A Two‐Site Model of Interacting ATP Sites<sup>a</sup>

Thoenges, Detlef; Linnertz, Holger; Schoner, Wilhelm

doi:10.1111/j.1749-6632.1997.tb52262.x

Cited by 8 publications

(11 citation statements)

References 14 publications

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“…Dual effects were also found on ATP hydrolysis by C 12 E 8 -solubilized and purified Na,K-ATPase with K 0.5 values of about 0.1 and 4 µM for the high-affinity site and of about 0.1 and 0.5 mM for the lowaffinity site, both with negative cooperativity, suggesting that the enzyme environment or oligomer formation is very important for substrate hydrolysis (21)(22)(23)40). It should be emphasized that the contribution of the highaffinity site to the ATPase specific activity of the enzyme generally corresponds to values about 1 to 10% of the total activity, which hinders its kinetic characterization (21)(22)(23)39).…”

Section: Nak-atpase Solubilized Using Only C 12 Ementioning

confidence: 92%

“…It should be emphasized that the contribution of the highaffinity site to the ATPase specific activity of the enzyme generally corresponds to values about 1 to 10% of the total activity, which hinders its kinetic characterization (21)(22)(23)39). It is very difficult to determine whether these arose from the presence of more than one ATP site per (aß) protomer or from a single site whose function and affinity change during the catalytic cycle (21)(22)(23)(24)40), and the functional relationship between the two hydrolysis sites and their behavior during the reaction cycle remains to be explored.…”

Section: Nak-atpase Solubilized Using Only C 12 Ementioning

confidence: 99%

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Solubilization of Na,K-ATPase from rabbit kidney outer medulla using only C12E8

Santos

Lamas

Ciancaglini

2002

Braz J Med Biol Res

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SDS, C 12 E 8 , CHAPS or CHAPSO or a combination of two of these detergents is generally used for the solubilization of Na,K-ATPase and other ATPases. Our method using only C 12 E 8 has the advantage of considerable reduction of the time for enzyme purification, with rapid solubilization and purification in a single chromatographic step. Na,KATPase-rich membrane fragments of rabbit kidney outer medulla were obtained without adding SDS. Optimum conditions for solubilization were obtained at 4ºC after rapid mixing of 1 mg of membrane Na,K-ATPase with 1 mg of C 12 E 8 /ml, yielding 98% recovery of the activity. The solubilized enzyme was purified by gel filtration on a Sepharose 6B column at 4ºC. Non-denaturing PAGE revealed a single protein band with phosphomonohydrolase activity. The molecular mass of the purified enzyme estimated by gel filtration chromatography was 320 kDa. The optimum apparent pH obtained for the purified enzyme was 7.5 for both PNPP and ATP. The dependence of ATPase activity on ATP concentration showed high (K 0.5 = 4.0 µM) and low (K 0.5 = 1.4 mM) affinity sites for ATP, with negative cooperativity. Ouabain (5 mM), oligomycin (1 µg/ml) and sodium vanadate (3 µM) inhibited the ATPase activity of C 12 E 8 -solubilized and purified Na,KATPase by 99, 81 and 98.5%, respectively. We have shown that Na,KATPase solubilized only with C 12 E 8 can be purified and retains its activity. The activity is consistent with the form of (aß) 2 association.

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Section: Nak-atpase Solubilized Using Only C 12 Ementioning

confidence: 92%

Section: Nak-atpase Solubilized Using Only C 12 Ementioning

confidence: 99%

Solubilization of Na,K-ATPase from rabbit kidney outer medulla using only C12E8

Santos

Lamas

Ciancaglini

2002

Braz J Med Biol Res

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“…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)(2)(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.…”

mentioning

confidence: 99%

“…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).…”

mentioning

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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“…All these observations, including the observation of a positive cooperative effect of 2'-ODANS-8-N 3 -ATP in the inactivation of Na+/K+-ATPase [7] led to the conclusion that two simultaneously existing ATP sites need to cooperate during ATP-driven Na+/ K + transport [7,8]. Unfortunately, the available data cannot differentiate between the possibility that two ATP-binding sites exist on a single a subunit and the possibility that the sodium pump works as a functional dimer (ocß) 2 with two cooperating ATP sites.…”

Section: Introductionmentioning

confidence: 99%

Quenching of 7‐chloro‐4‐nitrobenzo‐2‐oxa‐1,3‐diazole‐modified Na⁺/K⁺‐ATPase reveals a higher accessibility of the low‐affinity ATP‐binding site

1997

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7-Chloro-4-nitrobenzo-2-oxa-l,3-diazole (NBD-C1) labeled Na + /K + -ATPase covalently with two different inactivation constants (AT; = 2.5 IJM; K' = 10 \iM). It apparently modified the two different ATP-binding sites of the enzyme since it decreased the activity of the E 2 ATP site, i.e. the Inactivated para-nitrophenylphosphatase activity, in an enzyme whose high-affinity EiATP site had been blocked by fluorescein 5'isothiocyanate (FITC). It also reduced the activity of the Ei ATP site, i.e. the Na + -activated protein phosphorylation, in an enzyme whose low-affinity E 2 ATP site had been blocked by Co(NH 3 ) 4 P04. Fluorescence quenching experiments with KI, CsCl and MnCl 2 of the NBD-Cl-labeled Na + /K + -ATPase revealed two differently accessible types of fluorophores depending on the ATP site: The E 2 ATP site apparently differs from the EiATP site in that it is more open because the fluorophore labeling in the E 2 ATP site was sterically better accessible for quenchers.

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A Two‐Site Model of Interacting ATP Sites^a

Cited by 8 publications

References 14 publications

Solubilization of Na,K-ATPase from rabbit kidney outer medulla using only C12E8

Solubilization of Na,K-ATPase from rabbit kidney outer medulla using only C12E8

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

Quenching of 7‐chloro‐4‐nitrobenzo‐2‐oxa‐1,3‐diazole‐modified Na⁺/K⁺‐ATPase reveals a higher accessibility of the low‐affinity ATP‐binding site

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A Two‐Site Model of Interacting ATP Sitesa

Cited by 8 publications

References 14 publications

Solubilization of Na,K-ATPase from rabbit kidney outer medulla using only C12E8

Solubilization of Na,K-ATPase from rabbit kidney outer medulla using only C12E8

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

Quenching of 7‐chloro‐4‐nitrobenzo‐2‐oxa‐1,3‐diazole‐modified Na+/K+‐ATPase reveals a higher accessibility of the low‐affinity ATP‐binding site

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A Two‐Site Model of Interacting ATP Sites^a

Quenching of 7‐chloro‐4‐nitrobenzo‐2‐oxa‐1,3‐diazole‐modified Na⁺/K⁺‐ATPase reveals a higher accessibility of the low‐affinity ATP‐binding site