The isolated H4‐H5 cytoplasmic loop of Na,K‐ATPase overexpressed in Escherichia coli retains its ability to bind ATP

Obšil, Tomáš; Mérola, Fabienne; Lewit-Bentley, Anita; Amler, Evžen

doi:10.1016/s0014-5793(98)00373-1

Cited by 16 publications

(12 citation statements)

References 16 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…2), gave no indication for a second site. TNP-ATP binding was suppressed by the presence of ATP and ADP but not by AMP, as previously reported [34,35] (data not shown).…”

Section: Resultssupporting

confidence: 89%

The phosphatase activity of the isolated H₄‐H₅ loop of Na⁺/K⁺ ATPase resides outside its ATP binding site

Krumscheid

Ettrich²,

Sovová³

et al. 2004

European Journal of Biochemistry

View full text Add to dashboard Cite

The structural stability of the large cytoplasmic domain (H 4 -H 5 loop) of mouse a 1 subunit of Na + /K + ATPase (L354-I777), the number and the location of its binding sites for 2¢-3¢-O-(trinitrophenyl) adenosine 5¢-triphosphate (TNP-ATP) and p-nitrophenylphosphate (pNPP) were investigated. C-and N-terminal shortening revealed that neither part of the phosphorylation (P)-domain are necessary for TNP-ATP binding. There is no indication of a second ATP site on the P-domain of the isolated loop, even though others reported previously of its existence by TNP-N 3 ADP affinity labeling of the full enzyme. Fluorescein isothiocyanate (FITC)-anisotropy measurements reveal a considerable stability of the nucleotide (N)-domain suggesting that it may not undergo a substantial conformational change upon ATP binding. The FITC modified loop showed only slightly diminished phosphatase activity, most likely due to a pNPP site on the N-domain around N398 whose mutation to D reduced the phosphatase activity by 50%. The amino acids forming this pNPP site (M384, L414, W411, S400, S408) are conserved in the a 1)4 isoforms of Na + /K + ATPase, whereas N398 is only conserved in the vertebrates' a 1 subunit. The phosphatase activity of the isolated H 4 -H 5 loop was neither inhibited by ATP, nor affected by mutation of D369, which is phosphorylated in native Na + /K + ATPase.

show abstract

“…2), gave no indication for a second site. TNP-ATP binding was suppressed by the presence of ATP and ADP but not by AMP, as previously reported [34,35] (data not shown).…”

Section: Resultssupporting

confidence: 89%

The phosphatase activity of the isolated H₄‐H₅ loop of Na⁺/K⁺ ATPase resides outside its ATP binding site

Krumscheid

Ettrich²,

Sovová³

et al. 2004

European Journal of Biochemistry

View full text Add to dashboard Cite

show abstract

“…Several reports described the overexpression of soluble domains of P‐type ATPases in E. coli [5–7]. Though they have demonstrated the binding of nucleotides, they have not apparently succeeded in obtaining large amounts of fully active enzymes.…”

Section: Discussionmentioning

confidence: 99%

“…This approach has particular advantages if one aims at crystallization of the enzyme in different conformations and of mutants. Though overexpression of the soluble domains of P‐type ATPases has already been performed, crystallization has been unsuccessful presumably due to problems in the refolding process of denatured proteins [5,6] or cleavage of fusion proteins [7].…”

Section: Introductionmentioning

confidence: 99%

Soluble P‐type ATPase from an archaeon, Methanococcus jannaschii

2000

View full text Add to dashboard Cite

MJ0968 has been proposed to be an ancestor of P-type ATPase, because its primary structure is highly homologous to that of the core catalytic domain of P-type ATPase. However it completely lacks amino acid sequences that possibly constitute transmembrane domains. To examine if MJ0968 is indeed a P-type ATPase, it was overexpressed in Escherichia coli and purified. It did show ATPase activity, autophosphorylation and inhibition by vanadate. All these properties support the idea that MJ0968 is indeed a soluble P-type ATPase.z 2000 Federation of European Biochemical Societies.

show abstract

“…The product was purified on agarose gel. This vector was digested with Bam HI and Eco RI and subcloned into the linearized pGEX‐2T at the site location downstream from the GST coding sequence as described in Obšil et al 4 The ligated DNA was transformed into competent Escherichia coli DH5α cells. The stop codon at K605 was introduced using a Stratagene Quick‐Change Kit for site‐directed mutagenesis.…”

Section: Methodsmentioning

confidence: 99%

“…1 The α subunit contains 10 transmembrane segments with a large cytoplasmic loop, which is located between helices H 4 and H 5 , where the ATP binding site and the phosphorylation site are localized 2. This H 4 –H 5 loop was shown to preserve a rigid and self‐supporting structure3 and is able to bind nucleotide triphosphates 4. Recently, the tertiary structure of the H 4 –H 5 loop of pig kidney Na + /K + ‐ATPase was revealed by homology modeling 5.…”

Section: Introductionmentioning

confidence: 99%

Secondary and tertiary structure of nucleotide‐binding domain of αsubunit of Na⁺/K⁺‐ATPase

et al. 2002

Self Cite

View full text Add to dashboard Cite

The nucleotide-binding domain of the alpha subunit of mouse brain Na+/K+-ATPase was expressed and isolated from Escherichia coli cells. A model structure was constructed by comparative modeling with and without docked ATP. This was compared with the secondary structure determination from UV circular dichroism and Raman spectroscopy. Thus, we support the quality of the model and the correct folding of the recombinant protein. ATP binding was followed by Raman difference spectroscopy, and its influence on the secondary structure of the N domain seems to not be significant.

show abstract

The isolated H₄‐H₅ cytoplasmic loop of Na,K‐ATPase overexpressed in Escherichia coli retains its ability to bind ATP

Cited by 16 publications

References 16 publications

The phosphatase activity of the isolated H₄‐H₅ loop of Na⁺/K⁺ ATPase resides outside its ATP binding site

The phosphatase activity of the isolated H₄‐H₅ loop of Na⁺/K⁺ ATPase resides outside its ATP binding site

Soluble P‐type ATPase from an archaeon, Methanococcus jannaschii

Secondary and tertiary structure of nucleotide‐binding domain of αsubunit of Na⁺/K⁺‐ATPase

Contact Info

Product

Resources

About

The isolated H4‐H5 cytoplasmic loop of Na,K‐ATPase overexpressed in Escherichia coli retains its ability to bind ATP

Cited by 16 publications

References 16 publications

The phosphatase activity of the isolated H4‐H5 loop of Na+/K+ ATPase resides outside its ATP binding site

The phosphatase activity of the isolated H4‐H5 loop of Na+/K+ ATPase resides outside its ATP binding site

Soluble P‐type ATPase from an archaeon, Methanococcus jannaschii

Secondary and tertiary structure of nucleotide‐binding domain of αsubunit of Na+/K+‐ATPase

Contact Info

Product

Resources

About

The isolated H₄‐H₅ cytoplasmic loop of Na,K‐ATPase overexpressed in Escherichia coli retains its ability to bind ATP

The phosphatase activity of the isolated H₄‐H₅ loop of Na⁺/K⁺ ATPase resides outside its ATP binding site

The phosphatase activity of the isolated H₄‐H₅ loop of Na⁺/K⁺ ATPase resides outside its ATP binding site

Secondary and tertiary structure of nucleotide‐binding domain of αsubunit of Na⁺/K⁺‐ATPase