Portal de Periódicos da CAPES

Influence of Intramembrane Electric Charge on Na,K-ATPase

1995; Elsevier BV; Volume: 270; Issue: 9 Linguagem: Inglês

10.1074/jbc.270.9.4244

1083-351X

Irena Kłodos, Natalya U. Fedosova, Liselotte Plesner,

Photoreceptor and optogenetics research

Effects of lipophilic ions, tetraphenylphosphonium (TPP+) and tetraphenylboron (TPB−), on interactions of Na+ and K+ with Na,K-ATPase were studied with membrane-bound enzyme from bovine brain, pig kidney, and shark rectal gland.Na+ and K+ interactions with the inward-facing binding sites, monitored by eosin fluorescence and phosphorylation, were not influenced by lipophilic ions.Phosphoenzyme interactions with extracellular cations were evaluated through K+-, ADP-, and Na+-dependent dephosphorylation. TPP+ decreased: 1) the rate of transition of ADP-insensitive to ADP-sensitive phosphoenzyme, 2) the K+ affinity and the rate coefficient for dephosphorylation of the K-sensitive phosphoenzyme, 3) the Na+ affinity and the rate coefficient for Na+-dependent dephosphorylation. Pre-steady state phosphorylation experiments indicate that the subsequent occlusion of extracellular cations was prevented by TPP+. TPB− had opposite effects.Effects of lipophilic ions on the transition between phosphoenzymes were significantly diminished when Na+ was replaced by N-methyl-D-glucamine or Tris+, but were unaffected by the replacement of Cl− by other anions.Lipophilic ions affected Na-ATPase, Na,K-ATPase, and p-nitrophenylphosphatase activities in accordance with their effects on the partial reactions.Effects of lipophilic ions appear to be due to their charge indicating that Na+ and K+ access to their extracellular binding sites is modified by the intramembrane electric field. Effects of lipophilic ions, tetraphenylphosphonium (TPP+) and tetraphenylboron (TPB−), on interactions of Na+ and K+ with Na,K-ATPase were studied with membrane-bound enzyme from bovine brain, pig kidney, and shark rectal gland. Na+ and K+ interactions with the inward-facing binding sites, monitored by eosin fluorescence and phosphorylation, were not influenced by lipophilic ions. Phosphoenzyme interactions with extracellular cations were evaluated through K+-, ADP-, and Na+-dependent dephosphorylation. TPP+ decreased: 1) the rate of transition of ADP-insensitive to ADP-sensitive phosphoenzyme, 2) the K+ affinity and the rate coefficient for dephosphorylation of the K-sensitive phosphoenzyme, 3) the Na+ affinity and the rate coefficient for Na+-dependent dephosphorylation. Pre-steady state phosphorylation experiments indicate that the subsequent occlusion of extracellular cations was prevented by TPP+. TPB− had opposite effects. Effects of lipophilic ions on the transition between phosphoenzymes were significantly diminished when Na+ was replaced by N-methyl-D-glucamine or Tris+, but were unaffected by the replacement of Cl− by other anions. Lipophilic ions affected Na-ATPase, Na,K-ATPase, and p-nitrophenylphosphatase activities in accordance with their effects on the partial reactions. Effects of lipophilic ions appear to be due to their charge indicating that Na+ and K+ access to their extracellular binding sites is modified by the intramembrane electric field. INTRODUCTIONLipophilic ions like tetraphenylphosphonium (TPP+) 1The abbreviations used are: TPP+tetraphenylphosphoniumTPB−tetraphenylboronNa,K-ATPase(Na++ K+)-stimulated adenosine triphosphataseK-pNPPaseK+-stimulated p-nitrophenylphosphataseE1Na,K-ATPase form with high affinity toward ATP and Na+E2Na,K-ATPase form with high affinity toward K+ and low affinity to ATPEPphosphoenzymeE1P(Nam)phosphoenzyme with m occluded Na+ ions, resistant to K+ and sensitive to ADPEPADP-sensADP-sensitive phosphoenzymeEPADP-insensphosphoenzyme insensitive to ADPE2Pphosphoenzyme sensitive to K+EPK-sensphosphoenzyme sensitive to K+EPK-insensK-insensitive phosphoenzymeE2P(Kn)phosphoenzyme with n occluded K+ ionsNMGN-methyl-D-glucamineMES4-morpholineethanesulfonic acid. and tetraphenylboron (TPB−) partition into cell membranes (Andersen et al., 1978; Flewelling and Hubbell, 1986a, 1986b). The ions are located a few angstroms below the membrane surface (Andersen et al., 1978), and they modify the electrostatic potential profile inside the membrane dielectric (Andersen et al., 1978; Flewelling and Hubbell, 1986a; 1986b). It has been shown by Bühler et al.(1991), Läuger (1991a), and Stürmer et al.(1991) that these ions affect the reactivity of membrane-bound Na,K-ATPase toward Na+ and K+. The authors concluded from experiments with fluorescent, potential-sensitive aminostyrylpyridinium dyes that TPP+ and TPB− affect the release of Na+ from and the binding of K+ to external sites of Na,K-ATPase. The authors therefore suggested that the external cation binding sites are formed as deep wells, i.e. K+ binds to and Na+ is released from “sites that are located inside the membrane dielectric” (Läuger, 1991a). Since the binding of Na+ at the cytoplasmic side of Na,K-ATPase caused relatively small changes in the fluorescence response, the authors concluded that the cytoplasmic sites are located close to the lipid/water interface (Läuger, 1991a).In the present work we applied biochemical techniques supported by measurements of conformational changes by eosin fluorescence to describe which partial reactions in the Na,K-ATPase reaction cycle are affected by lipophilic ions. The studies were performed with broken membrane Na,K-ATPase preparations; thus, the lipophilic ions had access to both sides of the membrane and their distribution in the membrane was not modified by a trans-membrane potential. To ease the understanding of the experimental design, the following reaction scheme is used as a frame of reference: E1P(Nam)⇀↽mNa0E2P⇀↽nKoE2PKnATP↑↓ADP Pi↑↓E1Nam⇀↽mNaiE1⇀↽nKiE1Kn⇀↽E2(Kn) SCHEME 1In this very simplified scheme, E1 denotes the form of Na,K-ATPase characterized by a high affinity to ATP and Na+, while E2 denotes the form with a high affinity to K+ but a relatively low affinity to ATP and Na+ (Glynn, 1985; Läuger, 1991b). The lower row of the scheme shows reaction of Na+ and K+ with the cytoplasmic sites, while the upper row illustrates reaction of cations with the extracellular sites. The phosphoenzyme E1P(Nam) has m occluded Na+ ions and reacts with ADP resynthesizing ATP. E2P binds K+ with a very high affinity, dephosphorylates, and forms an intermediate with n occluded K-ions, E2(Kn).The effect of lipophilic ions on the following partial reactions were studied: transitions between E2(Kn) and E1Nam were characterized using eosin fluorescence as an indicator of conformation (Skou and Esmann, 1983); the steps leading to formation of phosphoenzymes were studied in experiments where the pre-steady state time course of E32P formation was measured, and the individual phosphoenzyme forms were characterized in chase experiments with ADP or K+. The entire reaction cycle was characterized through measurements of the steady state (1Andersen O.S. Feldberg S. Nakadomari H. Levy S. McLaughlin S. Biophys. J. 1978; 21: 35-70Abstract Full Text PDF PubMed Scopus (124) Google Scholar) phosphorylation level, (2Apell H.-J. J. Membr. Biol. 1989; 110: 103-114Crossref PubMed Scopus (63) Google Scholar) distribution of ADP- and K-sensitive phosphointermediates, (3Beauge L. Berberian G. Biochim. Biophys. Acta. 1983; 727: 336-350Crossref PubMed Scopus (25) Google Scholar) ATP and pNPP hydrolysis activity at varying concentrations of Na+ or K+ or both. The effects of lipophilic ions on the overall reaction are compared to their effects on the partial reactions. Preliminary results have been published (Klodos & Plesner, 1992).MATERIALS AND METHODSEnzyme PreparationNa,K-ATPase from bovine brain was prepared according to Klodos et al.(1975). Na,K-ATPase from pig kidney was prepared according to J⊘rgensen(1974) as modified by Jensen et al.(1984). Na,K-ATPase from shark rectal gland was prepared as described by Skou and Esmann(1979).The Na,K-ATPase activity of bovine brain, pig kidney, and shark rectal gland enzymes, measured at 37°C and under standard conditions (Ottolenghi, 1975, but without bovine serum albumin) were 4-5, 27, and 25 units•(mg of protein)-1, respectively. The maximum phosphorylation site concentrations, measured as described (Klodos et al., 1981), were 0.45-0.55 nmol•(mg of protein)-1 for the brain enzyme, 2.7 nmol•(mg of protein)-1 for the pig kidney enzyme, and 2.5 nmol•(mg of protein)-1 for shark rectal gland Na,K-ATPase.The protein amount was determined according to Lowry et al. (1951), as described by Jensen and Ottolenghi (1983a), using bovine serum albumin as standard.Measurement of Eosin FluorescenceThe experiments were performed in a SPEX Fluorolog-2 spectrofluorometer at 20°C, the excitation wavelength was 530 nm, the slit was 0.1 nm, and the light path was 1 cm. A Schott RG550 filter was used as emission cut-off filter.The fluorescence response of 0.5 μM eosin to binding of K+ and subsequently to addition of Na+ was measured in 10 mM HEPES, 10 mM MES, 10 mM EDTA buffer, pH 7.5, which favors a form of Na,K-ATPase with high affinity to eosin. The pH was adjusted with N-methyl-D-glucamine. The fluorescence of unspecifically bound eosin was measured in the presence of 375 μM ADP, which prevents specific binding of eosin (Esmann, 1992).TPPCl and NaTPB dissolved in dimethyl sulfoxide were added to final concentrations of 300 and 50 μM, respectively (dimethyl sulfoxidefinal = 0.05%). The pig kidney Na,K-ATPase (30 μg of protein•ml-1) in 2 ml of buffer solution was thermostated and continually stirred. All additions were from hand-held Hamilton syringes. For further analysis, data were expressed in percent of the starting level of fluorescence in each individual experiment.Phosphorylation-Dephosphorylation ExperimentsThe experiments were performed at 0°C and pH 7.4 with 300 μg of protein•ml-1. TPP+ and TPB−, when present, were added from freshly prepared solutions in water at least 10 min prior to the start of the phosphorylation. The phosphorylation medium also contained 0.1 mM MgCl2, 30 mM imidazole buffer, pH 7.4, at 0°C and varying [NaCl]. The phosphorylation was started by the addition of 25 μM [γ-32P]ATP. After varying periods of time, the reaction was either stopped by addition of 10% trichloroacetic acid (final concentration 5%) or dephosphorylation was initiated by the addition of a chase. The chase contained NaCl, buffer, and MgCl2 in the same concentrations as the phosphorylation medium and, in addition, either unlabeled ATP (final concentration 1 mM), unlabeled ATP and KCl (final concentrations were 1 mM and usually 20 mM respectively), or unlabeled ATP and ADP (final concentrations 1 and 2.5 mM). The dephosphorylation was stopped by addition of 10% trichloroacetic acid (final concentration 5%) at the times indicated. The amount of acid-stabile E32P was determined according to Klodos et al.(1981), and the results are presented in the figures after subtraction of EP levels, obtained after dephosphorylation for 5 min. The values in the figures are the mean of at least three experiments ± S.E.Measurement of ATPase ActivityThe [γ-32P]ATP hydrolysis was measured in 30 mM histidine buffer, pH 7.4, at 37°C in the presence of 3 mM ATP, 3 mM MgCl2, and [NaCl] and [KCl] given in the figures. 32Pi was determined according to Lindberg and Ernster(1956).Measurement of pNPP HydrolysisThe pNPPase activity was measured according to Ottolenghi(1975), but without bovine serum albumin in the incubation medium. The buffer was 30 mM imidazole buffer, pH 7.5, at 37°C. The reaction medium contained 20 mM MgCl2 and 10 mM pNPP. The pNPPase activity was measured as function of [KCl] at a constant salt concentration of the medium, 150 mM, replacing KCl with choline chloride, NMGCl, or NaCl. Identical results were obtained with choline chloride and NMGCl. The experiments in the presence of both KCl and NaCl were performed with or without 100 μM ATP. The amount of the released p-nitrophenol was determined from the optical density at 410 nm.Data ProcessingThe data were analyzed using the computer program “Plot 5.31” written by Bliss Forbush III, Dept. of Cellular and Molecular Physiology, School of Medicine, Yale University, applying a linear or nonlinear least squares analysis.ReagentsATP and ADP were purchased as sodium salts from Boehringer Mannheim, Germany. ATP used in the phosphorylation experiments was converted to its Tris salt by chromatography on a Dowex 1 column (from Sigma), and [γ-32P]ATP was purified on DEAE-Sephadex G-25 (N⊘rby and Jensen, 1971). ADP was purified by chromatography on a Dowex 50W H+ column, and the eluate was adjusted to pH 7.1 at 20°C with 2-amino-2-methyl-1,3-propanediol. p-Nitrophenyl phosphate (pNPP) purchased as sodium salt from Merck, Darmstadt, Germany, was purified by chromatography on Dowex 50W H+. The eluate was adjusted with Tris to pH 7.4 at 37°C. N-Methyl-D-glucamine (NMG) was purchased from Sigma. Eosin was obtained from Gurt, Chadwell Heath, Essex, UK. TPPCl and NaTPB were gifts from Dr. H.-J. Apell, Universität Konstanz, or purchased from Sigma. TPPCl and NaTPB were dissolved in dimethyl sulfoxide or in water. The water solutions of TPPCl and NaTPB were prepared on the day of the experiment. All other reagents were reagent grade.RESULTSThe description of the results follows the reaction : binding of Na+ and K+ to the dephosphoenzyme, phosphorylation, characterization of phosphointermediates, Na,K-ATPase and pNPPase activities, and, finally, a series of experiments aiming at clarification whether the effects of lipophilic ions are due to their charge. It should be noted that: 1) because of its higher partitioning into the lipid phase (Flewelling and Hubbell, 1986a, 1986b) TPB− was used in lower concentrations than that of TPP+, and 2) whenever K+ was present only the effect of TPP+ was tested, as TPB− is a strong chelator of K+ (Flaschka and Barnard, 1960).Interconversions between Dephosphoenzyme Forms: E1Nam↔ E2(Kn) TransitionConformational transitions of the Na,K-ATPase following cation binding to nonphosphorylated enzyme forms were monitored by eosin fluorescence according to Skou and Esmann(1983), who showed that the fluorescence of eosin specifically bound to the enzyme is higher than the fluorescence of eosin in the solution or of nonspecifically bound eosin. Specifically bound eosin was released from its high affinity binding site by the addition of ADP (Esmann, 1992). 375 μM ADP used in the reported experiments was more than sufficient to prevent specific eosin binding. The fluorescence level observed in the presence of ADP was equal to that measured in the presence of saturating K+, where E2(Kn), which did not bind eosin specifically, was the only form present. Neither TPP+ nor TPB− had any effect on the affinity of specific eosin binding measured as equilibrium eosin binding (not shown). Both ions affected eosin fluorescence: TPP+ caused an increase in the fluorescence of nonspecifically bound eosin, i.e. it increased the background fluorescence, while TPB− increased the quantum yield of the fluorescence only of specifically bound eosin (Fedosova and Jensen, 1994).In our experiments, specific eosin binding was induced by buffer (10 mM HEPES + 10 mM MES + 10 mM EDTA, pH 7.5 at 20°C). The subsequent addition of 4 mM Na+ did not result in an increase in fluorescence, indicating that already in the absence of Na+ the specific eosin binding was maximal. The difference between the level of fluorescence in the presence of buffer, Na+, or both, and the level in the presence of ADP was equal to the maximal fluorescence increase caused by the eosin binding to the enzyme (ΔFmax). ΔFmax corresponded therefore to the complete transition of the enzyme from eosin-bound E1 to the eosin-free E2 form.In the absence of Na+, the addition of K+ induced a decay of specific fluorescence which could be fitted adequately with a monoexponential function (Fig. 1, inset). The rate coefficient of the decay (kobs) and the equilibrium fluorescence decrease (ΔF/F0) were derived. ΔF/F0 versus [K+] was found to be a hyperbola: ΔF/F0 = (ΔFmax/F0)/(1 + K0.5,K/[K+]), where K0.5,K = 5.2 ± 0.9 μM (not shown). Similar values were previously found in different types of experiments (cf. Esmann, 1992). The rate coefficient of fluorescence decrease, kobs, in the same range of potassium concentrations, showed a linear dependence on [K+] (Fig. 1A) indicating a low affinity K+ binding.Thus, we observed the same contradiction between affinities estimated from equilibrium and transient experiments as described previously by Karlish et al.(1978) and Glynn and Karlish(1982), who proposed the following scheme to explain this discrepancy: E1+K+⇀↽KD,KE1K⇀↽k-1k+1E2K SCHEME 2They assumed that a low affinity K+ binding to the E1 form is followed by a conformational transition, poised heavily in favor of the E2 form (cf. reviews by Glynn(1985) and Glynn and Karlish (1990)). The rate coefficient of disappearance of the E1 form upon K+ addition is equal to kobs = k-1+ k+1/(1 + KD,K/[K+]) and becomes kobs = k-1+ k+1•[K+]/KD,K for [K+] ≪ KD,K. Glynn et al. (1987) showed that the time course of deocclusion and the conformational change are closely correlated. Thus, k-1 in the model is the rate constant for K+ deocclusion from the intracellular sites.In our experiments, the rate coefficient of the specific eosin fluorescence decrease in response to K+, kobs, displayed a linear dependence on [K+] (Fig. 1A). The rate constant k-1 for the E2→ E1 transition was estimated to be 0.03 ± 0.012 s-1 by extrapolation of the straight line to 0 [K+]. It is in agreement with the data on deocclusion and conformational transition rate constants measured by different techniques (Glynn, 1985; Glynn et al., 1987).Since KD,K = K0.5,K•(k+1/k-1), and as k+1 is about 300 s-1 (Steinberg and Karlish, 1989) and k-1 = 0.03 s-1, an approximate value of the dissociation constant for K+, KD,K, was estimated to be in the millimolar range.When 4 mM Na+ was present, i.e. the starting point was E1Nam, K+ addition was also followed by a monoexponential decay. The rate constant k-1 for the K+ deocclusion was unchanged, but the slope of kobs versus [K+] was decreased by a factor of 80 (Fig. 1B) and the K0.5,K value obtained from equilibrium measurements was 421 μM (Fig. 2). The simplest reaction scheme compatible with these results is E1Na⇀↽KD,NaE1⇀↽KD,KE1K⇀↽k-1k+1E2K SCHEME 3Figure 2:Equilibrium fluorescence change as function of K+ in the presence of 4 mM NaCl. Eosin fluorescence was measured in 4 mM NaCl, 10 mM HEPES, 10 mM MES, 10 mM EDTA, pH 7.5, and different concentrations of K+ in the absence (circles) or in the presence of 300 μM TPP+ (squares). Eosin fluorescence upon addition of K+ is expressed as percentage of the starting level of fluorescence. The values are the mean of three experiments ± S.E. The curve is a fit of the experimental data to the equation: ΔF/F0 = (ΔFmax/F0)/(1 + K0.5,K/[K+]), as described under “Materials and Methods.” The K0.5,K = 421 ± 0.9 μM.View Large Image Figure ViewerDownload Hi-res image Download (PPT)In this model, Na+ and K+ compete for the binding to the E1 form, but neither the K+ occlusion, defined as E1K to E2K transition, nor deocclusion are affected by Na+. TPP+ had no effect on the K+ binding in the presence of Na+ (Fig. 1B and Fig. 2).The experiments on Na+ binding in the absence of K+ were performed at low buffer concentration (1 mM HEPES + 1 mM MES) where no specific eosin binding was observed. It has been previously shown that in low ionic strength medium the enzyme has the characteristics of an E2 conformation (Skou and Esmann, 1980; Glynn and Richards, 1982; Jensen and Ottolenghi, 1983b; Klodos and Ottolenghi, 1985). Na+ addition to the medium was followed by a fluorescence increase which reflected the formation of the sodium-bound form of the enzyme. Neither TPP+ nor TPB− affected the Na+ dependence of the specific fluorescence increase (not shown). Thus, TPP+ affected neither Na+ nor K+ binding nor K+ deocclusion.Formation of PhosphoenzymeThe amount of EP was measured after a 2-s phosphorylation period at NaCl concentrations varying from 0 to 100 mM. We reported previously a marginal effect of the lipophilic ions on the formation of phosphoenzyme under these conditions (Fig. 2 in Klodos and Plesner(1992)).When the ionic strength was kept constant with NMGCl or TrisCl, no effect of lipophilic ions on the formation of EP was observed (Fig. 3). The same result was obtained when ionic strength of the medium was increased by addition of 100 mM NMGCl or 100 mM TrisCl (not shown). Tris+ decreased the apparent affinity to Na+, and the Na+ dependence curve became S-shaped. The half-saturating [NaCl] in the presence of TrisCl was 2.7 ± 0.12 mM (n = 6), and only 0.33 ± 0.02 mM (n = 6) with NMGCl in the medium. With 100 mM NMGCl, in the absence of added NaCl, 4-12% of the enzyme was phosphorylated (not shown), but it is not clear whether this phosphorylation was caused by NMG+ itself or by traces of Na+.Figure 3:Effect of TPP+ and TPB− on the formation of phosphoenzyme. Bovine brain Na,K-ATPase was incubated for 10 min at 20°C in 30 mM imidazole buffer (pH 7.4 at 0°C), 0.1 mM MgCl2, and varying [NaCl] and [NMGCl] (left panel) or [NaCl] and [TrisCl] (right panel), in the absence of lipophilic ions (circles) and in the presence of 300 μM TPP+ (squares) or 33 μM TPB− (triangles). The sum of [NaCl] and [NMGCl] or [TrisCl] was 100 mM. The samples were subsequently cooled to 0°C. 25 μM [γ-32P]ATP (final) was added, while concentrations of other components remained unchanged. The enzyme was phosphorylated at 0°C. After 2 s, the phosphorylation was stopped and the amount of phosphoenzyme was measured as described under “Materials and Methods.” The figure shows the amount of phosphoenzyme formed at 0-10 mM NaCl.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Na-dependent DephosphorylationNa+-dependent dephosphorylation of the phosphoenzyme was examined in the absence of ADP or KCl. The experiments were performed at 10-300 mM NaCl, and the chase contained 1 mM unlabeled ATP. The Na+ dependence of the dephosphorylation rate coefficients, obtained by a monoexponential fit of the dephosphorylation data, is shown in Fig. 4.Figure 4:Effect of lipophilic ions on Na-dependent dephosphorylation. Dependence of dephosphorylation rate constant on [NaCl]. Bovine brain Na,K-ATPase was phosphorylated for 60 s at 0°C as described under “Materials and Methods” at varying [NaCl] in the absence of lipophilic ions (circles), with 300 μM TPP+ (squares) or 33 μM TPB− (triangles). At zero time, a chase containing unlabeled ATP (1 mM final) was added. The dephosphorylation was stopped and the first order rate constants were estimated as described under “Materials and Methods.”View Large Image Figure ViewerDownload Hi-res image Download (PPT)In the absence of lipophilic ions, the dephosphorylation was stimulated by NaCl in the concentration range from 10 to 150 mM, similar to previous data by Hara and Nakao(1981) and N⊘rby et al.(1983). The stimulation appears to be due to a stimulation of dephosphorylation of the K-sensitive EP. It has been shown previously that the K-insensitive EP dephosphorylates directly (N⊘rby at al., 1983), and its transition to the K-sensitive EP is inhibited by NaCl (Klodos et al., 1994). However, because of a low relative amount of K+-insensitive EP (less than 12% of the total EP, Fig. 9), neither of the two processes could significantly affect the dephosphorylation of this phosphoform. Thus, NaCl stimulation of the Na+-dependent dephosphorylation was due to Na+ acting as a replacement, albeit poor, for K+ in activating the dephosphorylation of K-sensitive EP. As shown in Fig. 4, the rate coefficient of Na+-dependent dephosphorylation was increased by 33 μM TPB− at [NaCl] lower than 150 mM, whereas it was decreased by 300 μM TPP+ at all NaCl concentrations.Figure 9:TPP+ effect on K+-stimulated dephosphorylation of bovine brain Na,K-ATPase. The experiments were performed as in Fig. 8 with bovine brain Na,K-ATPase at varying [NaCl] in the absence (circles) or in the presence of 300 μM TPP+ (squares). [KCl] in the chase was 20 mM (final). A biexponential function EPt= EPO,K-sens•exp(-kfast•t) + EPO,K-insens•exp(-kslow•t) was fitted to the data like those in Fig. 8 as described under “Materials and Methods.” The relative amount of K-insensitive phosphoenzyme, EPO,K-insens, the rate coefficient of the decay of EPK-insens, kslow, and the rate coefficient of the decay of EPK-sens, kfast, are shown as function of [NaCl] in panels A, B, and C, respectively.View Large Image Figure ViewerDownload Hi-res image Download (PPT)ADP-dependent DephosphorylationNeither TPP+ nor TPB− in concentrations up to 600 μM and 100 μM, respectively, influenced the steady state EP level at [NaCl] ≥ 10 mM (not shown). In a series of experiments, EP was formed in the presence of 10 to 500 mM NaCl with or without lipophilic ions for 60 s and subsequently chased with 2.5 mM ADP.The addition of ADP in the chase produced rapid decay of the ADP-sensitive EP and exposed the slowly decaying ADP-insensitive EP (Fig. 5A). The biphasic dephosphorylation was analyzed in the following scheme ↽kb1EPADPscna⇀↽kb2kf2EPADP-inscna⇀kf3 SCHEMA 4Figure 5:Effect of lipophilic ions on ADP-dependent dephosphorylation. The experiments were performed with bovine brain Na,K-ATPase as in Fig. 4 at varying [NaCl] in the absence of lipophilic ions (circles) and in the presence of 300 μM TPP+ (squares) or 33 μM TPB− (triangles). At zero time, a chase solution containing unlabeled ATP (1 mM final) and ADP (2.5 mM final) was added. Dephosphorylation was stopped at the times shown. Panel A, dephosphorylation time course at 100 mM NaCl. A biexponential function EPt = EPO,ADP-sens•exp(-kfast•t) + EPO,ADP-insens•exp(-kslow•t) was fitted to the data like those in panel A as described under “Materials and Methods.” EPO,ADP-sens, the relative amount of ADP-sensitive phosphoenzyme, and kslow, the rate coefficient of decay in the slow phase, are shown as functions of [NaCl] in panels B and C, respectively.View Large Image Figure ViewerDownload Hi-res image Download (PPT)where EPADP-sens is a rapidly and EPADP-insens is a slowly decaying phosphoenzyme form(s). EPADP-sens and EPADP-insens are not synonymous with the classical ADP-sensitive, K-insensitive E1P or the ADP-insensitive, K-sensitive E2P, but signify operational quantities of phosphoforms characterized by their, rapid or slow, decay. kb1 is the rate coefficient of ADP-dependent decay, kf2 and kb2 are the rate constants of forward and backward transitions between the ADP-sensitive and the ADP-insensitive phosphointermediates, and kf3 is the rate coefficient of dephosphorylation of the ADP-insensitive EP. The forward transition is accompanied by dissociation of at least 1 Na+ (Yoda and Yoda, 1987; Glynn, 1988; J⊘rgensen, 1991, 1994; Goldshleger et al., 1994). Both the backward transition, accompanied by the binding of NaCl (Post et al., 1975; N⊘rby et al., 1983; N⊘rby and Klodos, 1988), and the dephosphorylation of the ADP-insensitive phosphointermediate, reflected by the rate coefficient of the Na+-dependent dephosphorylation (Fig. 4) (Klodos et al.(1981); cf. N⊘rby and Klodos (1988)), are dependent on [NaCl]. The rate coefficient of the decay in the slow phase is equal to the sum of kb2 and kf3.In the absence of lipophilic ions, an increase in [NaCl] resulted in: 1) an increase in the steady state amount of ADP-sensitive EP (Fig. 5, A and B), and 2) an increase in the rate of the slow decay (Fig. 5C). Both observations are in agreement with previously published data (Hara and Nakao, 1981; N⊘rby et al., 1983; Klodos and N⊘rby, 1987).TPP+ caused a large decrease in both the steady state amount of ADP-sensitive EP and in the rate of the slow phase at all NaCl concentrations (Fig. 5). The opposite was seen with TPB−, which caused a somewhat smaller, but significant, increase in both the steady state amount of EPADP-sens and a significant increase in the slope of the slow phase (Fig. 5). In other words, the effect of TPP+ was similar to that of a decrease in the concentration of NaCl and the effect of TPB− to an increase in [NaCl].Similar effects of lipophilic ions were observed in experiments with Na,K-ATPase from pig kidney (Fig. 6) and shark rectal gland (not shown). Although the steady state proportion of ADP-sensitive EP measured, in the presence of 100 mM NaCl in the medium, was lower with these enzymes than with brain Na, K-ATPase (compare Figure 5:, Figure 6: at 100 mM NaCl, cf. also Klodos & N⊘rby(1987) and Klodos et al.(1994)), qualitative effects of lipophilic ions on both the steady state level of ADP-sensitive EP and the slope of the slow phase were the same as with the brain Na,K-ATPase.Figure 6:ADP-stimulated dephosphorylation of pig kidney Na,K-ATPase: effect of lipophilic ions. The experiment was the same as in Fig. 5A except the source of enzyme. circles, NaCl alone; squares, +300 μM TPP+; open triangles, +33 μM TPB-; or filled triangles, 100 μM TPB-.View Large