Characterization of conformational changes in (Na,K) ATPase labeled with fluorescein at the active site.

TLDR: The collected data support and extend the previous suggestion that K+ ions bound initially at a low-affinity site in state E1 are trapped in the occluded form E2· (K) by the conformational change poised far (Kc≈1000) in the direction of E2 · (K).

Abstract: Conformational changes have been studied in (Na,K) ATPase labeled at or near the ATP binding region with fluorescein following incubation with fluorescein isothiocyanate (FITC). One or two fluorescein groups are bound per ATPase molecule. (Na,K) ATPase activity, phosphorylation from ATP, and nucleotide binding are abolished in labeled enzyme, but phosphorylation from inorganic phosphate or K-phosphatase activity are only partially inactivated. The fluorescein groups are incorporated only into the 96 KD catalytic chain of the (Na,K) ATPase, and presence of ATP during the incubation with FITC protects against the incorporation and inhibition of enzymic activity. Upon trypsin treatment of labeled membranes the fluorescein appears first in a 58 KD fragment and eventually is released into the medium. The fluorescein-labeled (Na,K) ATPase shows a large quenching of fluorescence (15–20%) on conversion of the E1 or E1 · Na conformation in cation-free or Na+-rich media to the E2 · (K) form in K+ (or congeners Tl+, Rb+, Cs+, NH 4 + ) rich media. Cation titrations suggest that K+ and Na+ ions compete at a single binding site and stabilize E1 · Na or E2 · (K) respectively;K K≈0.23 mM,K Na≈1.2 mM. The rate of the conformational transition E2 · (K) → E1 · Na is slow,k=0.3 sec−1, but contrary to previous experience [7, 8] ATP does not stimulate this rate. The rate of the transitions E1 + K+ → E2 · (K) rises sharply with K+ concentration and shows saturation behavior, from which ak max≈286 sec−1 andK k≈74 mM are deduced. The data support and extend the previous suggestion that K+ ions bound initially at a low-affinity (probably cytoplasm oriented) site in state E1 are trapped in the occluded form E2 · (K) by the conformational change poised far (K c≈1000) in the direction of E2 · (K). It is proposed in addition that at least two binding sites for K+ exist at the cytoplasmic surface of isolated (Na,K) ATPase in state E1 but a large difference in affinities precludes detection in fluorescence titrations of more than one site. A variety of ligands in addition to K+ produce fluorescence-quenched or E2 forms of the labeled (Na,K) ATPase. These include Mg2+ plus inorganic phosphate, without or with K+ ions (E2P or E2P · K) or with ouabain (E2-ouabain or E2P · ouabain). Na+ ions antagonize these effects. The collected data support the notion that there may be many subspecies of the E1 and E2 forms (either phosphorylated or nonphosphorylated) with different numbers of Na+ and/or K+ ions bound or occluded, each subspecies having a characteristic ability to catalyze reactions and/or transport cations. The relationship between the conformational changes in fluorescein-labeled enzyme and the subunit structure of the (Na,K) ATPase is discussed with particular reference to “half of the site” models for ATP hydrolysis.