Abstract
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.
Highlights
Summary—The results described above show that TNP-8N3ADP photoinactivates fluorescein isothiocyanate (FITC)-modified Na,K-ATPase, either membrane-bound or as soluble protomers, and that this process is preceded by probe binding which is both reversible and specific
These findings confirm our earlier observation that each ␣ unit can simultaneously bind fluorescein covalently and TNP-ADP at equilibrium [23]
The inference is that each ␣ protomer should present one catalytic ATP site and one allosteric site, which is available for TNP-8N3-ADP
Summary
C12E8, octaethylene glycol monododecyl ether; FITC, fluorescein 5Ј-isothiocyanate; pNPP, p-nitrophenyl phosphate; pNP, p-nitrophenol; TNP-ADP, 2Ј(3Ј)-O-(2,4,6-trinitrophenyl) adenosine 5Ј-diphosphate; TNP-8N3-ADP, 2Ј(3Ј)-O-(2,4,6-trinitrophenyl)8-azidoadenosine 5Ј-diphosphate; TNBS, 2,4,6-trinitrobenzenesulfonic acid; NIPI, N-(2-nitro-4-isothiocyanophenyl)-imidazole; E1, ubiquitin-activating enzyme; E2, ubiquitin carrier protein. In a bid to incorporate an affinity probe in the available site, we have found that TNP-8N3-ADP can photoinactivate FITC-modified ␣ protomers after binding at equilibrium It follows that FITC and TNP-8N3-ADP must be covalently attached to the same minimal enzyme unit. A preliminary communication has been presented [28]
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