Affinity labelling with MgATP analogues reveals coexisting Na⁺ and K⁺ forms of the α‐subunits of Na⁺/K⁺‐ATPase

Abstract

To test the hypothesis that Na + /K + -ATPase works as an (ab) 2 -diprotomer with interacting catalytic a-subunits, tryptic digestion of pig kidney enzyme, that had been inactivated with substitution-inert MgATP complex analogues, was performed. This led to the demonstration of coexisting C-terminal Na + -like 80-kDa as well as K + -like 60-kDa peptides and N-terminal 40-kDa peptides of the a-subunit. To localize the ATP binding sites on tryptic peptides, studies with radioactive MgATP complex analogues were performed: Co(NH 3 ) 4 -8-N 3 -ATP specifically modified the E 2 ATP (low affinity) binding site of Na + /K + -ATPase with an inactivation rate constant (k 2 ) of 12 £ 10 23´m in 21 at 37 8C and a dissociation constant (K d ) of 207^28 mm. Tryptic digestion of the [g + -ATPase is an integral membrane protein which transports Na + and K + ions against an electrochemical gradient; such transport is presumably connected to an oscillation of the enzyme between two major conformational states, namely, the E 1 Na + and the E 2 K + conformations and these E 1 and E 2 states probably have different affinities for ATP. The pumping is often considered to evolve from a conformational change of a single ATP site of the catalytic a-subunit existing, either as a high affinity E 1 ATP site (from which Na + export starts by phosphorylation) or as a low affinity E 2 ATP site (which is involved in K + import) [1]. A number of studies seem to refute the formerly favoured idea that Na + /K + pumping is the result of the conformational change of a single catalytic a-subunit and its single ATP binding site as proposed by the so-called Albers±Post model. Hence, this model excludes simultaneously existing and cooperating ATP binding sites [2,3] and the finding that specific labelling of the E 1 ATP or the E 2 ATP binding sites does not block labelling and partial activities of the other remaining empty site [4±9]. In addition, the demonstration of superphosphorylation', i.e. more than 2 mol phosphate incorporated per catalytic a-subunit [10], as well as the observation of a phosphorylation from, P i , during Na + -ATPase activity [11] is consistent with a model in which Na + /K + -ATPase is phosphorylated at both ATP binding sites [12,13].All of the above data support the conclusion that Na + /K + transport involves the interaction and cooperation of two ATP binding sites residing on two different catalytic a-subunits [14,15]. Consistent with this, by Fo Èrster energy transfer studies, we recently differentiated between an E 1 ATP site labelled by fluorescein isothiocyanate and an E 2 ATP site labelled by erythrosine isothiocyanate and determined that the two ATP binding sites are 6.45 nm apart. Moreover, the distance between the ouabain binding sites labelled by fluorescent ouabain derivatives is 4.9 nm. These distances are obviously too large to assume that both ATP and ouabain binding sites are on the same catalytic a-subunit [16]. Hence, it was concluded that Na + / K + -ATPase works as an (ab) 2 diprotomer of cooperating as...

Na, K-ATPase α3 is a death target of Alzheimer patient amyloid-β assembly

“…Simple arithmetic calculations thus give an ASPD:NAKα3 ratio ranging from 1:1-1:3. However, because past studies have shown that the NAK pump exists predominantly as (NAKα3) 2 :(NAKβ1) 2 heterotetramer in membranes (35,36) and ASPD appeared to bind almost all NAKα3-containing NAK pumps in membranes (Fig. 3E), we infer, using Occam's razor (37), that the most likely ratio is 1:2 (i.e., 1:1 binding of ASPD with NAKα3 dimer).…”

Rat renal glucose transporter SGLT1 exhibits zonal distribution and androgen-dependent gender differences

“…The specific antibody (rSGLT1-Ab) was affinity purified from the immune serum via the antigenic peptide-coupled support (9). The use of a polyclonal anti-Na-K-ATPase ␣-subunit antibody has been described (2,6). A monoclonal antibody against ␣-actin was purchased commercially (Chemicon, Temecula, CA).…”

Proteoliposomes in nanobiotechnology

“…It has been demonstrated that the αβ form of the Na,K-ATPase is capable of both ATP hydrolysis and active ion transport (Ward and Cavieres 1993, Martin et al 2000Miles et al 2011). However, accumulating evidence suggests that the enzyme normally self associates as (αβ) 2 dimers (Santos et al 2002(Santos et al , 2006bThoenges and Schoner 1997;Antolovic et al 1999;Costa et al 2003;Santos and Ciancaglini 2003) or as (αβ) 4 tetramers (Mahaney et al 1990;Donnet et al 2001;Taniguchi et al 2001).…”