Structural dynamics and oligomeric interactions of Na+,K(+)-ATPase as monitored using fluorescence energy transfer

Amler, Evžen; Abbott, Alan J.; Ball, William J.

doi:10.1016/s0006-3495(92)81859-3

Cited by 43 publications

(48 citation statements)

References 47 publications

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“…After the last centrifugation step, the protein was resuspended in 0.3 ml of 20 mM Tris/HCl buffer (pH 7.25) (final concentration 1.8 mg/ml). All lifetime measurements of FITC-labeled Na ϩ /K ϩ -ATPase were performed in the presence of 5 g/ml antifluorescein antibodies to correct for contributions by free and nonspecifically attached FITC molecules (34).…”

Section: Kinetic Analysis Of the Inactivation Of K ϩ -Activated P-nitmentioning

confidence: 99%

“…Steady-state fluorescence data were collected in quartz cuvettes on a Perkin-Elmer LS-5 fluorometer equipped with monochromators (34). Excitation and emission wavelengths were 500 and 520 nm, respectively, for FITC and FEDO, 362 and 471 nm for AO, and 530 and 555 nm for ErITC, respectively.…”

Section: Determination Of the Amount Of Fitc Eritc Ao Fedo And Comentioning

confidence: 99%

“…Quantum yield of bound FITC was compared with free FITC in ethanol, and bound ErITC was compared with free ErITC in water. Although the former increases about 10%, the quantum yield of the latter increases four times (34). For the determination of the molar FITC/ErITC ratio per mol of ␣-subunit, we labeled Na ϩ /K ϩ -ATPase at a specific activity of 25-27 units/mg of protein first with 10 M FITC at pH 7.25 and subsequently with 2 M ErITC (as described above).…”

Section: Determination Of the Amount Of Fitc Eritc Ao Fedo And Comentioning

confidence: 99%

“…For the determination of the molar FITC/ErITC ratio per mol of ␣-subunit, we labeled Na ϩ /K ϩ -ATPase at a specific activity of 25-27 units/mg of protein first with 10 M FITC at pH 7.25 and subsequently with 2 M ErITC (as described above). Steady-state fluorescence of the protein-bound fluorescein was corrected for unspecific fluorescence (outside the E 1 ATP binding site) by the addition of 5 g/ml antifluorescein antibodies (34). The molar concentration of Na ϩ /K ϩ -ATPase and its ␣-subunit was calculated from the protein concentration using the molecular weight of 113 kDa for the ␣-and 55 kDa for the ␤-subunit (3).…”

Section: Determination Of the Amount Of Fitc Eritc Ao Fedo And Comentioning

confidence: 99%

“…Affinity labeling of the low affinity E 2 ATP site needs the availability of specific ATP derivatives as probes (4). Since many derivatives of fluorescein (pyrene, eosin, or erythrosin) bind to the E 1 ATP site as well (32)(33)(34)(35), the possibility arose that fluorescent pseudo-ATP derivatives may exist that label the E 2 ATP site as well. Provided specific labeling of the E 2 ATP binding site by a fluorescent pseudo-ATP derivative can be achieved, Förster energy transfer measurements may give information on the distance of this E 2 ATP site to the E 1 ATP site, since the latter can be labeled specifically by FITC at Lys 501 (26,27,36).…”

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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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Section: Kinetic Analysis Of the Inactivation Of K ϩ -Activated P-nitmentioning

confidence: 99%

Section: Determination Of the Amount Of Fitc Eritc Ao Fedo And Comentioning

confidence: 99%

Section: Determination Of the Amount Of Fitc Eritc Ao Fedo And Comentioning

confidence: 99%

Section: Determination Of the Amount Of Fitc Eritc Ao Fedo And Comentioning

confidence: 99%

mentioning

confidence: 99%

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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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Data

Byrne¹,

Byrne²,

Coker³

et al. 2013

FRET – Förster Resonance Energy Transfer

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ATP‐binding to P‐type ATPases as revealed by biochemical, spectroscopic, and crystallographic experiments

Kubala

2006

Proteins

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P-type ATPases form a large family of cation translocating ATPases. Recent progress in crystallography yielded several high-resolution structures of Ca(2+)-ATPase from sarco(endo)plasmic reticulum (SERCA) in various conformations. They could elucidate the conformational changes of the enzyme, which are necessary for the translocation of cations, or the mechanism that explains how the nucleotide binding is coupled to the cation transport. However, crystals of proteins are usually obtained only under conditions that significantly differ from the physiological ones and with ligands that are incompatible with the enzyme function, and both of these factors can inevitably influence the enzyme structure. Biochemical (such as mutagenesis, cleavage, and labeling) or spectroscopic experiments can yield only limited structural information, but this information could be considered relevant, because measurement can be performed under physiological conditions and with true ligands. However, interpretation of some biochemical or spectroscopic data could be difficult without precise knowledge of the structure. Thus, only a combination of both these approaches can extract the relevant information and identify artifacts. Briefly, there is good agreement between crystallographic and other experimental data concerning the overall shape of the molecule and the movement of cytoplasmic domains. On the contrary, the E1-AMPPCP crystallographic structure is, in details, in severe conflict with numerous spectroscopic experiments and probably does not represent the physiological state. Notably, the E1-ADP-AlF(4) structure is almost identical to the E1-AMPPCP, again suggesting that the structure is primarily determined by the crystal-growth conditions. The physiological relevance of the E2 and E2-P structures is also questionable, because the crystals were prepared in the presence of thapsigargin, which is known to be a very efficient inhibitor of SERCA. Thus, probably only crystals of E1-2Ca conformation could reflect some physiological state. Combination of biochemical, spectroscopic, and crystallographic data revealed amino acids that are responsible for the interaction with the nucleotide. High sequence homology of the P-type ATPases in the cytoplasmic domains enables prediction of the ATP-interacting amino acids also for other P-type ATPases.

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Structural dynamics and oligomeric interactions of Na+,K(+)-ATPase as monitored using fluorescence energy transfer

Cited by 43 publications

References 47 publications

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

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

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ATP‐binding to P‐type ATPases as revealed by biochemical, spectroscopic, and crystallographic experiments

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