Photoinactivation of fluorescein isothiocyanate-modified Na,K-ATPase by 2'(3')-O-(2,4,6-trinitrophenyl)8-azidoadenosine 5'-diphosphate. Abolition of E1 and E2 partial reactions by sequential block of high and low affinity nucleotide sites.
The Na,K-ATPase activity of the sodium pump exhibits apparent multisite kinetics toward ATP, a feature that is inherent to the minimal enzyme unit, the alpha beta protomer. We have argued that this should arise from separate catalytic and noncatalytic sites on the alpha beta protomer as fluorescein isothiocyanate (FITC) blocks a high affinity ATP site on all alpha subunits and yet the modified Na, K-ATPase retains a low affinity response to nucleotides (Ward, D. G., and Cavieres, J. D. (1996) J. Biol. Chem. 271, 12317-12321). We now find that 2'(3')-O-(2,4,6-trinitrophenyl)8-azido-adenosine 5'-diphosphate (TNP-8N3-ADP), a high affinity photoactivatable analogue of ATP, can inhibit the K+-phosphatase activity of the FITC-modified enzyme during assays in dimmed light. The inhibition occurs with a Ki of 140 microM at 20 mM K+; it requires the adenine ring as 2'(3')-O-(2,4 6-trinitrophenyl) (TNP)-UDP or TNP-uridine are less potent and 2,4,6-trinitrobenzene-sulfonate is ineffective. Under irradiation with UV light, TNP-8N3-ADP inactivates the K+-phosphatase activity of the fluorescein-enzyme and also its phosphorylation by [32P]Pi. The photoinactivation process is stimulated by Na+ or Mg2+, and is inhibited by K+ or excess TNP-ADP. In the presence of 50 mM Na+ and 1 mM Mg2+, TNP-8N3-ADP photoinactivates with a K0.5 of 15 microM. Furthermore, TNP-8N3-ADP photoinactivates the FITC-modified, solubilized alpha beta protomers, even more effectively than the membrane-bound fluorescein-enzyme. These results strongly suggest that catalytic and allosteric ATP sites coexist on the alpha beta protomer of Na,K-ATPase.
Ward
D G
DG
Transport ATPase Laboratory, Department of Cell Physiology and Pharmacology, University of Leicester, Leicester, LE1 9HN, United Kingdom.
Cavieres
J D
JD
D000244:Adenosine Diphosphate / Q000031:analogs & derivatives* / Q000494:pharmacology; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000494:pharmacology; D000494:Allosteric Regulation / Q000502:physiology; D000818:Animals; D001386:Azides / Q000494:pharmacology; D001665:Binding Sites / Q000502:physiology; D002384:Catalysis; D004791:Enzyme Inhibitors / Q000494:pharmacology; D016650:Fluorescein-5-isothiocyanate / Q000031:analogs & derivatives; D005456:Fluorescent Dyes / Q000378:metabolism; D007668:Kidney / Q000201:enzymology; D007700:Kinetics; D010766:Phosphorylation; D019920:Photoaffinity Labels / Q000494:pharmacology; D010782:Photolysis; D000254:Sodium-Potassium-Exchanging ATPase / Q000037:antagonists & inhibitors*; D013552:Swine; D014466:Ultraviolet Rays
D001386:Azides; D004791:Enzyme Inhibitors; D005456:Fluorescent Dyes; D019920:Photoaffinity Labels; C058475:2',3'-O (2,4,6-trinitrophenyl)-8-azidoadenosine diphosphate; C017040:8-azidoadenosine 5'-triphosphate; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D000254:Sodium-Potassium-Exchanging ATPase; D016650:Fluorescein-5-isothiocyanate