Linked Life Data

7608186

Source:http://linkedlifedata.com/resource/pubmed/id/7608186

Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
27
pubmed:dateCreated
1995-8-16
pubmed:abstractText
Modification of aspartic acid 369 in the sheep alpha 1 Na+,K(+)-ATPase to asparagine results in a membrane-associated form of Na+,K(+)-ATPase that can bind [3H]ouabain with high affinity in the presence of Mg2+ alone (KD = 20.4 +/- 2.6 nM). Ouabain binding to the D369N mutant is not stimulated by inorganic phosphate, confirming that Asp369 is both the catalytic phosphorylation site and the only Pi interaction site which stimulates ouabain binding. Cation inhibition of Mg(2+)-stimulated ouabain binding to the D369N mutant demonstrated that three Na+ and two K+ ions inhibit [3H]ouabain binding and suggests that this inhibition must occur via a cation-sensitive conformational change which does not directly involve dephosphorylation of the enzyme. In the presence of 10 mM Mg2+, ATP stimulates ouabain binding to the wild type protein, (AC50 = 21.4 +/- 2.7 microM) but inhibits the binding to the D369N mutant (IC50 = 2.52 +/- 0.17 microM) indicating that the mutation does not destroy the high affinity site for MgATP but does change the nature of the protein conformation normally induced by a nucleotide-Na+,K(+)-ATPase interaction. Increasing the Mg2+ from 1 to 10 mM did not alter the AC50 or IC50 values for ATP and reveals that the Mg2+ interaction which stimulates ouabain binding in the absence of nucleotide involves a distinct divalent cation site not associated with the binding of the magnesium-nucleotide complex. Thus, altering the catalytic phosphorylation site of Na+,K(+)-ATPase does not affect the expression of the ouabain-sensitive protein in the membrane fraction of NIH 3T3 cells and does not disrupt the binding of Na+, K+, Mg2+, ouabain, or ATP to the enzyme. However, the D369N substitution does inhibit the formation of a nucleotide-protein complex with high affinity for ouabain.
pubmed:grant
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
pubmed:status
MEDLINE
pubmed:month
Jul
pubmed:issn
0021-9258
pubmed:author
pubmed:issnType
Print
pubmed:day
7
pubmed:volume
270
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
16206-12
pubmed:dateRevised
2007-11-15
pubmed:meshHeading
pubmed:year
1995
pubmed:articleTitle
Amino acid replacement of Asp369 in the sheep alpha 1 isoform eliminates ATP and phosphate stimulation of [3H]ouabain binding to the Na+, K(+)-ATPase without altering the cation binding properties of the enzyme.
pubmed:affiliation
Department of Molecular Genetics, Biochemistry, and Microbiology, University of Cincinnati, College of Medicine, Ohio 45267-0524, USA.
pubmed:publicationType
Journal Article, Research Support, U.S. Gov't, P.H.S.