2′-O-Dansyl Analogs of ATP Bind with High Affinity to the Low Affinity ATP Site of Na+/K+-ATPase and Reveal the Interaction of Two ATP Sites during Catalysis

Abstract

Na؉ /K ؉ -transport through mammalian cell membranes by Na ؉ /K ؉ -ATPase (EC 3.6.1.37) needs the interaction of ATP sites with different binding affinities during catalysis: one with catalytic (high affinity site) and one with regulatory properties (low affinity site). To find affinity labels for the latter one, the effects of 2-Odansylated ATP analogs on Na -ATPase binds ATP with high affinity to the sodium exporting E 1 -form (E 1 ATP binding site) and is consequently phosphorylated. After the release of sodium at the outer cell side and the following dephosphorylation, the enzyme needs a second binding of ATP. Therefore, ATP binds with low affinity to the E 2 -form (E 2 ATP binding site) of Na ϩ /K ϩ -ATPase and enhances the rate-limiting step of deocclusion of potassium during import (2, 3). In contrast to expectations deriving from this single ATP site model with its subsequent formation of the ATP sites, use of substitution-inert MgATP complex analogs has led to the postulate of a coexistence (in time and at different places) of both ATP sites (4). The Repke-Schön-Stein model (5) attempts to explain such a situation by shifting the energy excess of the sodium-transporting subunit to the potassium-transporting subunit. Each subunit follows a whole Albers-Post cycle but 180°out of phase. The bicyclic model of Plesner, on the other hand, gets its power from two ATP binding sites whose partial activities (Na ϩ -ATPase, K ϩ -phosphatase) are lower than the overall reaction (Na ϩ /K ϩ -ATPase). A single subunit does not have to pass all of the intermediates of the Albers-Post circle, but the sum fulfills all steps required for a whole turnover (6). However, there is still a lot of discussion about the intermediates shared by the partial reactions and the overall reaction (7,8).Substitution-inert MgATP complex analogs like CrATP 1 or CoATP are helpful tools to dissect the overall Na ϩ /K ϩ -ATPase activity by specific modifications of either the E 1 ATP site or the E 2 ATP site (9, 10). The activities of the E 1 ATP binding site (for example ATP/ADP exchange and "frontdoor phosphorylation") are unaffected by the inactivation of the E 2 ATP binding site by Co(NH 3 ) 4 PO 4 (11). Similarly, CrAMP-PCP, which inactivates the E 1 ATP site but is unable to phosphorylate it, does not affect activities of the E 2 ATP site, namely 86 Rb ϩ occlusion, K ϩ -activated phosphatase activity, and "backdoor phosphorylation" (12, 13). Although substitution-inert metal ATP complexes are on the one hand helpful tools to get information on basic prop-

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

“…Both the spectral shift and the appearance of fluorescence, were a clear indication of NBD-Cl modifications of the enzyme. This was observed when the enzyme was labeled either with or without Co(NH 3 ) 4 ATP or Cr(H 2 0) 4 Ado/ , P[CH 2 ]P. The former compound is known as a specific inhibitor of the E 2 ATP site [2,5,7] while the latter blocks specifically the EiATP site [2,3]. The absorption or fluorescence spectra of enzyme-bound NBD-Cl did not differ significantly if the labeling of the enzyme was done in the presence of Co(NH3) 4 ATP or Cr(H 2 0) 4 AdoPP[CH 2 ]P (EjATP site or E 2 ATP site is blocked).…”

“…Frontdoor phoshorylation from [Y-32 P] was studied as a function of the exposure time of the enzyme at 37°C with NBD-Cl. For experimental details see Section 2 and [5,7]. Mean values of three experiments are shown.…”

“…All these observations, including the observation of a positive cooperative effect of 2'-ODANS-8-N 3 -ATP in the inactivation of Na+/K+-ATPase [7] led to the conclusion that two simultaneously existing ATP sites need to cooperate during ATP-driven Na+/ K + transport [7,8]. Unfortunately, the available data cannot differentiate between the possibility that two ATP-binding sites exist on a single a subunit and the possibility that the sodium pump works as a functional dimer (ocß) 2 with two cooperating ATP sites.…”

“…The inactivated enzyme was centrifuged at 100 000 Xg, washed in 20 mM Tris/HCl (pH 7.25) and incubated in 20 mM Tris/HCl, 15 mM NaCl with 100 uM or 1 mM NBD-Cl. After different incubation times at 37°C the enzyme was washed twice with 20 mM Tris/HCl (pH 7.25) and frontdoor phosphorylation was estimated according to Thoenges and Schoner [7]. Background labeling was subtracted using the value of a sample where 1 U of Na + /K + -ATPase had been quenched with 250 u.1 of 25% trichloroacetic acid before phosphorylation from [y-32 P]ATP started.…”

“…The time course of inactivation of Na + /K + -ATPase was followed by transferring aliquots of 50 ul to the coupled optical test [15]. The initial rates of inactivation were calculated and analyzed according to the method of Piszkiewics et al [7,17].…”