Abstract
ATP hydrolysis by Na+/K+-ATPase proceeds via the interaction of simultaneously existing and cooperating high (E1ATP) and low (E2ATP) substrate binding sites. It is unclear whether both ATP sites reside on the same or on different catalytic alpha-subunits. To answer this question, we looked for a fluorescent label for the E2ATP site that would be suitable for distance measurements by Förster energy transfer after affinity labeling of the E1ATP site by fluorescein 5'-isothiocyanate (FITC). Erythrosin 5'-isothiocyanate (ErITC) inactivated, in an E1ATP site-blocked enzyme (by FITC), the residual activity of the E2ATP site, namely K+-activated p-nitrophenylphosphatase in a concentration-dependent way that was ATP-protectable. The molar ratios of FITC/alpha-subunit of 0.6 and of ErITC/alpha-subunit of 0.48 indicate 2 ATP sites per (alpha beta)2 diprotomer. Measurements of Förster energy transfer between the FITC-labeled E1ATP and the ErITC-labeled or Co(NH3)4ATP-inactivated E2ATP sites gave a distance of 6.45 +/- 0.64 nm. This distance excludes 2 ATP sites per alpha-subunit since the diameter of alpha is 4-5 nm. Förster energy transfer between cardiac glycoside binding sites labeled with anthroylouabain and fluoresceinylethylenediamino ouabain gave a distance of 4.9 +/- 0.5 nm. Hence all data are consistent with the hypothesis that Na+/K+-ATPase in cellular membranes is an (alpha beta)2 diprotomer and works as a functional dimer (Thoenges, D., and Schoner, W. (1997) J. Biol. Chem. 272, 16315-16321).
Highlights
ATP hydrolysis by Na؉/K؉-ATPase proceeds via the interaction of simultaneously existing and cooperating high (E1ATP) and low (E2ATP) substrate binding sites
We looked for a fluorescent label for the E2ATP site that would be suitable for distance measurements by Forster energy transfer after affinity labeling of the E1ATP site by fluorescein 5-isothiocyanate (FITC)
The pumping mechanism may be described by conformational changes of a single ATP site of the catalytic ␣-subunit between a high affinity E1ATP1 site and a low affinity E2ATP site [1,2,3]
Summary
E1ATP site, nucleotide binding site of Naϩ/Kϩ-ATPase with high affinity for ATP; E2ATP site, nucleotidebinding site of Naϩ/Kϩ-ATPase with low affinity for ATP; AO, anthroylouabain; ErITC, erythrosin 5Ј-isothiocyanate; FEDO, fluoresceinylethylenediamine ouabain; FITC, fluorescein 5Ј-isothiocyanate; TNP-ATP, 2Ј,3Ј-O-(2Ј,4Ј,6Ј-trinitrophenyl)adenosine 5Ј-triphosphate. Provided specific labeling of the E2ATP binding site by a fluorescent pseudo-ATP derivative can be achieved, Forster energy transfer measurements may give information on the distance of this E2ATP site to the E1ATP site, since the latter can be labeled by FITC at Lys501 [26, 27, 36]. Energy transfer from the E1ATP-labeled fluorescein to the E2ATP-labeled erythrosin (as well to the Co2ϩ ions sitting in the E2ATP site) was so low that it is unlikely that both ATP sites reside on the same catalytic ␣-subunit
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