Linked Life Data

9341234

Source:http://linkedlifedata.com/resource/pubmed/id/9341234

Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
43
pubmed:dateCreated
1997-11-17
pubmed:abstractText
The kinetics of Na+-dependent partial reactions of the Na+,K+-ATPase were investigated via the stopped-flow technique using the fluorescent labels RH421 and BIPM. After the enzyme is mixed with MgATP, both labels give almost identical kinetic responses. Under the chosen experimental conditions two exponential time functions are necessary to fit the data. The dominant fast phase, 1/tau1 approximately 180 s-1 (saturating [ATP] and [Na+], pH 7.4 and 24 degrees C), is attributed to phosphorylation of the enzyme and a subsequent conformational change (E1ATP(Na+)3 --> E2P(Na+)3 + ADP). The rate of the phosphorylation reaction measured by the acid quenched-flow technique was 190 s-1 at 100 microM ATP, suggesting that phosphorylation controls the kinetics of the RH421 signal and that the conformational change is very fast (>/=600 s-1). The rate of the RH421 signal was optimal at pH 7.5. The Na+ concentration dependence of 1/tau1 showed half-saturation at a Na+ concentration of 8-10 mM with positive cooperativity involved in the occupation of the Na+ binding sites. The apparent dissociation constant of the high affinity ATP binding site determined from the ATP concentration dependence of 1/tau1 was 7.0 (+/-0.6) microM, while the apparent Kd for the low affinity site and the rate constant for the E2 to E1 conformational change evaluated in the absence of Mg2+ were 143 (+/-17) microM and </= 28 s-1. At RH421 concentrations in the micromolar range, a decrease in the value of 1/tau1 is observed. On the basis of rapid quenched-flow measurements, this inhibition can be attributed to a reaction step subsequent to phosphorylation. This accounts for previously observed kinetic discrepancies between RH421 and BIPM.
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
pubmed:status
MEDLINE
pubmed:month
Oct
pubmed:issn
0006-2960
pubmed:author
pubmed:issnType
Print
pubmed:day
28
pubmed:volume
36
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
13406-20
pubmed:dateRevised
2007-11-15
pubmed:meshHeading
pubmed-meshheading:9341234-Adenosine Triphosphate, pubmed-meshheading:9341234-Animals, pubmed-meshheading:9341234-Cations, Monovalent, pubmed-meshheading:9341234-Dose-Response Relationship, Drug, pubmed-meshheading:9341234-Fluorescent Dyes, pubmed-meshheading:9341234-Hydrogen-Ion Concentration, pubmed-meshheading:9341234-Kidney Medulla, pubmed-meshheading:9341234-Kinetics, pubmed-meshheading:9341234-Maleimides, pubmed-meshheading:9341234-Phosphorylation, pubmed-meshheading:9341234-Protein Conformation, pubmed-meshheading:9341234-Pyridinium Compounds, pubmed-meshheading:9341234-Sodium, pubmed-meshheading:9341234-Sodium-Potassium-Exchanging ATPase, pubmed-meshheading:9341234-Spectrometry, Fluorescence, pubmed-meshheading:9341234-Styrenes, pubmed-meshheading:9341234-Sulfhydryl Reagents, pubmed-meshheading:9341234-Swine
pubmed:year
1997
pubmed:articleTitle
Stopped-flow kinetic investigations of conformational changes of pig kidney Na+,K+-ATPase.
pubmed:affiliation
Department of Biophysical Chemistry, Max-Planck-Institut für Biophysik, Kennedyallee 70, D-60596 Frankfurt am Main, Germany,
pubmed:publicationType
Journal Article, Research Support, Non-U.S. Gov't