conformations. The E 1 and E 2 states have different affinities for ATP. The pumping mechanism may be described by conformational changes of a single ATP site of the catalytic ␣-subunit between a high affinity E 1 ATP 1 site (from where Na ϩ export starts by phosphorylation) and a low affinity E 2 ATP site (which is involved in K ϩ import) (1-3). Yet a model assuming consecutive changes of a single ATP site during the catalytic process, the so-called Albers-Post model, is inconsistent with the recent kinetic demonstration of simultaneously existing and cooperating ATP binding sites (4, 5) and the finding that specific labeling of the E 1 ATP or the E 2 ATP sites does not block labeling and partial activities of the other empty site (6 -13). The recent demonstration of a "superphosphorylation," i.e. that at least 2 mol of phosphate can be incorporated into the catalytic ␣-subunit per mol of ouabain binding sites (14), is consistent with the coexistence of phosphorylated intermediates (E 1 P, E*P, and E 2 P (15)) at different places in an oligomeric enzyme. Also, the observations of phosphorylation from P i during Na ϩ -ATPase activity (16) and in an FITC-treated enzyme (10) are consistent with the possibility that Na ϩ /K ϩ -ATPase is phosphorylated from both ATP sites (12, 17).Since two ATP binding sites of Na ϩ /K ϩ -ATPase cooperate during ATP hydrolysis (4, 5), it is of great interest to obtain more detailed information on the mutual interaction of both ATP sites in the absence (8,10,18,19) and presence of the transported cations (20) and on their distance. There are a great number of experiments favoring the idea that both ATP sites reside on different ␣-subunits (21, 22). But studies with detergent-solubilized Na ϩ /K ϩ -ATPase seem to contradict this assumption (13,23,24). Ward and Cavieres (13, 24) demonstrated that the detergent-solubilized putative (␣␤) promoter of Na ϩ /K ϩ -ATPase shows negative cooperativity of ATP hydrolysis. This finding is in conflict with the assumption of cooperating catalytic ␣-subunits during ATP hydrolysis but supports the possibility of two interacting ATP sites residing on the same catalytic ␣-subunit. The amino acid sequence forming the high affinity E 1 ATP site has been defined by affinity...

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

Cited by 21 publications

References 68 publications

The carbohydrate moieties of the beta-subunit of Na+, K(+)-ATPase: their lateral motions and proximity to the cardiac glycoside site

The carbohydrate moieties of the beta-subunit of Na+, K(+)-ATPase: their lateral motions and proximity to the cardiac glycoside site

Structural basis for E1–E2 conformational transitions in Na, K-pump and Ca-pump proteins

Molecular Distance Measurements Reveal an (αβ)2Dimeric Structure of Na+/K+-ATPase

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