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J Biol Chem, Vol. 274, Issue 45, 31792-31796, November 5, 1999

Acid-labile ATP and/or ADP/P_i Binding to the Tetraprotomeric Form of Na/K-ATPase Accompanying Catalytic Phosphorylation-Dephosphorylation Cycle^*

Takeshi Yokoyama, Shunji Kaya, Kazuhiro Abe, Kazuya Taniguchi^§, Tsuyoshi Katoh^¶, Michio Yazawa^¶, Yutaro Hayashi, and Sven Mårdh^**

From  Biological Chemistry and ^¶ Bioorganic Chemistry, Division of Chemistry, Graduate School of Science, Hokkaido University, Sapporo 060-0810, Japan, the  Department of Biochemistry, Kyorin University School of Medicine, Mitaka 181-1611, Japan, and the ^** Department of Biomedicine and Surgery, Faculty of Health Sciences, Linköping University, Linköping S-581 85, Sweden

	ABSTRACT

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The Na/K-ATPase has been shown to bind 1 and 0.5 mol of ³²P/mol of -chain in the presence [-³²P]ATP and [-³²P]ATP, respectively, accompanied by a maximum accumulation of 0.5 mol of ADP-sensitive phosphoenzyme (NaE1P) and potassium-sensitive phosphoenzyme (E2P). The former accumulation was followed by the slow constant liberation of P_i, but the latter was accompanied with a rapid ~0.25 mol of acid-labile P_i burst. The rubidium (potassium congener)-occluded enzyme (~1.7 mol of rubidium/mol of -chain) completely lost rubidium on the addition of sodium + magnesium. Further addition of ~100 µM [-³²P]ATP and [-³²P]ATP, both induced 0.5 mol of ³²P-ATP binding to the enzyme and caused accumulation of ~1 mol of rubidium/mol of -chain, accompanied by a rapid ~0.5 mol of P_i burst with no detectable phosphoenzyme under steady state conditions. Electron microscopy of rotary-shadowed soluble and membrane-bound Na/K-ATPases and an antibody-Na/K-ATPase complex, indicated the presence of tetraprotomeric structures ()₄. These and other data suggest that Na/K-ATP hydrolysis occurs via four parallel paths, the sequential appearance of (NaE1P:E·ATP)₂, (E2P:E·ATP:E2P:E·ADP/P_i), and (KE2:E·ADP/P_i)₂, each of which has been previously referred to as NaE1P, E2P, and KE2, respectively.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION REFERENCES

It is generally accepted that Na/K-dependent ATP hydrolysis (1) occurs via the sequential appearance of phosphoenzymes and dephosphoenzymes (Scheme I), accompanied by the active transport of 3 sodium and 2 potassium ions across the membranes (2-6). The maximum ligand binding or occlusion and phosphorylation capacity per catalytic subunit (-chain) reported are generally taken as evidence for the functional subunit being a protomer (7-9). However, the maximum phosphorylation capacity of these enzymes has been unequivocally shown to be 0.5 mol/mol of -chain during sodium-dependent ATP hydrolysis (10) even though the binding stoichiometry is 1 mol of ouabain/mol. Data concerning conformational changes of Na/K-ATPase in real time (11) and a quarter site phosphorylation of the -chain by ATP (10) and P_i (12-14) as well as other kinetic data (15, 16) support the hypothesis (11) that four different ATP binding sites to the tetraprotomeric enzyme form, ()₄, induce out of phase conformational changes in Na/K-ATPase (14, 17). Therefore, questions arise as to the stoichiometry of ATP binding to the phosphorylated or dephosphorylated (cation-occluded) enzyme states during ATP hydrolysis as well as the gross molecular structure of the enzyme.

	MATERIALS AND METHODS

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Methods for the purification of Na/K-ATPase from kidneys of pig (14) and dog (18) and for the estimation of the amount of EP from [-³²P]ATP (15) and bound ³²P (19) have been described previously except that the enzyme (2~4 mg of protein/ml) was incubated with 50 µl of a pH 7.4 solution containing 25 mM imidazole HCl, 0.1 mM EDTA-Tris, 25 mM sucrose, 2 M NaCl, or changing the concentrations of NaCl to maintain the ionic strength at 2 M with choline chloride or 16 mM sodium and 0.43 mM MgCl₂ and various concentrations of [- or -³²P]ATP and 50 mM [³H]glucose to estimate water space. Approximately 25 µl of the mixture was applied to double membranes, an upper Bio-Rad nitrocellulose membrane with a pore size of 0.45 µm with the lower type AA Millipore filter with a pore size of 0.80 µm under vacuum. After 5~10 s, the Millipore filters were incubated with 1 ml of a solution containing cold 5 mM ATP-Tris and 50 mM glucose. An aliquot of both the sample and the reaction mixture before filtration was taken and mixed with scintillation mixture and counted. To obtain maximum rubidium occlusion, pig enzyme preparations giving ~2 nmol of phosphoenzyme/mg of protein were incubated (0.5 mg of protein/ml) with 100 µl of a buffer solution at pH 7.4 containing 25 mM imidazole HCl, 0.1 mM EDTA-Tris, 25 mM sucrose with 320 µM ⁸⁶RbCl without or with 10 mM RbCl at 25 °C. After 60 min, the samples were diluted with 20 ml of ice-cold buffer as described above except 320 µM ⁸⁶Rb was replaced with 30 mM KCl with or without 10 mM RbCl. The rubidium-occluded enzymes were isolated on a Millipore membrane (20) and measured as described previously (19). To estimate rubidium occlusion via E2P, pig kidney enzymes were also incubated with 320 µM ⁸⁶RbCl in the presence of variable concentrations of ATP in the presence of 16 mM NaCl + 1 mM CaCl₂ or 160 mM NaCl + 0.43 (or 4) mM MgCl₂ for 10 s at 0 °C.

	RESULTS AND DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION REFERENCES

Fig. 1 shows that the amount of bound ³²P in the presence of 0.43 mM magnesium + 2 M sodium with increasing concentrations of [-³²P]ATP and [-³²P]ATP increased to give saturation at ~1 and ~0.5 mol/mol of -chain (K_0.5 = 30~20), respectively. The maximum amount (10) of NaE1P¹ (14, 17, 21), estimated as trichloroacetic acid-stable E³²P formed from [-³²P]ATP under steady state conditions, was 0.5 mol/mol of -chain (K_0.5 = 7 µM) as has been shown previously (10). These data show that 1 mol of ³²P bound in the presence of [-³²P]ATP was due to 0.5 mol each of NaE1P and trichloroacetic acid-labile enzyme-bound ATP or P_i. Thus, 0.5 mol of ³²P bound in the presence of [-³²P]ATP was due to the trichloroacetic acid-labile enzyme-bound ATP or ADP. A similar stoichiometry of ³²P binding and phosphorylation was also observed (not shown) in the dog kidney enzyme, giving ~3.5 nmol of phosphoenzyme/mg of protein as in the case of the pig kidney enzyme. These data can be explained by the presence of a diprotomer, designated as NaE1P:E·ATP or NaE1P:E·ADP/P_i. The binding of ³²P is enzymatic, as evidenced by the fact that the amount of ³²P bound to pig enzymes at ~10 s after the addition of 100 µM [-³²P]ATP and [-³²P]ATP, respectively, decreased to 3 and ~20%, after a 15-min incubation at 0 °C.

View larger version (25K): [in this window] [in a new window]   Fig. 1.   Concentration dependence of [-32P]ATP and [-32P]ATP on the amount of trichloroacetic acid-labile bound 32P and trichloroacetic acid stable phosphoenzyme (EP). Bound 32P in the presence of [-32P]ATP (closed circles) or [-32P]ATP (open circles) was estimated from the difference between the counts of the reaction mixture and the counts of the filtrate in the Millipore filter. The amounts of 32P bound/mg of protein were corrected as mole/mole -chain from the maximum amount of EP to be 0.5 mole/mole -chain (11). The x axis shows the initial concentrations of ATP added. Data shown in this paper are the means ± S.D. for three or four samples.

To investigate the presence of bound ³²P in E2P, the concentrations of sodium were reduced from 2 M to 16 mM to increase the fraction of E2P (14, 17, 21). Fig. 1, inset, shows that the effect of changing sodium concentrations at a constant ionic strength of 2 M maintained with choline chloride had a negligible effect on the stoichiometry of bound ³²P, which reached nearly 1 mol/mol of -chain in the presence of [-³²P]ATP.

The phosphoenzyme in the presence of 16 mM sodium + 1984 mM choline chloride and 100 µM [-³²P]ATP with magnesium was shown to be a mixture of ~0.25 mol of NaE1P and E2P each, and the amount of ³²P bound in the presence of [-³²P]ATP was ~0.5 mol/mol of -chain. These data suggest the accumulation of 0.25 mol of either NaE1P:E·ATP or NaE1P:E·ADP/P_i and 0.25 mol of either E2P:E·ATP or E2P:E·ADP/P_i. In other words the nonphosphorylated -chain of E2P also bound ATP or ADP/P_i as in the case of NaE1P.

To investigate whether bound ³²P is present in a rubidium-occluded enzyme (RbE2), rubidium occlusion was measured under various conditions as described under "Materials and Methods." A rubidium-occluded enzyme (Fig. 2, closed squares), which is formed directly by incubating the enzyme with 0.32 mM rubidium, lost occluded rubidium nearly completely upon the addition of 160 mM sodium with 0.43 or 4 mM magnesium or 16 mM sodium with 1 mM calcium. With increasing concentrations of ATP from 0 to 5 mM, the amount of rubidium occlusion increased to nearly 1 mol and then appeared to decrease to a constant level of ~0.5 mol of rubidium/mol of -chain, especially in the presence of 16 mM sodium + 1 mM calcium.

View larger version (22K): [in this window] [in a new window]   Fig. 2.   Concentration dependence of ATP on the amount of occluded rubidium. Rubidium occlusion of pig kidney enzymes was measured as described under "Material and Methods".

For both ³²P binding in the presence of 100 µM [-³²P]ATP and [-³²P]ATP, the amount of phosphoenzyme, and that of rubidium occlusion, were measured in the presence of 1 mM calcium, 16 mM sodium, and 0.32 mM rubidium. Both ³²P bindings were shown to be ~0.5 mol/mol of -chain with 0.02 mol of E³²P and ~1 mol for the occluded Rb/-chain. These data mean the stoichiometry of rubidium occlusion/EP dephosphorylation, ~2, as shown in the case of another potassium congener, 2 thallium-occluded/EP-dephosphorylated (20). They are consistent with the view that 2 mol of rubidium occlusions occur in 1 mol of the -chain, which was phosphorylated to form E2P just prior the binding of rubidium. These data suggest the presence of stoichiometric amounts of both RbE2 and trichloroacetic acid-labile bound ³²P, such for RbE2:E·ATP or RbE2:E·ADP/P_i in the presence of ~100 µM ATP with sodium and calcium or magnesium.

To measure trichloroacetic acid-labile P_i more directly, a time course for the phosphorylation and liberation of P_i was followed by quenching the reaction with trichloroacetic acid. The addition of ATP in the presence of 2 M sodium with magnesium (Fig. 3A) induced the accumulation of NaE1P (14, 17, 21), which was followed by a slow steady liberation of P_i as also shown at 25 °C (21). These data indicate the presence of the trichloroacetic acid-labile binding of ATP, but not of P_i, to the nonphosphorylated -chains of NaE1P such as NaE1P:E·ATP.

View larger version (25K): [in this window] [in a new window]   Fig. 3.   Time course of EP formation and PI liberation in the accumulation condition of NaE1P, E2P, and RbE2 via E2P. Pig kidney enzymes (1 mg/ml) were incubated with 50 µl of imidazole HCl buffer containing 0.43 mM MgCl2 with 2 M NaCl, 16 mM NaCl, and 16 mM NaCl + 320 µM RbCl, respectively, for the ATP-induced accumulation of NaE1P (A), E2P (B), and RbE2 (C). The time course of phosphorylation (solid squares) and Pi liberation (open symbols) at 0 °C were followed after the addition of 50 µl of the buffer containing 200 µM [-32P]ATP. The reaction was terminated by trichloroacetic acid, and trichloroacetic acid-labile Pi was separated by charcoal (32), and the amount of both Pi and EP were measured. The amount of Pi burst was estimated from the extrapolation of the time course of Pi liberation to time 0. The v/EP was estimated from the slope and the maximum amount of EP, 0.5 mol/mol of -chain. Inset, 5 s after the addition of [-32P]ATP, the reaction mixture was diluted with 2 ml of a solution containing the same concentration of ligands present in the phosphorylation reaction except the enzyme was absent and [-32P]ATP was replaced with 100 µM nonradioactive ATP-Tris. The reaction was terminated with trichloroacetic acid. An apparent first order rate constant of EP breakdown was estimated.

Fig. 3B shows the addition of ATP in the presence of 16 mM sodium with magnesium-induced accumulation of E2P (14, 17, 21), which was accompanied by a rapid clear P_i burst estimated from the extrapolation of P_i liberation at time 0, 0.18 ± 0.01 mol/mol of -chain. The maximum amount of P_i burst, estimated from the burst sizes observed via the additions of 25, 50, 100, and 200 µM ATP, appeared to give 0.24 mol/mol of -chain with an apparent K_m of 43 µM. A similar P_i burst has also been shown by the addition of 32 µM ATP at 25 °C (21). These considerations suggest the presence of both 0.25 mol of trichloroacetic acid-labile ATP and ADP/P_i binding to nonphosphorylated -chains of E2P, such as E2P:E·ATP:E2P:E·ADP/P_i.

To investigate the effect of bound ATP and/or ADP/P_i on sodium-dependent ATP hydrolysis, the rate constant of EP breakdown and the turnover number, v/EP, was compared. Fig. 3B and the inset show that the rate constant and v/EP are, respectively, around 0.07/s and 0.18 ± 0.01/s. A value of v/EP, 0.15 ± 0.01/s, was also obtained in a similar experiment. The rate constant is approximately half the value of v/EP. These data clearly indicate that ATP hydrolysis occurs via both E2P and enzyme-bound ATP and/or ADP/P_i.

Fig. 3C shows that the addition of ATP to the enzyme in the presence of 16 mM sodium, 0.43 mM magnesium, and 0.32 mM rubidium induced a rapid burst of P_i, followed by a slow steady increase of P_i production without detectable phosphoenzyme. The magnitude of the burst, 0.42 mol/mol of -chain, was close to the value of the maximum amount of EP in the absence of rubidium, 0.5 mol/mol of -chain. These data show that the trichloroacetic acid-labile binding of ADP/P_i occurs to the non-rubidium-occluded -chain of the enzyme in the form of RbE2:E·ADP/P_i. The data suggest that ATP hydrolysis occurs via KE2:E·ADP/P_i, which is considered to be a KE2·P_i complex, following the breakdown of E2P (Scheme I), as shown from the initial burst of P_i production (22).

View larger version (17K): [in this window] [in a new window]   Scheme I.   Sequential model for Na/K-ATPase: Post-Albers mechanism.

The data described above and other data (10-15) strongly suggest a tetraprotomeric structure for the enzyme. To obtain direct evidence for this, both solubilized and membrane-bound Na/K-ATPases were rotary-shadowed and observed by electron microscopy. Purified membrane-bound dog kidney Na/K-ATPase was solubilized (18) with octaethylene glycol dodecyl ether (C₁₂E₈) in the presence of 100 mM potassium acetate and subjected to gel filtration at 0 °C. The ATP binding capacity² appeared as three different peaks with molecular weights of 7.5 × 10⁵-1.5 × 10⁶, 3.0 × 10⁵, and 1.5 × 10⁵. When the first peak fraction was examined, tetrameric, dimeric, and monomeric structures, which may correspond to tetraprotomer, diprotomer, and protomer (), respectively, were observed (Fig. 4, A-D). The protomer showed a pear-like shape ~21 nm in length and ~11 nm in the width at the widest portion and appeared to be a unitary structure of the diprotomer and tetraprotomer. The percentages of these structures were 32, 39, and 27%, respectively, and higher oligomeric structures constituted only 2% (n = 151). The tetrameric, dimeric, and monomeric molecules were 29, 49, and 21%, respectively, for the second fraction (n = 200) and 18, 38, and 43%, respectively, for the third fraction (n = 208). When the structure of the molecules contained in the first peak fraction were fixed by cross-linking with a zero-length cross-linker, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC), the morphology of these structures was unchanged (Fig. 4, B and E), but their relative amounts were different; the higher oligomeric and tetrameric structures increased to 18 and 39%, respectively, and the monomeric structure decreased to 5% (n = 153). Therefore, it is possible that native enzyme could exist in a tetrameric form, which tends to dissociate into dimeric and monomeric forms when solubilized.

View larger version (46K): [in this window] [in a new window]   Fig. 4.   Rotary-shadowed molecules of Na/K-ATPases. Samples of Na/K-ATPase preparations were diluted to ~10 µg/ml with 60% glycerol (33), sprayed onto mica, rotary-shadowed with platinum, and observed with a Hitachi H-800 electron microscope operated at 75 kV (34). The solubilized dog kidney enzyme was used directly (A-D) or after cross-linking with EDC (E) to fix its structure. The membrane-bound pig enzymes without (F-I) or with (J) Anti-FITC antibodies (Molecular Probes, Inc.) were treated with EDC and then solubilized with C12E8. The FITC-labeled enzyme (~0.9 mol labeled at Lys-501/mol of -chain) was prepared as described previously (10). The EDC treatment of these enzymes was carried out on ice overnight with 5 mM EDC in the presence of 10 mM imidazole (pH 7.0) and was terminated with 10 mM dithiothreitol (34). A, monomeric, dimeric, and tetrameric structures of solubilized enzyme in the first peak fractions on gel filtration are indicated by single, double, and triple arrowheads, respectively; B, E, F, and I, the enzyme molecules with a tetrameric appearance; C and G, enzyme molecules with a dimeric appearance; D and H, enzyme molecules with a monomeric appearance. E, enzyme fixed by cross-linking with EDC; I, FITC-labeled enzyme; J, FITC-labeled enzyme complexed with anti-FITC antibodies. A structure judged as most probable by eye for its magnified image was drawn under each each electron micrograph in B-J. The rotary shadowed antidodies observed as triangula or fat "Y"-shaped structures, as reported previously (35, 36), were drawn as dotted structures in J. The scale bar for B-J is indicated at the bottom.

Electron microscopy of the membrane-bound pig enzyme, which had been treated with EDC prior to solubilization with C₁₂E₈, also showed tetrameric, dimeric, and monomeric structures, which were morphologically similar to those observed for the dog enzyme (Fig. 4, A-E and F-H). To confirm that the molecules observed were, in fact, Na/K-ATPase molecules, the enzyme (10) containing ~0.9 mol/mol of -chain of Lys-501 labeled with fluorescein 5'-isothiocyanate (FITC) was complexed with anti-FITC antibodies (Fig. 4, I and J). The antibodies showed the presence of tetramer with 1-4 molecules of the antibodies attached to the wider end of the pear-like structure, which also indicated that the wider end reflected the cytosolic side (Fig. 4J). No antibody binding was observed for the unlabeled enzyme.

The tetraprotomeric nature of Na/K-ATPase was proposed nearly 20 years ago (12, 13, 23). A quarter, half and third to fourth, and full site reactivity have been demonstrated by several independent data, collected by us. 1) The stoichiometries of ATP binding (15, 24), rubidium occlusion (Fig. 2) and phosphorylation by ATP (10), P_i (14), acetyl phosphate (16), and p-nitrophenyl phosphate (14) and the ratio of ouabain binding to phosphoenzyme (15, 24); 2) half-site reactivities of NaE1P to acetate (16) and p-nitrophenol (15), namely a quarter site reactivity of the -chain (10); 3) the fact that ATP induced four different conformational changes out of phase (11); 4) ³²P binding and the burst size of P_i (Fig. 3, B and C); and 5) electron microscopy (Fig. 4). The experiments described in this paper not only show direct biochemical evidence for the presence of a tetraprotomer structure of Na/K-ATPase during ATP hydrolysis but also indicate quite new aspects of the enzyme forms. All reaction intermediates detectable in the Post-Albers scheme (Scheme I) bind ATP and/or ADP/P_i.

Further experiments are required to clarify the role of tetrameric structure in Na/K-ATP hydrolysis, although changes in the subunit interaction by physiological ligands were already suggested (4, 5, 10, 11, 18).

To explain the data described above, we propose a tetramer mechanism for ATP hydrolysis (Fig. 5). ATP binding is followed by four parallel paths, which occur at each of the two half-sites for phosphorylation-dephosphorylation and for ATP hydrolysis without acid-stable phosphoenzyme via (NaE1P:E·ATP)₂, (E2P:E·ATP:E2P:E·ADP/P_i), and (KE2:E·ADP/P_i)₂, respectively. The sequential formation of E2P from NaE1P and KE2 from E2P (Scheme I) is accompanied by, respectively, hydrolysis of half of the trichloroacetic acid-labile bound ATP to ADP/P_i and of another half of the bound ATP to ADP/P_i (Fig. 5). The finding related to the stoichiometric amount of trichloroacetic acid-labile ATP and ADP/P_i binding and the P_i burst via nonphosphorylated protomer(s) explain the long standing discrepancies between the turnover number of the enzyme, v/EP, and the rate constant for the breakdown of EP (5, 20) as well as the origin of the P_i burst (21, 22, 25).

View larger version (33K): [in this window] [in a new window]   Fig. 5.   Hypothetical model of tetraprotomeric Na/K-ATPase during ATP hydrolysis. Large open circles and triangles, respectively, designate the sodium-bound and potassium-occluded protomer of Na/K-ATPase and the small circles and triangles, respectively, designate 3 sodium and 2 potassium ions, and the corresponding closed symbols designate occluded cations. Shaded symbols designate the difference in the reactivity of NaE1P to acetate (16) or p-nitrophenol (15) and ATP sensitivity of the rubidium occluded form of the enzyme (Fig. 2). Trichloroacetic acid-labile ATP or ADP/Pi bound to protomers are also shown. Open circles with ATP- or ADP/Pi-bound forms might either contain or occlude sodium, and the KE2 form in the presence of 3~5 mM ATP with magnesium and sodium may decrease to a quarter (Fig. 2) with increase in the amount of bound ATP or ADP/Pi or phosphoenzyme (37). These remain to be determined. Under nonphosphorylated conditions as shown in the dotted parentheses in the figure, the enzyme bound or occluded a maximum of 3 sodium or 2 potassium ions/mol of -chain, respectively (8, 9).

Dynamic changes in stoichiometry for the binding of ligands and/or the occlusion of Na/K-ATPase (Fig. 2 and see Refs. 5, 6, and 26) suggest oligomeric interactions between protomers (4-11, 18, 26-31), which might induce different phosphorylation site stoichiometry by synthetic substrates such as chromium tetraaquaadenosine 5'-triphosphate (9) and p-nitrophenyl phosphate (15) and by chymotrypsin cleavage (31).

If the simultaneous presence of the sodium- and rubidium-bound form of the enzyme becomes clear as an intermediate of ATP hydrolysis such as the RbE2:NaE1·ADP/P_i complex, the determination of one of the two transport mechanisms, i.e. simultaneous or consecutive would be crucial. Further experiments, such as the binding stoichiometry of magnesium, sodium, and potassium to these subunits as shown as E·ATP and E·ADP/P_i (Fig. 5) and the ATP-induced subunit interactions will be required for better understanding the molecular mechanism of ATP hydrolysis.

	FOOTNOTES

^* This work was supported in part by grants-in-aid for Scientific Research and an International Scientific Research Program from the Ministry of Education, Science, Sports and Culture of Japan and for the promotion of science from the Novartis Foundation and The Swedish Medical Research Council (Project 4X-4965).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^§ To whom correspondence should be addressed: Biological Chemistry, Graduate School of Science, Hokkaido University, Sapporo 060-0810, Japan. Tel.:/Fax: 81-11-736-2074; E-mail: ktan@ccms1.hucc. hokudai.ac.jp.

² Y. Hayashi, unpublished data.

	ABBREVIATIONS

The abbreviations used are: NaE1P, sodium-occluded ADP-sensitive phosphoenzyme; NaEATP, Na/K-ATPase complexed with sodium and ATP in the presence of magnesium; E2P, potassium-sensitive phosphoenzyme; RbE2, rubidium-occluded enzyme; KE2, potassium-occluded enzyme; E·ATP, trichloroacetic acid-labile ATP-Na/K-ATPase complex; E·ADP/P_i, trichloroacetic acid-labile ADP/P_i-Na/K-ATPase complex; (NaE1P:E·ATP)₂, (E2P:E·ATP:E2P:E·ADP/P_i), and (KE2:E·ADP/P_i)₂, teteraprotomer form of each enzyme state, which has been previously referred to as NaE1P, E2P, and KE2, respectively; C₁₂E₈, octaethylene glycol dodecyl ether; EDC, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide; FITC, 5'-isothiocyanate.

	REFERENCES

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