Ligand binding to (Na,K)-ATPase labeled with 5-iodoacetamidofluorescein.
The equilibrium binding of sodium, potassium, and adenine nucleotides to dog kidney (Na,K)-ATPase was studied by measuring changes in the fluorescence of enzyme labeled with 5-iodoacetamidofluorescein (5-IAF). The intensity of the fluorescence emission at 520 nm of the bound fluorescein (excited at 490 nm) is increased by ATP, adenyl-5'-yl imidodiphosphate (AMP-PNP), ADP (but not AMP), and Na+, and decreased by K+, Rb+, NH+4, and LI+. Thus the fluorescence effects correlate with the ability of these groups of ligands to stabilize E1 and E2 conformations, respectively. The Na+-induced increase in fluorescence has two components: a slow, high-affinity increase of approximately 7% (K0.5 = 0.16 mM) with positive cooperativity; and a large (approximately 15%), rapid, low-affinity (K0.5 = 34 mM) increase that is not cooperative. The K0.5 for the high-affinity effect is decreased by oligomycin and increased by K+. ATP effects on the fluorescence follow Michaelis-Menten kinetics and are of high affinity (K0.5 = 0.12 microM); K+ increases the K0.5 for ATP, AMP-PNP, and ADP but does not induce cooperative behavior. K+ itself decreases the fluorescence signal by about 9%, with high affinity (K0.5 = 5 microM), showing Michaelis-menten behavior in the absence of other ligands, while with ATP, Na+, or Mg2+ present, K+ effects are cooperative and of lower affinity.
D000255:Adenosine Triphosphate / Q000494:pharmacology; D000818:Animals; D002414:Cations, Monovalent / Q000494:pharmacology; D004285:Dogs; D005452:Fluoresceins; D005453:Fluorescence; D005456:Fluorescent Dyes; D007679:Kidney Medulla / Q000201:enzymology; D007700:Kinetics; D008024:Ligands / Q000378:metabolism; D009840:Oligomycins / Q000494:pharmacology; D010766:Phosphorylation; D011188:Potassium / Q000494:pharmacology; D011485:Protein Binding; D012964:Sodium / Q000494:pharmacology; D000254:Sodium-Potassium-Exchanging ATPase / Q000378:metabolism*
D002414:Cations, Monovalent; D005452:Fluoresceins; D005456:Fluorescent Dyes; D008024:Ligands; D009840:Oligomycins; C015296:5-iodoacetamidofluorescein; D000255:Adenosine Triphosphate; D012964:Sodium; D000254:Sodium-Potassium-Exchanging ATPase; D011188:Potassium