Switch to
| Predicate | Object |
|---|---|
| rdf:type | |
| lifeskim:mentions | |
| pubmed:issue |
40
|
| pubmed:dateCreated |
1997-10-22
|
| pubmed:abstractText |
Substitution of alanine for Ser775 in a ouabain-resistant alpha1 sheep isoform causes a 30-fold decrease in apparent affinity for K+ as an activator of the Na,K-ATPase, as well as an increase in apparent affinity for ATP (Arguello, J. M., and Lingrel, J. B (1995) J. Biol. Chem. 270, 22764-22771). This study was carried out to determine whether Ser775 is a direct cation-ligating residue or whether the change in apparent affinity for K+ is secondary to a conformational alteration as evidenced in the change in ATP affinity, with the following results. Kinetics of K+(Rb+) influx into intact cells show that the change is due to a change in K+ interaction at the extracellular surface. The K+ dependence of formation of K+-occluded enzyme (E2(K)) and of the rate of formation of deoccluded enzyme from E2(K) indicate that the Ser775 --> Ala mutation results in a marked increase (>/=30-fold) in rate of release of K+ from E2(K). The high affinity Na+-like competitive antagonist 1,3-dibromo2,4,6-tris-(methylisothiouronium)benzene (Br2TITU), which interacts with the E1 conformation and blocks cytoplasmic cation binding (Hoving, S., Bar-Shimon, M., Tijmes, J. J. , Tal, D. M., and Karlish, S. J. D. (1995) J. Biol. Chem. 270, 29788-29793), inhibits Na+-ATPase of the mutant less than the control enzyme. With intact cells, Br2TITU acts as a competitive inhibitor of extracellular K+ activation of both the mutant and control enzymes. In this case, the mutant was more sensitive to inhibition. With vanadate as a probe of conformation, a difference in conformational equilibrium between the mutant and control enzymes could not be detected under turnover conditions (Na+- ATPase) in the absence of K+. These results indicate that the increase in apparent affinity for ATP effected by the Ser775 --> Ala mutation is secondary to a change in intrinsic cation affinity/selectivity. The large change in affinity for extracellular K+ compared with cytoplasmic Na+ and to Br2TITU binding supports the conclusion that the serine hydroxyl is either part of the K+-gate structure or a direct cation-ligating residue that is shared by at least one Na+ ion, albeit with less consequence on rate constants for Na+ binding or release compared with K+.
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| pubmed:grant | |
| pubmed:language |
eng
|
| pubmed:journal | |
| pubmed:citationSubset |
IM
|
| pubmed:chemical |
http://linkedlifedata.com/resource/pubmed/chemical/1,3-dibromo-2,4,6-tris-(methylisothi...,
http://linkedlifedata.com/resource/pubmed/chemical/Adenosine Triphosphate,
http://linkedlifedata.com/resource/pubmed/chemical/Alanine,
http://linkedlifedata.com/resource/pubmed/chemical/Cations, Monovalent,
http://linkedlifedata.com/resource/pubmed/chemical/Enzyme Inhibitors,
http://linkedlifedata.com/resource/pubmed/chemical/Isothiuronium,
http://linkedlifedata.com/resource/pubmed/chemical/Macromolecular Substances,
http://linkedlifedata.com/resource/pubmed/chemical/Ouabain,
http://linkedlifedata.com/resource/pubmed/chemical/Potassium,
http://linkedlifedata.com/resource/pubmed/chemical/Recombinant Proteins,
http://linkedlifedata.com/resource/pubmed/chemical/Rubidium,
http://linkedlifedata.com/resource/pubmed/chemical/Serine,
http://linkedlifedata.com/resource/pubmed/chemical/Sodium-Potassium-Exchanging ATPase
|
| pubmed:status |
MEDLINE
|
| pubmed:month |
Oct
|
| pubmed:issn |
0021-9258
|
| pubmed:author | |
| pubmed:issnType |
Print
|
| pubmed:day |
3
|
| pubmed:volume |
272
|
| pubmed:owner |
NLM
|
| pubmed:authorsComplete |
Y
|
| pubmed:pagination |
24987-93
|
| pubmed:dateRevised |
2007-11-15
|
| pubmed:meshHeading |
pubmed-meshheading:9312104-Adenosine Triphosphate,
pubmed-meshheading:9312104-Alanine,
pubmed-meshheading:9312104-Animals,
pubmed-meshheading:9312104-Binding Sites,
pubmed-meshheading:9312104-Cations, Monovalent,
pubmed-meshheading:9312104-Enzyme Inhibitors,
pubmed-meshheading:9312104-HeLa Cells,
pubmed-meshheading:9312104-Humans,
pubmed-meshheading:9312104-Isothiuronium,
pubmed-meshheading:9312104-Kinetics,
pubmed-meshheading:9312104-Macromolecular Substances,
pubmed-meshheading:9312104-Mutagenesis, Site-Directed,
pubmed-meshheading:9312104-Ouabain,
pubmed-meshheading:9312104-Point Mutation,
pubmed-meshheading:9312104-Potassium,
pubmed-meshheading:9312104-Recombinant Proteins,
pubmed-meshheading:9312104-Rubidium,
pubmed-meshheading:9312104-Serine,
pubmed-meshheading:9312104-Sheep,
pubmed-meshheading:9312104-Sodium-Potassium-Exchanging ATPase,
pubmed-meshheading:9312104-Time Factors,
pubmed-meshheading:9312104-Transfection
|
| pubmed:year |
1997
|
| pubmed:articleTitle |
Evidence that Ser775 in the alpha subunit of the Na,K-ATPase is a residue in the cation binding pocket.
|
| pubmed:affiliation |
Department of Medicine, McGill University, Montreal, Quebec, Canada H3G 1A4.
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| pubmed:publicationType |
Journal Article,
Research Support, U.S. Gov't, P.H.S.,
Research Support, Non-U.S. Gov't
|