Document Detail


Metal fluoride complexes of Na,K-ATPase: characterization of fluoride-stabilized phosphoenzyme analogues and their interaction with cardiotonic steroids.
MedLine Citation:
PMID:  21708939     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
The Na,K-ATPase belongs to the P-type ATPase family of primary active cation pumps. Metal fluorides like magnesium-, beryllium-, and aluminum fluoride act as phosphate analogues and inhibit P-type ATPases by interacting with the phosphorylation site, stabilizing conformations that are analogous to specific phosphoenzyme intermediates. Cardiotonic steroids like ouabain used in the treatment of congestive heart failure and arrhythmias specifically inhibit the Na,K-ATPase, and the detailed structure of the highly conserved binding site has recently been described by the crystal structure of the shark Na,K-ATPase in a state analogous to E2·2K(+)·P(i) with ouabain bound with apparently low affinity (1). In the present work inhibition, and subsequent reactivation by high Na(+), after treatment of shark Na,K-ATPase with various metal fluorides are characterized. Half-maximal inhibition of Na,K-ATPase activity by metal fluorides is in the micromolar range. The binding of cardiotonic steroids to the metal fluoride-stabilized enzyme forms was investigated using the fluorescent ouabain derivative 9-anthroyl ouabain and compared with binding to phosphorylated enzyme. The fastest binding was to the Be-fluoride stabilized enzyme suggesting a preformed ouabain binding cavity, in accord with results for Ca-ATPase where Be-fluoride stabilizes the E2-P ground state with an open luminal ion access pathway, which in Na,K-ATPase could be a passage for ouabain. The Be-fluoride stabilized enzyme conformation closely resembles the E2-P ground state according to proteinase K cleavage. Ouabain, but not its aglycone ouabagenin, prevented reactivation of this metal fluoride form by high Na(+) demonstrating the pivotal role of the sugar moiety in closing the extracellular cation pathway.
Authors:
Flemming Cornelius; Yasser A Mahmmoud; Chikashi Toyoshima
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Publication Detail:
Type:  Journal Article; Research Support, Non-U.S. Gov't     Date:  2011-06-27
Journal Detail:
Title:  The Journal of biological chemistry     Volume:  286     ISSN:  1083-351X     ISO Abbreviation:  J. Biol. Chem.     Publication Date:  2011 Aug 
Date Detail:
Created Date:  2011-08-22     Completed Date:  2011-10-18     Revised Date:  2013-06-28    
Medline Journal Info:
Nlm Unique ID:  2985121R     Medline TA:  J Biol Chem     Country:  United States    
Other Details:
Languages:  eng     Pagination:  29882-92     Citation Subset:  IM    
Affiliation:
Department of Biomedicine, University of Aarhus, Aarhus C, Denmark. fc@biophys.au.dk
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MeSH Terms
Descriptor/Qualifier:
Animals
Cardiotonic Agents / chemistry*,  metabolism
Crystallography, X-Ray
Enzyme Stability
Fish Proteins / chemistry*,  metabolism
Fluorides / chemistry*,  metabolism
Ouabain / chemistry*,  metabolism
Protein Binding
Sharks
Sodium-Potassium-Exchanging ATPase / chemistry*,  metabolism
Chemical
Reg. No./Substance:
0/Cardiotonic Agents; 0/Fish Proteins; 0/Fluorides; 630-60-4/Ouabain; EC 3.6.3.9/Sodium-Potassium-Exchanging ATPase
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