Holger Linnertz

Molecular Distance Measurements Reveal an (αβ)2Dimeric Structure of Na+/K+-ATPase HIGH AFFINITY ATP BINDING SITE AND K+-ACTIVATED PHOSPHATASE RESIDE ON DIFFERENT α-SUBUNITS

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 α-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/α-subunit of 0.6 and of ErITC/α-subunit of 0.48 indicate 2 ATP sites per (αβ)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 α-subunit since the diameter of α 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 (αβ)2 diprotomer and works as a functional dimer (Thoenges, D., and Schoner, W. (1997) J. Biol. Chem.272, 16315–16321).

Erythrosin 5′-isothiocyanate labels Cys549 as part of the low-affinity ATP binding site of Na+/K+-ATPase1

The high‐affinity E1ATP site of Na+/K+‐ATPase labeled with fluorescein 5′‐isothiocyanate and its E2ATP site labeled with erythrosin 5′‐isothiocyanate (ErITC), as was shown recently [Linnertz et al. (1998) J. Biol. Chem. 273, 28813–28821], reside on separate and adjacent catalytic α subunits. This paper provides evidence that specific labeling of the E2ATP binding site with ErITC resulted in a modification of the Cys549 residue in the tryptic fragment with the sequence Val545‐Leu‐Gly‐Phe‐Cys549‐His550. Hence, Cys549 is part of or close to the low‐affinity E2ATP binding site of Na+/K+‐ATPase.

The origin of the diphenylhexatriene short lifetime component in membranes and solvents

Abstract Fluorescence decay of 1,6-diphenyl-1,3,5-hexatriene (DPH) was found to be bicomponental in lipid bilayers and isotropic solvents. Mean lifetime values of both components correlated with the permittivity of the solvents. Decomposition of time-resolved spectra of DPH in phospholipid vesicles revealed a significant red shift of the spectrum of the short-lived component with respect to the spectrum of the long-lived component. This indicated that part of DPH was in a hydrophilic environment, which was supported by fluorescence energy transfer experiments. We showed that the increased fraction of the short-lived component in lipid bilayers originates from the population of DPH at the membrane–water interface.

Quenching of 7‐chloro‐4‐nitrobenzo‐2‐oxa‐1,3‐diazole‐modified Na+/K+‐ATPase reveals a higher accessibility of the low‐affinity ATP‐binding site

7‐Chloro‐4‐nitrobenzo‐2‐oxa‐1,3‐diazole (NBD‐Cl) labeled Na+/K+‐ATPase covalently with two different inactivation constants (K i=2.5 μM; K i′=10 μM). It apparently modified the two different ATP‐binding sites of the enzyme since it decreased the activity of the E2ATP site, i.e. the K+‐activated para‐nitrophenylphosphatase activity, in an enzyme whose high‐affinity E1ATP site had been blocked by fluorescein 5′isothiocyanate (FITC). It also reduced the activity of the E1ATP site, i.e. the Na+‐activated protein phosphorylation, in an enzyme whose low‐affinity E2ATP site had been blocked by Co(NH3)4PO4. Fluorescence quenching experiments with KI, CsCl and MnCl2 of the NBD‐Cl‐labeled Na+/K+‐ATPase revealed two differently accessible types of fluorophores depending on the ATP site: The E2ATP site apparently differs from the E1ATP site in that it is more open because the fluorophore labeling in the E2ATP site was sterically better accessible for quenchers.

A two-site model of interacting ATP sites.

The Albers-Post model of the sodium pump explains the mechanism of Na+/K+activated ATP hydrolysis as events of a single ATP binding site that exists consecutively in two different conformations fulfilling two different tasks: The high ufSlnity ATP binding site is involved in the Na+ export that occurs via the formation of a phosphointermediate. On release of ADP and Na+ it is consequently converted into a low energy phosphointermediate which occludes K+ on phosphate hydrolysis. The release of K+ at the inner membrane site needs the binding of ATP at a low ufSlnity ATP binding site. An essential of this single site model is that the low affinity ATP site (EzATP site) is subsequently converted to the high aftinity ATP site (EIATP site).l However, much data obtained mostly by the use of substitution-inert MgATP-complex analogs show that high and low affinity ATP sites coexist.2 It is not yet definitively proven whether the catalytic a subunit contains a second ATP site,3 but doubling of phosphorylation sites under certain conditions favors the concept of an (a@)* dimer with cooperating ATP binding site^.^^^ If high and low affinity ATP sites coexist in time and space, their cooperation during Na+/K+-activated ATP hydrolysis and cation transport must follow a two-site competitive model. Therefore, to obtain information on the existence of such kinetics we used substitution-inert MgATP complex analogs as well as fluorescent ATP derivatives.