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

Linnertz, Holger; Urbanova, Petra; Amler, Evžen

doi:10.1016/s0014-5793(97)01460-9

Cited by 9 publications

(6 citation statements)

References 19 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…It is surprising, therefore, that FITC is a label of the E 1 ATP site and not of the E 2 ATP site, whereas ErITC binds to and modifies both ATP sites. The idea has been put forward more recently that the E 2 ATP site has a broader binding pocket than the E 1 ATP site, since after modification of the E 2 ATP site with 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole chloride, the latter site was easily accessible to a number of fluorescence quenchers (54). This explanation, however, cannot be applied to the differential labeling of ATP sites by ErITC and FITC, since the more bulky ErITC should not bind to the E 1 ATP site, which is what actually happens (Table I).…”

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

Self Cite

View full text Add to dashboard Cite

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...

show abstract

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

Self Cite

View full text Add to dashboard Cite

show abstract

“…The external quencher KI (15) has limited access to the fluorophores attached to the ODN in the lipid-coated particles. Thus, the lipid-coated particles should show less quenching than the free ODN or naked polyplexes.…”

Section: Protection Of Odn From Ki Quenchingmentioning

confidence: 99%

Liposome-Encapsulated Polyethylenimine/Oligonucleotide Polyplexes Prepared by Reverse-Phase Evaporation Technique

Bickel

2012

AAPS PharmSciTech

View full text Add to dashboard Cite

Abstract. Liposome-encapsulated polyplex system represents a promising delivery system for oligonucleotide-based therapeutics such as siRNA and asODN. Here, we report a novel method to prepare liposome-encapsulated cationic polymer/oligonucleotide polyplexes based on the reverse-phase evaporation following organic extraction of the polyplexes. The polyplexes of polyethylenimine and oligonucleotide were first formed in aqueous buffer at an N/P ratio of 6. The overall positively charged polyplexes were then mixed with the anionic phospholipids in overall organic media. The overall organic environment and electrostatic interaction between anionic phospholipids and positively charged polyplexes resulted in inverted micelle-like particles with the polyplexes in the core. After phase separation, the hydrophobic particles were recovered in organic phase. Reverse-phase evaporation of the organic solvent in the presence of hydrophilic polymer-grafted lipids resulted in a stable aqueous dispersion of hydrophilic lipid-coated particles with the polyplex in the core. Transmission electron microscopy visualization revealed spherical structures with heavily stained polyplex cores surrounded by lightly stained lipid coats. The lipid-coated polyplex particles showed colloidal stability, complete protection of the loaded oligonucleotide molecules from enzymatic degradation, and high loading efficiency of more than 80%. Thus, this technique represents an alternative method to prepare lipid-coated polyplex particles as a delivery system of oligonucleotide therapeutics.

show abstract

“…After the last centrifugation step, the protein was resuspended in 0.3 ml of 20 mM Tris‐HCl buffer of pH 7.25 (final concentration 1.8 mg/ml). The partial activities and specificity of the labeled compounds were measured as described previously [2, 3, 11, 19]. Notably, 5′‐isothiocyanates like FITC or ErITC are reportedly potent labels of both Cys and Lys residues at pH 7.25 [20].…”

Section: Methodsmentioning

confidence: 99%

Erythrosin 5′‐isothiocyanate labels Cys⁵⁴⁹ as part of the low‐affinity ATP binding site of Na⁺/K⁺‐ATPase¹

et al. 1998

Self Cite

View full text Add to dashboard Cite

show abstract

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

Cited by 9 publications

References 19 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

Liposome-Encapsulated Polyethylenimine/Oligonucleotide Polyplexes Prepared by Reverse-Phase Evaporation Technique

Erythrosin 5′‐isothiocyanate labels Cys⁵⁴⁹ as part of the low‐affinity ATP binding site of Na⁺/K⁺‐ATPase¹

Contact Info

Product

Resources

About

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

Cited by 9 publications

References 19 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

Liposome-Encapsulated Polyethylenimine/Oligonucleotide Polyplexes Prepared by Reverse-Phase Evaporation Technique

Erythrosin 5′‐isothiocyanate labels Cys549 as part of the low‐affinity ATP binding site of Na+/K+‐ATPase1

Contact Info

Product

Resources

About

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

Erythrosin 5′‐isothiocyanate labels Cys⁵⁴⁹ as part of the low‐affinity ATP binding site of Na⁺/K⁺‐ATPase¹