Fluorescence measurements of nucleotide association with the Na(+)/K(+)-ATPase.
Biochim Biophys Acta
; 1794(11): 1549-57, 2009 Nov.
Article
in En
| MEDLINE
| ID: mdl-19595797
ABSTRACT
The Na(+)/K(+)-ATPase, a membrane-associated ion pump, uses energy from the hydrolysis of ATP to pump 3 Na(+) ions out of and 2 K(+) into cells. The dependence of ATP hydrolysis on ATP concentration was measured using a fluorescence coupled-enzyme assay. The dependence on concentration of nucleotide association with the ATPase was examined using ADP and ATP-induced quenching of the fluorescence of ATPase labeled with Cy3-maleimide (Cy3-ATPase) or Alexa Fluor 546 carboxylic acid, succinimidyl ester (AF-ATPase). The kinetics of ATP hydrolysis in the presence of Na(+) and K(+) exhibited negative cooperativity with a Hill coefficient (n(H)) of 0.66 and a half-maximal concentration (K(0.5)) of 61 microM; in the absence of K(+), n(H) was 0.58 and K(0.5) was 13 microM. Nucleotide-induced fluorescence quenching exhibited negative cooperativity with an n(H) of 0.3-0.5. These results suggest that negative cooperativity observed in ATP hydrolysis is attributable to negative cooperativity in nucleotide association to the ATPase. Interaction between AF-ATPase and ATP labeled with Alexa Fluor 647 (AF-ATP) showed significant Förster resonance energy transfer (FRET). These results indicate that the ATPase exists as oligoprotomeric complexes in this preparation, and that this aggregation has significant effects on enzyme function.
Full text:
1
Collection:
01-internacional
Database:
MEDLINE
Main subject:
Adenosine Triphosphate
/
Sodium-Potassium-Exchanging ATPase
Type of study:
Risk_factors_studies
Limits:
Animals
Language:
En
Journal:
Biochim Biophys Acta
Year:
2009
Document type:
Article
Affiliation country:
Estados Unidos