Fluorescence measurements of nucleotide association with the Na(+)/K(+)-ATPase.
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.
Pratap
Promod R
PR
University of North Carolina at Greensboro, Department of Physics and Astronomy, P.O. Box 26170, Greensboro, NC 27402-6170, USA. pratapp@uncq.edu
Mikhaylyants
Lydia O
LO
Olden-Stahl
Natalie
N
D000244:Adenosine Diphosphate / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism*; D000818:Animals; D002232:Carbocyanines / Q000737:chemistry; D004372:Ducks; D005453:Fluorescence; D005456:Fluorescent Dyes; D011188:Potassium / Q000378:metabolism; D011806:Quinolinium Compounds / Q000737:chemistry; D012491:Salt Gland / Q000201:enzymology; D012964:Sodium / Q000378:metabolism; D000254:Sodium-Potassium-Exchanging ATPase / Q000378:metabolism*
C481052:Alexa fluor 546; D002232:Carbocyanines; D005456:Fluorescent Dyes; D011806:Quinolinium Compounds; C089187:cyanine dye 3; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D012964:Sodium; D000254:Sodium-Potassium-Exchanging ATPase; D011188:Potassium