Stimulation of p-nitrophenylphosphatase activity of Na+ ⁄ K+-ATPase by NaCl with oligomycin or ATP Haruo Homareda1 and Makoto Ushimaru2 Department of Biochemistry, Kyorin University School of Medicine, Mitaka, Tokyo, Japan Department of Chemistry, Kyorin University School of Medicine, Mitaka, Tokyo, Japan Keywords diprotomer; Na+ ⁄ K+-ATPase; oligomycin; p-nitrophenylphosphate (pNPP); p-nitrophenylphosphatase (pNPPase) Correspondence H Homareda, Department of Biochemistry, Kyorin University School of Medicine, Mitaka, Tokyo 181-8611, Japan Fax & Tel: +81 422 76 7651 E-mail: homareda@kyorin-u.ac.jp (Received July 2004, revised 24 October 2004, accepted 19 November 2004) doi:10.1111/j.1742-4658.2004.04496.x It is known that the addition of NaCl with oligomycin or ATP stimulates ouabain-sensitive and K+-dependent p-nitrophenylphosphatase (pNPPase) activity of Na+ ⁄ K+-ATPase We investigated the mechanism of the stimulation The combination of oligomycin and NaCl increased the affinity of pNPPase activity for K+ When the ratio of Na+ to Rb+ was 10 in the presence of oligomycin, Rb+-binding and pNPPase activity reached a maximal level and Na+ was occluded Phosphorylation of Na+ ⁄ K+-ATPase by p-nitrophenylphosphate (pNPP) was not affected by oligomycin Because oligomycin stabilizes the Na+-occluded E1 state of Na+ ⁄ K+-ATPase, it seemed that the Na+-occluded E1 state increased the affinity of the phosphoenzyme formed from pNPP for K+ On the other hand, the combination of ATP and NaCl also increased the affinity of pNPPase for K+ and activated ATPase activity Both activities were affected by the ligand conditions Oligomycin noncompetitively affected the activation of pNPPase by NaCl and ATP Nonhydrolyzable ATP analogues could not substitute for ATP As NaE1P, which is the high-energy phosphoenzyme formed from ATP with Na+, is also the Na+-occluded E1 state, it is suggested that the Na+-occluded E1 state increases the affinity of the phosphoenzyme from pNPP for K+ through the interaction between a subunits Therefore, membrane-bound Na+ ⁄ K+-ATPase would function as at least an (ab)2-diprotomer with interacting a subunits at the phosphorylation step Na+ ⁄ K+-ATPase (Na+ ⁄ K+-exchanging ATPase; EC 3.6.3.9) is a membrane-integrated protein that actively transports Na+ from the inside of cells to the outside and transports K+ in the reverse direction, coupled with ATP hydrolysis Na+ ⁄ K+-ATPase has two conformations, the E1 conformational state and the E2 conformational state Na+ and ATP bind to E1 The NaE1ATP formed is phosphorylated to the high-energy phosphoenzyme with Na+ (NaE1P) and then transformed to the low-energy phosphoenzyme (E2P), accompanied by Na+ release E2P is K+dependently dephosphorylated to E2 + Pi [1] Because Na+ ⁄ K+-ATPase can hydrolyze p-nitrophenylphos- phate (pNPP), a reaction that is ouabain-sensitive and K+-dependent, the pNPPase activity is presumed to be a partial reaction of Na+ ⁄ K+-ATPase [2–5] About 30 years ago, it was reported that NaCl with oligomycin or ATP stimulated K+-dependent pNPPase activity, although NaCl and ATP individually inhibited the activity and oligomycin had little effect [6–11] Oligomycin and ATP are an inhibitor and a substrate for Na+ ⁄ K+-ATPase, respectively [2–5] Because it remains unclear why both the inhibitor and the substrate activate the K+-dependent pNPPase activity in the presence of Na+ [2], we investigated this question to clarify the activation mechanism Abbreviations AMPPCP, adenylyl-(b,c-methylene)-diphosphonate; E1P, high-energy phosphoenzyme; E2P, low-energy phosphoenzyme; EP, phosphoenzyme; K0.5, concentration giving half-maximal activation; pNPP, p-nitrophenylphosphate; pNPPase, p-nitrophenylphosphatase FEBS Journal 272 (2005) 673–684 ª 2005 FEBS 673 Stimulation of pNPPase activity of Na+ ⁄ K+-ATPase In the first part of this paper, we describe the effect of oligomycin with NaCl on pNPPase activity The ligand combination induced high-affinity K+-dependent pNPPase activity When the ratio of Na ⁄ Rb in the presence of oligomycin was 10, the binding of Rb+, a congener of K+, and pNPPase activity reached a maximal level and Na+ remained occluded Today, it is well known that oligomycin occludes Na+ within the Na+ ⁄ K+-ATPase molecule, so that this antibiotic inhibits Na+ transport and Na+ ⁄ K+-ATPase activity but not K+-dependent pNPPase activity [12–19] Therefore, the present data suggest that the Na+occluded E1 state increased the affinity of pNPPase for K+ In the second part, the effect of ATP with NaCl on pNPPase activity is described The ligand combination induced high-affinity K+-dependent pNPPase activity and ATPase activity at the same time Variation of the ligand conditions affected both activities ADP and adenylyl (b,c-methylene)diphosphonate (AMPPCP) could not substitute for ATP Because NaE1P, which is formed from ATP with Na+, is also the Na+-occluded E1 state [20], the present results suggest that the Na+-occluded E1 state increases the affinity of the phosphoenzyme (EP) from pNPP for K+ We explain the present data using a model in which membranebound Na+ ⁄ K+-ATPase functions as an (ab)2-diprotomer with interacting a subunits H Homareda and M Ushimaru pNPP at pH 8.4 and 37 °C The K0.5 was similar to that at pH 7.4 (data not shown) PNPPase activity in the presence of oligomycin and NaCl The effect of oligomycin with NaCl on pNPPase activity was observed in the presence of 0–3 mm KCl (Fig 1) In the presence of mm KCl without oligomycin, NaCl gradually inhibited pNPPase activity (Fig 1A) The addition of 10 lm oligomycin, which appears to be the maximal concentration in an aqueous solution including 1% (v ⁄ v) ethanol [19], strengthened the inhibitory effect of NaCl on pNPPase activity in the presence of NaCl up to 20 mm In the presence of NaCl at concentrations of more than 20 mm, however, the pNPPase activity with oligomycin was higher than that without the antibiotic In the presence of mm KCl, pNPPase activity with oligomycin was higher than that without the antibiotic in the presence of NaCl at concentrations of more than mm (Fig 1B) In the presence of 0.3 mm KCl, the inhibitory effect of Na+ was absent in the absence of oligomycin (Fig 1C) The addition of oligomycin stimulated the activity fivefold in the presence of 10–30 mm NaCl These results show that, when the Results Effect of pH on ouabain-insensitive pNPPase activity Although the ouabain-insensitive ATPase activity of the Na+ ⁄ K+-ATPase preparation used in this study was less than 5% in the presence of 0.1 mm ouabain, 16% of the total pNPPase activity was ouabain-insensitive at pH 7.4 To find experimental conditions in which the ouabain-insensitive pNPPase activity was minimized, pNPPase activity was measured at pH 6.4, 7.4 and 8.4 The ouabain-insensitive activity was 32%, 16% and 10% at pH 6.4, 7.4 and 8.4, respectively, as shown by Nagai et al [21] In addition to the change in pH, the ouabain concentration was increased from 0.1 to mm to completely depress the increase in the ouabain-insensitive activity due to increasing the KCl concentration On the other hand, increasing the Na+ concentration had no effect on the ouabain-insensitivity of pNPPase activity The concentration giving halfmaximal activation (K0.5) of pNPPase for K+ was mm, and the Vmax was 2.8 lmolỈmin)1Ỉmg)1 in the presence of 10 mm KCl, mm MgCl2 and 2.5 mm 674 Fig Effect of oligomycin on pNPPase activity in the presence of NaCl and KCl Ouabain-sensitive pNPPase activity in the absence (s) or presence (n) of 10 lM oligomycin was assayed in a mixture containing lg (A) or 10 lg (B–D) Na+ ⁄ K+-ATPase, the standard ligands [5 mM MgCl2, 50 mM Tris ⁄ Tes (pH 8.4 at 23 °C), 2.5 mM pNPP, mM EDTA, and with or without mM ouabain], 1% ethanol, 0–300 mM NaCl, and (A) mM KCl, (B) mM KCl, (C) 0.3 mM KCl, or (D) mM KCl Data represent the means of two independent experiments FEBS Journal 272 (2005) 673–684 ª 2005 FEBS H Homareda and M Ushimaru ratio of Na ⁄ K was higher than 4, oligomycin activated pNPPase In the absence of KCl, oligomycin slightly enhanced pNPPase activity in the presence of NaCl at concentrations of more than 10 mm (Fig 1D) Figure shows the activation of pNPPase by KCl in the presence of NaCl with or without oligomycin In the absence of oligomycin, increasing the NaCl concentration decreased pNPPase activity (Fig 2A) In the presence of oligomycin, increasing the NaCl concentration obviously increased the affinity of pNPPase for K+ (Fig 2B) In the presence of 10 lm oligomycin and 30 mm NaCl, the K0.5 for K+ was 0.3 mm The combination of 10 lm oligomycin and 10 mm NaCl started to demonstrate an activation of pNPP hydrolysis with two phases This activation was clearly confirmed by the combination of 30 mm NaCl and 10 lm oligomycin (Fig 2B) The K0.5 and Vmax were 0.3 mm and 0.8 lmolỈmin)1Ỉmg)1 for the high-affinity K+dependent pNPPase activity, and were mm and 1.6 lmolỈmin)1Ỉmg)1 for the low-affinity K+-dependent pNPPase activity, respectively (Fig 3) Fig Activation of pNPPase activity by KCl in the presence of NaCl with or without oligomycin The pNPPase activity in the absence (A) or presence (B) of 10 lM oligomycin was assayed in a mixture containing 10 lg Na+ ⁄ K+-ATPase, the standard ligands, 1% ethanol, 0–3 mM KCl and (s), mM (n), mM (h), 10 mM (b), 30 mM (d), 100 mM (m) or 300 mM NaCl (j) Data represent the means of two determinations FEBS Journal 272 (2005) 673–684 ª 2005 FEBS Stimulation of pNPPase activity of Na+ ⁄ K+-ATPase Fig Activation of pNPPase by KCl in the presence of NaCl and oligomycin The pNPPase activity was assayed in a mixture containing lg Na+ ⁄ K+-ATPase, the standard ligands, 1% ethanol, 0–30 mM KCl, and 10 lM oligomycin (m) or 30 mM NaCl plus 10 lM oligomycin (n) Plots and bars represent the means ± SD from three determinations The relation between pNPPase activity and ion-binding pNPPase activity and ion binding were measured in reaction mixtures containing 0.1 mm RbCl, 0.5 mm pNPP and 0–10 mm NaCl (Fig 4) In this experiment, the MgCl2 concentration was reduced to mm, and the reaction temperature lowered to °C because a high Mg2+ concentration inhibited the binding of Na+ and K+ [14,15] and a low temperature increased the affinities of Na+ ⁄ K+-ATPase for Na+ and K+ (H Homareda, unpublished data) Specific (ouabain-sensitive) Na+ binding was very low in the absence of oligomycin, which increased the affinity of Na+ ⁄ K+-ATPase for Na+ [14] The resultant increase in Na+ binding was regarded as Na+ occlusion In the absence of oligomycin, NaCl gradually inhibited pNPPase activity and Rb+ binding (Fig 4A,B) In the presence of oligomycin, the binding curve of Rb+ and the activation curve of pNPPase showed a convex shape The peak of both Rb+ binding and pNPPase activity occurred at mm NaCl A plausible explanation is that the activation of pNPPase by oligomycin with NaCl is due to the increase in K+ affinity On the other hand, Na+ occlusion was preserved under the ligand conditions used (Fig 4C) Therefore, the Na+-occluded E1 state of Na+ ⁄ K+-ATPase seemed to induce the high-affinity K+-dependent pNPPase activity Phosphorylation from pNPP We examined whether the phosphorylation by pNPP was affected by oligomycin or other ligands in the 675 Stimulation of pNPPase activity of Na+ ⁄ K+-ATPase H Homareda and M Ushimaru Table Phosphorylation of Na+ ⁄ K+-ATPase from pNPP, Pi and ATP For phosphorylation by pNPP or Pi, Na+ ⁄ K+-ATPase was incubated for 10 at 37 °C in the presence of mM MgCl2, 0.5 mM ouabain, 50 mM Tris ⁄ Tes (pH 8.4 at 23 °C), mM [32P]pNPP or 0.3 mM 32Pi with or without the ligands shown For phosphorylation by ATP, Na+ ⁄ K+-ATPase was incubated for 30 s at °C in the presence of mM MgCl2, 50 mM Tris ⁄ Tes (pH 8.4 at 23 °C), 0.1 mM [32P]ATP with or without 2.5 mM pNPP Data are presented as the means ± SD from three to six determinations nmolỈmg)1 mM [32P]pNPP + 0.5 mM ouabain + 10 lM oligomycin + 10 lM oligomycin + 10 mM NaCl + 10 lM oligomycin + 10 mM NaCl + 0.3 mM KCl + 0.3 mM ATP + 0.3 mM Pi 0.3 mM 32Pi + 0.5 mM ouabain + mM pNPP 0.1 mM [32P]ATP + 10 mM NaCl + 2.5 mM pNPP Fig pNPPase activity, Rb+ binding and Na+ occlusion in the same ligand condition (A) The pNPPase activity in the absence (s) or presence (n) of 10 lM oligomycin was assayed in a mixture containing 20 lg Na+ ⁄ K+-ATPase, mM MgCl2, 50 mM Tris ⁄ Tes (pH 8.4 at 23 °C), 0.5 mM pNPP, 0–10 mM NaCl, 0.1 mM RbCl, 1% ethanol, with or without mM ouabain The reaction was started by the addition of pNPP and followed for 90 at °C (B) Ouabain-sensitive 86Rb+ binding in the absence (s) or presence (n) of 10 lM oligomycin was assayed in a mixture containing 30 lg Na+ ⁄ K+-ATPase, mM MgCl2, 50 mM Tris ⁄ Tes (pH 8.4 at 23 °C), 0.5 mM pNPP, 0–10 mM NaCl, 0.1 mM 86RbCl, 1% ethanol, with or without 0.1 mM ouabain After the addition of pNPP, the mixture was centrifuged (C) Oligomycin-stimulated 22Na+ binding was assayed in a mixture containing 30 lg Na+ ⁄ K+-ATPase, mM MgCl2, 50 mM Tris ⁄ Tes (pH 8.4 at 23 °C), 0.5 mM pNPP, 0.3, or mM 22NaCl, 0.1 mM RbCl, 1% ethanol, with or without 10 lM oligomycin After the addition of pNPP, the mixture was centrifuged The detailed procedure is described in Experimental procedures Plots and bars in (A), (B) and (C) represent the means ± SD from three determinations presence of ouabain (Table 1) The amounts of EP were not affected by 10 lm oligomycin, 10 mm NaCl, 0.3 mm KCl or 0.3 mm ATP To determine whether the phosphorylation by [32P]pNPP was affected by contaminating 32Pi, which is nonenzymatically released from [32P]pNPP, or was unreactive 32Pi in the reaction mixture for [32P]pNPP synthesis, 0.3 mm nonradioactive 676 % 1.86 1.85 1.83 1.88 ± ± ± ± 0.05 0.03 0.04 0.02 100 99 98 101 1.63 1.21 1.81 0.65 1.05 1.00 ± ± ± ± ± ± 0.03 0.09 0.05 0.02 0.05 0.05 88 65 100 36 100 95 Pi was added to the reaction mixture If the phosphorylation was due to the contamination by 32Pi, EP must be significantly decreased by the addition of nonradioactive Pi The result showed that EP from mm [32P]pNPP was decreased to 65% by 0.3 mm Pi, whereas EP from 0.3 mm 32Pi was decreased to 36% by mm pNPP pNPPase activity decreased by only 15% in the presence of 0.3 mm Pi Therefore, the EP formed was not due to 32Pi The amount of EP from pNPP was 1.9 times greater than that from ATP This value was consistent with the ratio of EP from Pi or pNPP to EP from ATP [22–24] pNPPase activity in the presence of NaCl, KCl and ATP The effect of ATP with NaCl on pNPPase activity was observed in the presence of mm KCl (Fig 5A) In the absence of NaCl, increasing the ATP concentration from to mm decreased pNPPase activity Increasing the NaCl concentration without ATP inhibited pNPPase activity However, simultaneous addition of NaCl and ATP induced convex-shaped activation curves of pNPPase The combination of 10 mm NaCl and 0.1 mm ATP maximally activated pNPPase In the presence of ATP and KCl, increasing the NaCl concentration induced convex-shaped activation curves of pNPPase (Fig 5B) When 10 mm NaCl and 0.1 mm ATP were present, KCl at 3, and 0.3 mm activated pNPPase by 2.4-fold, 6.4-fold and 20-fold over reactions without NaCl, respectively Consequently, the combination of 10 mm NaCl, 0.3 mm KCl FEBS Journal 272 (2005) 673–684 ª 2005 FEBS H Homareda and M Ushimaru Stimulation of pNPPase activity of Na+ ⁄ K+-ATPase Fig K+-dependent activation curves for pNPPase in the presence of NaCl with or without ATP pNPPase activity in the absence (s) or presence (n) of 0.1 mM ATP was assayed in a mixture containing lg Na+ ⁄ K+-ATPase, the standard ligands, 10 mM NaCl and 0–30 mM KCl Plots and bars represent the means ± SD from three determinations Fig Effects of ATP and KCl on pNPPase activity in the presence of NaCl The pNPPase activity was assayed in a mixture containing lg Na+ ⁄ K+-ATPase, the standard ligands, 0–100 mM NaCl, and (A) mM KCl with (s), 0.01 mM (d), 0.1 mM (n) or mM ATP (m), or (B) 0.1 mM ATP with mM (s), mM (d), 0.3 mM (j) or mM KCl (h) In (B), b and c represent 0.1 mM AMPPCP and 0.1 mM ADP in the presence of 0.3 mM KCl, respectively Plots and bars represent the means ± SD from three determinations and 0.1 mm ATP maximally activated pNPPase The combination of mm NaCl and 0.1 mm ATP slightly activated pNPPase even in the absence of KCl ADP and AMPPCP could not substitute for ATP Figure shows that the combination of 0.1 mm ATP and 10 mm NaCl increases the affinity of pNPPase for K+, as shown by oligomycin with NaCl (Figs and 3) The combination decreased the K0.5 for K+ from to 0.2 mm, which was one-fifth of the K0.5, mm, under the usual conditions Competition between oligomycin and ATP Figure shows the competition between oligomycin and ATP Oligomycin decreased the Vmax without affecting the K0.5, suggesting that oligomycin was a noncompetitive inhibitor of ATP ATPase activity in the presence of NaCl, KCl and ATP The ligand combination of 10 mm NaCl, 0.3 mm KCl, 0.1 mm ATP and 2.5 mm pNPP activated ATPase in FEBS Journal 272 (2005) 673–684 ª 2005 FEBS Fig Effect of oligomycin on pNPPase activity in the presence of NaCl and ATP pNPPase activity was assayed in a mixture containing lg Na+ ⁄ K+-ATPase, the standard ligands, 10 mM NaCl, 0–30 mM KCl and 0.1 mM ATP (s), 10 lM oligomycin (n) or 0.1 mM ATP plus 10 lM oligomycin (h) Plots and bars represent the means ± SD from three determinations addition to pNPPase (Fig 8A) Omission of KCl significantly decreased both activities (Fig 8B) Increasing the NaCl concentration inactivated pNPPase more than ATPase irrespective of the absence and presence of KCl (Fig 8A,B) In the presence of 10 mm NaCl and 0.1 mm ATP, KCl concentrations up to mm simultaneously activated both activities with a K0.5 of 0.2 mm for K+ (Fig 9A) KCl concentrations greater than mm gradually inactivated ATPase On the other hand, omission of NaCl completely inactivated ATPase (Fig 9B) 677 Stimulation of pNPPase activity of Na+ ⁄ K+-ATPase Fig Na+-dependent activation curves for Na+ ⁄ K+-ATPase and pNPPase in the presence of ATP with or without KCl Ouabain-sensitive activities of ATPase and pNPPase in the presence (A) or absence (B) of 0.3 mM KCl were assayed in a mixture containing lg Na+ ⁄ K+-ATPase, the standard ligands, 0–300 mM NaCl and 0.1 mM ATP (n) or 0.1 mM [32P]ATP (m) n and m represent pNPPase and ATPase activity, respectively Plots and bars represent the means ± SD from three determinations In the presence of 10 mm NaCl, 0.3 mm KCl and 0.1 mm ATP, increasing the pNPP concentration increased pNPPase activity (Fig 10A) The K0.5 for pNPP was mm This was equivalent to the value under the usual conditions ATPase activity was decreased by high pNPP concentrations, although the decrease was not more than 50% On the other hand, increasing the ATP concentration had a complex effect on pNPPase (Fig 10B) ATP concentrations up to 0.3 mm activated pNPPase, whereas ATP concentrations greater than 0.3 mm completely inhibited it The activation curve of ATPase was biphasic From a double-reciprocal plot analysis, the K0.5 values for ATP were 0.14 and 2.0 mm and the Vmax values were 0.7 and 5.0 lmolỈmin)1Ỉmg)1 Discussion The affinity of pNPPase for K+ is an order of magnitude lower than that of Na+ ⁄ K+-ATPase for K+ [2,5] The combination of NaCl and oligomycin induced the high-affinity K+-dependent pNPPase 678 H Homareda and M Ushimaru Fig K+-dependent activation curves for Na+ ⁄ K+-ATPase and pNPPase in the presence or absence of NaCl The activities of ATPase and pNPPase in the presence (A) or absence (B) of 10 mM NaCl were assayed in a mixture containing lg Na+ ⁄ K+-ATPase, the standard ligands, 0–30 mM KCl, and 0.1 mM ATP (n) or 0.1 mM [32P]ATP (m) n and m represent pNPPase and ATPase activity, respectively Plots and bars represent the means ± SD from three determinations activity (Figs and 3) The K0.5 for K+ was 0.3 mm, which was equivalent to that, 0.2 mm, for Na+ ⁄ K+ATPase activity (Fig 9A) The increase in K+ affinity caused by oligomycin with Na+ was supported by the ion-binding experiment (Fig 4) When the ratio of Na ⁄ Rb in the presence of oligomycin was 10, Rb+ binding and pNPPase activity reached a maximal value, and the occluded Na+ was preserved (Fig 4) Because this antibiotic stabilizes the Na+-occluded E1 state of Na+ ⁄ K+-ATPase, it seemed that NaE1–oligomycin increased the affinity of pNPPase for K+ The NaE1–oligomycin complex is an arrested form [2–5,12] Therefore, enzyme states other than the complex must hydrolyze pNPP Scheiner-Bobis et al [25–27] and Linnertz et al [28] showed that fluorescein isothiocyanate, which blocks the high-affinity binding site for ATP, affects Na+ ⁄ K+-ATPase activity but not pNPPase activity and showed that Co(NH3)4ATP, which blocks the low-affinity site for ATP, preserves Na+dependent phosphorylation by ATP but inactivates pNPPase They suggested that Na+ ⁄ K+-ATPase FEBS Journal 272 (2005) 673–684 ª 2005 FEBS H Homareda and M Ushimaru Stimulation of pNPPase activity of Na+ ⁄ K+-ATPase the E1 and E2 states coexist depending on the ligand conditions (Fig 11) The consistency between the proposed model and the crystal structure is discussed in the last paragraph According to our model [model (2) in Fig 11], Na+-occluded E1–oligomycin and E2– oligomycin coexist in the presence of 10–30 mm NaCl and 10 lm oligomycin, as shown in Figs and E2– oligomycin is phosphorylated to E2P–oligomycin, which has low-affinity for K+, by pNPP, as shown in Table The Na+-occluded E1–oligomycin complex increases the affinity of E2P–oligomycin for K+ through the interaction between Na+-occluded E1 and E2P This assumption is supported by the finding that Na+ transforms the K+-insensitive E2P, which is formed from Pi and has low affinity for K+, to the K+-sensitive E2P, which has high affinity for K+ [38] Increasing the KCl concentration in the presence of NaCl and oligomycin induces the activation curve with two phases (Fig 3) As K+ binding competes with Na+ binding [15], it is likely that Na+-occluded E1 is transformed to KE2 by high K+ concentration, so that Fig 10 Effects of pNPP and ATP on pNPPase and ATPase activities The activities of ATPase and pNPPase were assayed in a mixture containing lg Na+ ⁄ K+-ATPase, mM MgCl2, 50 mM Tris ⁄ Tes (pH 8.4 at 23 °C), mM EDTA, 10 mM NaCl, 0.3 mM KCl, with or without mM ouabain and (A) 0–30 mM pNPP with 0.1 mM ATP (n) or 0.1 mM [32P]ATP (m), or (B) 2.5 mM pNPP with 0–3 mM ATP (n) or [32P]ATP (m) n and m represent pNPPase and ATPase activity, respectively Plots and bars represent the means ± SD from three determinations works as a functional (ab)2-diprotomer, in which the E1 state and the E2 state coexist [29] The effect of fluorescein isothiocyanate resembles the effect of oligomycin Furthermore, our data suggest that the interaction between NaE1 and E2 increased the affinity of pNPPase for K+ Mimura et al [30] showed that octaethylene glycol dodecyl ether-solubilized Na+ ⁄ K+ATPase forms a loosely associated diprotomer in the E1 state but a tightly associated one in the E2 state Nakamura et al [31] suggested a monomer–dimer transition model of Ca2+-ATPase, in which the cytoplasmic domains clap like a castanet duet, and Carvalho-Alves et al [32] proposed dimerization of the cytoplasmic domain of Ca2+-ATPase Abe et al [33] showed that H+,K+-ATPase functions as an oligomer in the membrane, although a monomer of these P-type ATPases has ATPase activity [34–37] Therefore, we attempted to explain the present data using the (ab)2diprotomer model with interactive a subunits, in which FEBS Journal 272 (2005) 673–684 ª 2005 FEBS Fig 11 Proposed models (1) Activation of pNPPase by K+ and pNPP (2) Activation of pNPPase by Na+, K+, pNPP and oligomycin (3) Activations of pNPPase and ATPase by Na+, K+, pNPP and ATP E1 (Na) represents the Na+-occluded E1 state E2(s)P and E2(i)P represent the K+-sensitive E2P and K+-insensitive E2P state, respectively lK and hK, (Na) and O represent KCl at low and high concentrations, occluded Na+ and oligomycin, respectively P(ATP) and P(pNPP) represent the phosphate transferred from ATP and pNPP, respectively M represents membrane The conformations of E, E1 (Na) and E2 are referred to the crystal structure of Ca2–E1, Ca2–E1–AMPPCP and E2–thapsigargin, respectively [49–51] The conformation of E2(s) is slightly different from the one of E2(i) The domains including M5, M7 and M10 face each other in a diprotomer The upper and lower side of the enzyme represent the external and internal side of cells, respectively Oligomycin is accessible to Na+ ⁄ K+-ATPase at the external side [52] K+ is not transported by pNPPase activity [53] 679 Stimulation of pNPPase activity of Na+ ⁄ K+-ATPase KE2 exhibits the low-affinity K+-dependent pNPPase activity The combination of ATP and NaCl also induced the high-affinity K+-dependent pNPPase activity (Fig 6) The K0.5 for K+ decreased from to 0.2 mm, which was equivalent to that for K+ in Na+ ⁄ K+-ATPase activity (Fig 9A) ADP and AMPPCP could not substitute for ATP (Fig 5B and [8]) The combination of ATP and NaCl activated both ATPase and pNPPase, showing that the equilibrium between the E1 and E2 states is dependent on the ligand conditions (Figs 8– 10) According to the Post-Albers scheme [1], Na+ and ATP bind to the E1 state The NaE1ATP formed is phosphorylated to NaE1P and then converted to E2P + Na+ Because NaE1P is the Na+-occluded E1 state [20] and the phosphoenzymes are in closer contact [39], it would be understood that NaE1P increases the affinity of E2P from pNPP for K+ Nandi et al [40,41] proposed a working model of Na+ ⁄ K+-ATPase and H+,K+-ATPase They postulated that pNPP is accessible to the pNPP hydrolytic sites at the internal and external surfaces However, Garrahan et al [42] used acetyl phosphate, a membrane-impermeable substrate, to show that the pNPP hydrolytic site locates on the internal side of the cell membrane Therefore, we presumed in a proposed model [model (3) in Fig 11] that the E1 state and the E2 state have a high-affinity binding site and a low-affinity binding site for ATP, respectively [25–28] and that pNPP is internally accessible at the low-affinity site for ATP in the E2 state but less accessible at the high-affinity site for ATP in the E1 state, because of a much lower affinity of pNPP for the high-affinity ATP site (Fig and Table 1) Our model facilitates understanding of the results shown in Figs 8–10 In the presence of high Na+, low K+, moderate pNPP and low ATP concentrations, both pNPPase and ATPase were activated, and higher Na+ concentrations inhibited pNPPase activity more than ATPase activity (Fig 8) In this case, ATP binds to the E1 state, which is phosphorylated to NaE1P On the other hand, pNPP accesses the low-affinity site for ATP in the E2 state, which is phosphorylated to E2P (Table 1) NaE1P increases the affinity of E2P for K+ Consequently, K+ binds to the high-affinity K+ site on K+-sensitive E2P and accelerates its dephosphorylation (Fig 4A) An excess of NaCl may competitively inhibit the effect of K+ on pNPPase from the external side [43] The combination of NaCl with ATP or oligomycin slightly enhanced pNPPase activity even in the absence of KCl (Figs 1D, 5B and 8B) Nagamune et al [44] have demonstrated ouabain-sensitive pNPPase activity in the absence of KCl, so these combinations may stimulate the activity 680 H Homareda and M Ushimaru As another possibility, it is likely that NaE1P formed by pNPP with Na+, as proposed by Yamazaki et al [22], and NaE1P formed by ATP with Na+ are spontaneously dephosphorylated Figure 9A shows that increasing the KCl concentration activated pNPPase but gradually inactivated ATPase Omission of NaCl activated pNPPase but not ATPase (Fig 9B) In this case, KCl concentrations up to mm activate both pNPPase and ATPase Increasing the KCl concentration over mm accelerates dephosphorylation of E2P from pNPP, whereas the high KCl concentration or the absence of Na+ disturbs the phosphorylation from ATP Figure 10A shows that increasing the pNPP concentration activated pNPPase but partly decreased ATPase activity In this case, pNPP incompletely inhibits the ATP binding to the high-affinity ATP site in the E1 state Figure 10B shows that increasing the ATP concentration activated ATPase, whereas pNPPase was activated by low ATP concentrations but inactivated by high ATP concentrations Because ATP concentrations up to 0.3 mm inhibit phosphorylation from pNPP little (Table 1), both activities are preserved ATP above 0.3 mm occupies the low-affinity ATP site in the E2 state, so that Na+ ⁄ K+-ATPase is activated but pNPPase activity is completely blocked Oligomycin noncompetitively affected pNPPase activity in the presence of Na+ and ATP (Fig 7) This antibiotic binds in the N-terminal domain of the a subunit [18], whereas ATP binds in the domain containing Lys501 of the a subunit [27] NaE1–oligomycin is an arrested form, whereas NaE1ATP is an active form in the ATP hydrolysis reaction Therefore, the differences in the binding sites and in the biochemical properties between oligomycin and ATP should lead to the noncompetitive effect of oligomycin on ATP Toyoshima et al [45,46] and Sørensen et al [47] have solved the crystal structures of the Ca2–E1, Ca2– nucleotide–E1 and thapsigarigin–E2 states in Ca2+ATPase at a high resolution The structures are classified into two groups depending on the structure of the cytoplasmic domain, i.e an open form and a compact form The Ca2–E1 state is an open form [45–47] Binding of the nucleotide converts it into a compact form [47] The compact form induced by binding of the ADP–AlF4– complex, one of the ATP analogues, resembles the E1P state and occludes Ca2+ [47] The thapsigargin–E2 state is a compact form, although its conformation is slightly different from that of the Ca2– nucleotide–E1 state [47] The crystal structure of Ca2+-ATPase is suggested to be similar to that of Na+ ⁄ K+-ATPase [48–50] Therefore, the conformations of the intermediates that appeared in this study were referred to the crystal structures of Ca2+-ATPase FEBS Journal 272 (2005) 673–684 ª 2005 FEBS H Homareda and M Ushimaru (Fig 11) The E state is drawn as an open form This state may loosely associate with another E state at the cytoplasmic domain, as shown by Carvalho-Alves et al [32] E2(i)P, which corresponds to K+-insensitive E2P, is drawn as a compact form Because the K+-activation curve of pNPPase showed a positive co-operativity [51], pNPPase probably works as a diprotomer [model (1) in Fig 11] The Na+-occluded E1 state is drawn as a compact form E2(s)P, which corresponds to K+-sensitive E2P, may be slightly different from the E2(i)P state because the affinity for K+ varies dependently with the configurations of the 4th, 5th and 6th transmembrane segments (M4, M5 and M6) [50] It is likely that Na+-occluded E1 associates with E2(s)P through M5, M7 and M10 because (a) these segments are linearly arranged in the crystal structure [49], (b) M7 and M10 are almost unmoved by large movement of the cytoplasmic domain [45–47], and (c) the association between the transmembrane domains including these segments is not disturbed by the cytoplasmic domain in a compact form [45–47] The conversion of the Ca2– E1 state into the Ca2+-occluded E1 state accompanies movement of M1 and M2 [45–47] Oligomycin binds to the domain including M1 and M2 of Na+ ⁄ K+-ATPase a subunit [18] Therefore, it is conceivable that arrest of the movement by oligomycin stabilizes the Na+occluded E1 state and inhibits Na+ transport Experimental procedures Materials ATP(Na)2 and AMPPCP were purchased from RocheDiagnostics (Penzberg, Germany) A portion of the ATP(Na)2 was converted into the sodium-free ATP form by passing it through a cation-exchange column Oligomycin B and ouabain were purchased from Sigma Chemical Company (St Louis, MO, USA) Oligomycin B was stored as a 1-mm solution in cold ethanol pNPP (ditris salt) was from ICN Biomedicals Inc (Aurora, OH, USA) 86RbCl and 22NaCl were obtained from Amersham Pharmacia Biotech (Amersham, Bucks, UK) [32P]ATP[cP] and 32Pi were obtained from PerkinElmer Life Sciences Japan (Tokyo, Japan) Other reagents were purchased from Wako Pure Chemicals Industries, Ltd (Osaka, Japan) Preparation of Na+ ⁄ K+-ATPase Microsomes were prepared from canine kidney outer medulla and treated with sodium deoxycholate, as described by Hayashi et al [54] More than 95% of the total Na+ ⁄ K+-ATPase activity (about lmol PiỈmin)1Ỉmg)1) was ouabain-sensitive under the usual conditions FEBS Journal 272 (2005) 673–684 ª 2005 FEBS Stimulation of pNPPase activity of Na+ ⁄ K+-ATPase Synthesis of [32P]pNPP [32P]pNPP was synthesized by the method of Guan & Dixon [55] 32Pi (33 MBq in 0.02 m HCl) and 0.2 mmol (20 mg) crystalline phosphoric acid were dissolved in dehydrated acetonitrile to use as starting materials The amount of pNPP (cyclohexylamine salt) synthesized was measured from the absorbance at 310 nm The amount and the initial specific radioactivity of [32P]pNPP were 51 lmol and 9000 cpmỈnmol)1, respectively Assay of pNPPase activity The standard reaction mixture (0.5 mL) was composed of lg Na+ ⁄ K+-ATPase, mm MgCl2, 50 mm Tris ⁄ Tes (pH 8.4 at 23 °C), mm EDTA, 2.5 mm pNPP and NaCl and KCl at the concentrations indicated in the figure legends, with or without 0.1 mm ATP, with or without 10 lm oligomycin, and with or without mm ouabain The control experiment for the oligomycin effect was performed in the presence of 1% ethanol without oligomycin The pNPPase reaction was started by addition of the enzyme, and the reaction was followed for 5–10 at 37 °C and stopped by addition of 2.5 mL 0.1 m NaOH The p-nitrophenol liberated was measured from the absorbance at 420 nm Ouabain-sensitive activity was determined from the difference between the activities in the presence and absence of mm ouabain When pNPPase activity was measured at °C, the amount of Na+ ⁄ K+-ATPase was changed to 20 lg and the concentrations of MgCl2 and pNPP to and 0.5 mm, respectively The incubation time was extended to 90 Assay of Na+ ⁄ K+-ATPase activity The same reaction mixture (0.5 mL) as that used for pNPPase activity was prepared, except that [32P]ATP[cP] (0.1 MBqỈlmol)1) was used instead of nonradioactive ATP The Na+ ⁄ K+-ATPase reaction was started by the addition of the enzyme, followed for or at 37 °C and stopped by the addition of 0.1 mL ice-cold 50% (w ⁄ v) trichloroacetic acid containing mm ATP and mm Pi To isolate the liberated 32Pi, 0.2 mL m H2SO4 ⁄ 40 mm ammonium molybdate solution and, next, 0.8 mL of an isobutyl alcohol ⁄ benzene solution was added to the mixture, which was mixed for 15 in a vortex mixer [56] The 32Pi isolated in the organic layer was measured with a liquid scintillation spectrophotometer Ouabain-sensitive activity was determined from the difference between the radioactivities in the presence and absence of ouabain Ion-binding assay 86 Rb+ binding and 22Na+ binding to Na+ ⁄ K+-ATPase were measured using the centrifugation method developed 681 Stimulation of pNPPase activity of Na+ ⁄ K+-ATPase by Matsui & Homareda [14–18], with slight modifications For the 86Rb+ binding, 30 lg Na+ ⁄ K+-ATPase was preincubated with or without 0.01 lmol ouabain at room temperature for 30 in a 80-lL reaction mixture composed of 0.1 lmol MgCl2, lmol Tris ⁄ Tes (pH 8.4 at 23 °C), 0–1 lmol NaCl, and lL ethanol with or without nmol oligomycin The reaction mixture was kept in ice water After 10 lL mm 86RbCl (1 MBqỈlmol)1) and then 10 lL mm pNPP had been added, the reaction mixture (100 lL) was immediately centrifuged at 368 000 g for at °C in an ultracentrifuge (Hitachi himac CS120FX; Tokyo, Japan) The supernatant was aspirated, and the inside wall of each tube was wiped carefully to remove any that remained Each pellet was dissolved in 0.1 mL m NaOH by warming at 60 °C for 20 min; then the entire solution was transferred to a counting vial, neutralized with 0.15 mL m HCl, which was used to wash the inside of the tube, and then mixed with a scintillator The radioactivity was measured using a liquid scintillation spectrophotometer Ouabain-sensitive binding was calculated from the difference between the radioactivities of the pellets in the presence and absence of ouabain For the 22Na+ binding, 30 lg Na+ ⁄ K+-ATPase was suspended in a 80-lL reaction mixture composed of lmol Tris ⁄ Tes (pH 8.4 at 23 °C), 0.03, 0.1 or 0.3 lmol 22NaCl (0.2 MBqỈlmol)1), lL ethanol with or without nmol oligomycin The incubation mixture was kept in ice-cold water After 10 lL mm RbCl and 10 lL mm pNPP ⁄ 10 mm MgCl2 had been added, the reaction mixture (100 lL) was immediately centrifuged Oligomycin-stimulated Na+ binding, which is regarded as the occluded Na+ [13], was calculated from the difference between the radioactivities of the pellets in the presence and absence of oligomycin H Homareda and M Ushimaru the difference between radioactivities of reaction mixtures with native and acid-denatured enzymes When the phosphorylation by Pi was examined, 0.1 lmol [32P]pNPP and 0.03 lmol Pi in the reaction mixture were replaced by 0.1 lmol pNPP and 0.03 lmol 32Pi, respectively The phosphorylation reaction was started by the addition of 32Pi with or without pNPP The reaction mixture (0.5 mL) for phosphorylation by ATP was composed of 50 lg Na+ ⁄ K+-ATPase, mm MgCl2, 50 mm Tris ⁄ Tes (pH 8.4 at 23 °C), 10 mm NaCl, 0.1 mm [32P]ATP[cP] (1 MBqỈlmol)1), with or without 2.5 mm pNPP The phosphorylation reaction was started by addition of the enzyme, followed for 30 s at °C and stopped by the addition of 0.1 mL ice-cold 5% (v ⁄ v) perchloric acid containing mm ATP and mm Pi The mixture was filtered through a membrane filter with a pore size of 0.45 lm The filter was washed three times with mL icecold 5% perchloric acid containing mm ATP and mm Pi, and the radioactivity on the filter was measured with a liquid scintillation spectrophotometer The amount of EP was calculated from the difference between radioactivities of reaction mixtures with native and acid-denatured enzymes Determination of protein and oligomycin concentration Protein and oligomycin concentrations were determined as described elsewhere [18,19] Acknowledgements We thank Drs R L Post, Y Fukushima and Y Tahara for critical reading and helpful suggestions, Mr S Mikkaichi for synthesis of [32P]pNPP, and Ms E Hagiwara for technical support Assay of phosphorylated intermediate To measure the phosphorylation by pNPP, 50 lg Na+ ⁄ K+-ATPase was preincubated with 0.05 lmol ouabain at room temperature for 15–30 in a 40-lL reaction mixture composed of 0.5 lmol MgCl2 and lmol Tris ⁄ Tes (pH 8.4 at 23 °C) Then, the ligands indicated in Table and lL ethanol with or without nmol oligomycin were added The reaction was started by the addition of 0.1 lmol [32P]pNPP with or without 0.03 lmol Pi (a reaction mixture of 100 lL), followed for 10 at 37 °C by the method of Inturrisi & Titus [57] and stopped by addition of mL ice-cold 5% (w ⁄ v) trichloroacetic acid The mixture was centrifuged at 14 000 g for The precipitate was washed three times with mL ice-cold 5% trichloroacetic acid containing mm pNPP and mm Pi and dissolved in 0.3 mL m NaOH by incubation at 60 °C for 10 After neutralization with HCl, the radioactivity of the precipitate was measured with a liquid scintillation spectrophotometer The amount of EP was calculated from 682 References Post RL (1979) A perspective on sodium and potassium ion transport adenosine triphosphatase In Cation Flux across Biomembranes (Mukohata Y & Packer L, eds), pp 3–19 Academic Press, New York Glynn IM & Karlish SJD (1975) The sodium pump Annu Rev Physiol 37, 13–55 Schwartz A, Lindenmayer GE & Allen JC (1975) The sodium-potassium adenosine triphosphatase: pharmacological, physiological and biochemical aspects Pharmacol Rev 27, 3–134 Cavieres JD (1977) The sodium pump in human 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intermediate J Biol Chem 266, 17026–17030 56 Martin JB & Doty DM (1949) Determination of inorganic phosphate Modification of isobutyl alcohol procedure Anal Chem 21, 965–967 57 Inturrisi CE & Titus E (1970) Ouabain-dependent incorporation of 32P from p-nitrophenyl phosphate into a microsomal phosphatase Mol Pharmacol 6, 99–107 FEBS Journal 272 (2005) 673–684 ª 2005 FEBS ... 675 Stimulation of pNPPase activity of Na+ ⁄ K+-ATPase H Homareda and M Ushimaru Table Phosphorylation of Na+ ⁄ K+-ATPase from pNPP, Pi and ATP For phosphorylation by pNPP or Pi, Na+ ⁄ K+-ATPase. .. Stimulation of pNPPase activity of Na+ ⁄ K+-ATPase Fig Na+- dependent activation curves for Na+ ⁄ K+-ATPase and pNPPase in the presence of ATP with or without KCl Ouabain-sensitive activities of ATPase... that oligomycin occludes Na+ within the Na+ ⁄ K+-ATPase molecule, so that this antibiotic inhibits Na+ transport and Na+ ⁄ K+-ATPase activity but not K+-dependent pNPPase activity [12–19] Therefore,