Linked Life Data

17511477

Source:http://linkedlifedata.com/resource/pubmed/id/17511477

Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
23
pubmed:dateCreated
2007-6-5
pubmed:abstractText
The kinetics of the E2 --> E1 conformational change of unphosphorylated Na+,K+-ATPase was investigated via the stopped-flow technique using the fluorescent label RH421 (pH 7.4, 24 degrees C). The enzyme was pre-equilibrated in a solution containing 25 mM histidine and 0.1 mM EDTA to stabilize the E2 conformation. When rabbit enzyme was mixed with 130 mM NaCl alone or with 130 mM NaCl and varying concentrations of Na2ATP simultaneously, a fluorescence decrease was observed. In the absence of ATP, the fluorescence decrease followed a biexponential time course, but at ATP concentrations after mixing of >or=50 microM, the fluorescence transient could be adequately fitted by a single exponential. On the basis of the agreement between theoretical simulations and experimental traces, we propose that in the absence of bound ATP the conformational transition occurs as a two step reversible process within a protein dimer, E2:E2 --> E2:E1 --> E1:E1. In the presence of 130 mM NaCl, the sum of the forward and backward rate constants for the E2:E2 --> E2:E1 and E2:E1 --> E1:E1 transitions were found to be 10.4 (+/-1.0) and 0.49 (+/-0.02) s-1, respectively. At saturating concentrations of ATP, however, the transition occurs in a single reversible step with the sum of its forward and backward rate constants equal to 35.2 (+/-0.3) s-1. It was found that ATP acting at a high affinity site (Kd approximately 0.25 microM), stimulated the reverse reaction, E1ATP --> E2ATP, in addition to its known allosteric low affinity (Kd approximately 71 microM) stimulation of the forward reaction, E2ATP --> E1ATP.
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
pubmed:status
MEDLINE
pubmed:month
Jun
pubmed:issn
0006-2960
pubmed:author
pubmed:issnType
Print
pubmed:day
12
pubmed:volume
46
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
7034-44
pubmed:dateRevised
2007-11-15
pubmed:meshHeading
pubmed:year
2007
pubmed:articleTitle
Allosteric effect of ATP on Na(+),K(+)-ATPase conformational kinetics.
pubmed:affiliation
School of Chemistry, University of Sydney, Sydney, NSW 2006, Australia, and Faculty of Biology, University of Konstanz, D-78435 Konstanz, Germany. r.clarke@chem.usyd.edu.au
pubmed:publicationType
Journal Article, Research Support, Non-U.S. Gov't