THE JOURNAL OF BIOLOGICAL CHEMISTRYVol. 257, No. 18, Issue of September 25, pp. 10659-10667, 1982Printed in U.S.A.

Conformational Change Accompanying Transition of ADP-sensitivePhosphoenzyme to Potassium-sensitive Phosphoenzyme of (Na*,K*)-ATPase Modified with N-[p-(2-Benzimidazolyl)phenyl]maleimide*

(Received for publication, April 14, 19821

Kazuya Taniguchit, Kuniaki Suzuki, and Shoichi Iida

From the Department of Pharmacology, School of Dentistry, Hokkaido University, Sapporo, 060, Japan

The addition of Mg2+ or ATP to (Na+,K+)-ATPase (EC3.6.1.3) of pig kidney modified with a sulfhydryl flu-orescent reagent N-[p-(2-benzimidazolyl)phenyl]mal-eimide simply reduced fluorescence in the presence ofNa+; however, the addition of both ligands to the en-zyme induced a reversible dynamic change. The direc-tion of the change was dependent on the concentrationof Na+ present. These dynamic changes in fluorescenceintensity both in the presence of low and high concen-trations of Na+ can be repeated by the re-addition ofATP but not by ADP. Addition of ouabain under theformer condition stabilized the fluorescence at thehighest level, but the addition of ouabain under thelatter condition increased the fluorescence from thelowest to the highest level. The phosphoenzyme formedunder the former condition was sensitive to K+ andinsensitive to ADP while the phosphoenzyme formedunder the latter condition was sensitive to ADP andinsensitive to K+ . The data indicate that the positiveand negative fluorescence changes were induced by theformation of K+-sensitive phosphoenzyme and ADP-sensitive phosphoenzyme, respectively. N-Ethylmal-eimide treatment partially inhibited the positivechange without affecting the negative change. Thesedata also indicate that the transition of ADP-sensitivephosphoenzyme to K+-sensitive phosphoenzyme ac-companied the largest fluorescence intensity changewhich was examined during the hydrolysis of ATP. Thedata obtained from the tryptophan fluorescence of boththe native and the modified enzyme suggest that themicro-environments of the tryptophan and the sulfhy-dryl residues are similar in the state of K+-sensitivephosphoenzyme but different in the state of ADP-sen-sitive phosphoenzyme.

It is proposed that the hydrolysis of ATP by (Na+,K+)-ATPase occurs via various phosphoenzymes (1-8). The differ-ent reactivities of these phosphoenzymes to physiologicalligands suggest the presence of different conformational statesof phosphoenzymes (1-8). Much evidence that ligands induceconformational changes has accumulated (1-20). To under-stand better the mechanism of energy coupling between hy-drolysis of ATP and (Na+,K+ ) transport, it is important tofind the conformational differences of various reaction inter-mediates. In particular, an understanding of the difference

* This work was supported by grants from the Ministry of Educa-tion, Science, and Culture of Japan The costs of publication of thisarticle were defrayed in part by the payment of page charges. Thisarticle must therefore be hereby marked "advertisement" in accord-ance with 18 U.S.C. Section 1734 solely to indicate this fact.

: To whom all correspondence should be addressed.

between E1P' and E2 P is important because ATP has beensynthesized from E2P formed from Pi (21). In that case, thereactivity of ADP to the phosphoenzyme appeared only afterhigh concentrations of Na+ were added. The above experimentsuggested that the binding of Na+ to the low affinity sites ofE2P energized the phosphoenzyme to form E1P (21). Thus,the change in the reactivity should be the difference of theconformational states of these phosphoenzymes. However,there is little direct evidence to show the conformational stateof EIP or the difference between E1P and E2P. Recently,decrease in the tryptophan fluorescence with increase in theNa+ concentration accompanying phosphorylation from ATPhas been reported to be the conformational difference betweenE2P and EIP (22). However, the concentrations of Na+ useddo not seem to be sufficient to shift the equilibrium betweenEIP and E2P to the former (21, 23-25). In the previous report(18), we showed the ATP-dependent reversible conforma-tional change of BIPM-modified (Na+,K+)-ATPase and sug-gested that the transient rise in the fluorescence reflects theformation of K+-sensitive phosphoenzymes. In the presentpaper, we show the ATP-dependent reversible fluorescenceintensity changes of both the BIPM and tryptophan residues(13, 15, 18), measure the amount of phosphoenzymes bychanging the concentration of Na+ , and examine the sensitiv-ity of these phosphoenzymes to ADP and K+ . The data showthat the transition from E1P to E2P accompanies the largestfluorescence intensity change of BIPM residues among otherpartial reactions examined.

MATERIALS AND METHODS

(Na+,K+)-ATPase was purified from pig kidney by the method ofHayashi et al. (26) in which sodium dodecyl sulfate treatment wasomitted. The preparation was further treated with NaI by the methodof Nakao et al. (27). (Na+,K+)-ATPase preparations purified by themethod of J0rgensen (28) modified by Hayashi and Post (29) werealso used. The specific activity of these enzyme preparations wasabout 600-2000 Amol/mg of protein/h. The treatment of BIPM wasstarted by the addition of BIPM containing 5% acetone as describedpreviously (18) except that the concentration of BIPM during thetreatment was reduced to 0.2 instead of 1 mM unless otherwise stated.After 45 min, 40 ml containing 80 ,tmol of,8-mercaptoethanol, 1 mmolof imidazole-HCI (pH 7.4), 1 mmol of sucrose, and 4 flmol of EDTA-Tris were added to stop the modification. The sample was centrifugedat 20,000 rpm for 45 min to pack the enzyme. The precipitates werethen suspended in 40 ml of 25 mM imidazole-HCl (pH 7.4), 25 mMsucrose, 0.1 mM EDTA containing 10 mg/ml of bovine serum albumin,which was added to remove BIPM, and the suspension was centri-fuged again as above. The resulting precipitates were suspended asabove except that bovine serum albumin was not included in the

'The abbreviations used are: EIP, ADP-sensitive phosphoenzyme;E2 P, K+-sensitive phosphoenzyme; BIPM, N-[p-(2-benzimidazolyl)-phenyl]maleimide; IH, imidazole-HCI; NaE,, Na+-bound enzyme;KE2, K+-bound enzyme.

10659

by guest on Novem

ber 17, 2020http://w

ww

.jbc.org/D

ownloaded from

Conformational Difference between E1 P and E2 P in (Na+,K+)-ATPase

solution and the suspension was centrifuged again. The precipitateswere suspended in 0.25 M sucrose, 1 mM EDTA-Tris (pH 7.4) to givea protein concentration of 4 to 5 mg/ml, divided into small aliquotsand stored at -75 C until use (18).

The fluorescence measurements were performed at 25 C with asample of 3.2 ml of 25 mM inidazole-HCI (pH 7.4), 25 mM sucrose, 0.1mM EDTA-Tris, and about 10 to 30 /g of ATPase protein/ml on ahomemade mixing device with a temperature-controlled circulatingbath (18). The fluorescence was excited at 308 nm and the emittedlight was detected at 365 nm, both at slit widths of 7 nm unlessotherwise stated.

(Na+,K+)-ATPase activity was measured by a coupled assay usingpyruvate kinase and lactate dehydrogenase (30). K+-dependent p-nitrophenylphosphatase activity was measured as described previ-ously (31). The amount of phosphoenzyme was estimated and meas-ured by the method of Post and Sen (32) unless otherwise stated.

Protein concentration was estimated by the method of Bradford(33) with a Bio-Rad protein assay kit using bovine plasma albumin asa standard.

The amount of BIPM which became bound to the protein wasestimated using the molar adsorption coefficient 28,000 at 313 nm(34). The number of sulfhydryl groups in the enzyme was estimatedwith 5,5'-dithiobis(2-nitrobenzoic acid) (35) in a Hitachi 557 dualbeam spectrophotometer with double beam mode at 25 C. Bothsample and reference cells contained 0.5 mi of 3% sdium dodecylsulfate containing 25 mM IH, 2 mM EDTA-Tris (pH 7.3). A solutionof 0.25 M sucrose containing 1 mM EDTA-Tris (0.125 Ml) with orwithout 70 to 100 pg of enzyme protein was added to the sample andthe reference cells, respectively. After 15 min, the reaction was startedby adding 0.25 ml of 10 mM 5,5'-dithiobis(2-nitrobenzoic acid) con-taining 25 mM imidazole-HC1, 2 mM EDTA-Tris (pH 7.3) to bothcells. Recorded at 412 nm, the difference of absorbance between thesample and the reference cells increased with time to give a maximumvalue at around 10 min. The number of thiol groups present in theenzyme was estimated from the maximum value using the molarabsorption coefficient 14,150 (35).

[y-32P]ATP was synthesized by the method of Post and Sen (32).5,5'-Dithiobis(2-nitrobenzoic acid) was obtained from Wako Pure

Chemicals, Ltd. (Osaka, Japan). Pyruvate kinase and lactate dehy-drogenase were obtained from Boehringer Mannheim Gmbh. Phos-phoenolpyruvate and NADH were obtained from Oriental Yeast Co.,Ltd. (Tokyo, Japan), and ATP was purchased from KYOWA Fer-mentation Co., Ltd. (Tokyo, Japan). All other chemicals were ofreagent grade.

RESULTS

Biochemical Properties of BIPM-treated Enzyme-In theprevious experiments (18), the enzyme was treated with 1 mM

BIPM, and the resulting preparations showed more or lessthan 2% fluorescence change during Na+-dependent hydroly-sis of ATP and contained 50 nmol of BIPM/mg of protein. Tomodify the enzyme more specifically, BIPM concentrationdependency of the fluorescence change was studied. Treat-ment with 1 or 10 ,pM BIPM for 45 min at 0 °C in the presenceof 5 mM KCI yielded preparations which showed little changeduring the hydrolysis of ATP. The amount of BIPM boundwas 2.3 and 8.4 nmol/mg of protein, respectively. Treatmentwith 200 /aM BIPM gave a preparation which showed thedynamic change as described below, and the amount of BIPMwhich became bound to the enzyme was about 35 nmol/mg ofprotein. The emission maxima of these preparations were 385,378, and 365 nm, respectively, and the spectral width de-creased in the same order. The data suggested that BIPM wasincorporated into more hydrophobic regions with increasingconcentration of BIPM. In the following experiments, thepreparations used were treated with 0.2 mM BIPM unlessotherwise stated. Some biochemical properties of a typicalpreparation are shown in Table I. As shown, the preparationretained 47, 69, and 65% of (Na+,K+)-ATPase activity, K+ -

dependent p-nitrophenylphosphatase activity, and ATP-de-pendent phosphorylation capacity, respectively. The values ofK0.5 were estimated from the concentrations of ligands to givea half-maximum effect on K-dependent p-nitrophenylphos-phatase activity. The activity was known to be inhibited byNa+ or high concentration of ATP, but the inhibition by Na+

could be partially recovered by low concentration of ATP(2-4). The K0.5 for Na+ and ATP (high affinity) was increasedand decreased to 165 and to 60% by the treatment. Theseresults represented the largest decrease and increase in theapparent affinity change induced by the treatment (Table I).

The excitation and emission spectra of a typical preparationare shown in Fig. 1, the maximum being 287 nm with ashoulder at 314 nm for the former (a) and 363 nm for thelatter (a'), respectively. The spectra of the native enzyme (b,b') and of BIPM in the presence of fi-mercaptoethanol (c, c')are also shown.

The number of sulfhydryl groups of the native enzyme wasdetermined by 5,5'-dithiobis(2-nitrobenzoic acid) to be 100nmol/mg of protein in the presence of 0.2% sodium dodecylsulfate. After BIPM treatment of the native enzyme, the value

TABLE I

Biochemical properties of BIPM-treated enzyme

The enzyme was treated with BIPM. The control preparation wasobtained as described above except that BIPM was omitted.(Na+,K+)-ATPase activity was measured in the presence of 1 Mg of(Na+,K+)-ATPase protein, 5 mM Mg2+, 4 mM ATP, 160 mM Na+, 16mM K+, 25 mM sucrose, 0.1 mM EDTA, 25 mM imidazole-HCl, pH 7.4,at 37 °C with or without 1 mM ouabain in a final volume of 0.75 mlusing a coupled system containing 1.5 mM phosphoenolpyruvate, 0.25mM NADH to which 10 /g/ml of pyruvate kinase and lactic dehydro-genase were added. The K+-dependent p-nitrophenylphosphatasereaction (37 °C at pH 7.4) was started by addition of 50 pl of anenzyme solution containing 2.4 pg of (Na+,K+)-ATPase protein, 12.5,pmol of sucrose, and 0.05 pmol of EDTA-Tris, pH 7.4, to 350 l1 of areaction mixture containing 16 mol of IH, 6.4 mol of p-nitrophen-ylphosphatase, 8 pmol of MgC12, and 16 pamol of KC1. After 10 min,the reaction was terminated by the addition of 1.25 mil of 2% sodiumdodecyl sulfate containing 2.5% Na 2CO3 and the activity was measuredas described (23). The phosphorylation reaction was started (0 °C) byaddition of 10 pl of a solution containing 2 nmol of [y-nP]ATP-Trisand 43 nmol of MgCl 2 (pH 7.4) to 90 pl of a reaction mixture containing

20 Ig of protein of (Na+,K+)-ATPase, 2.5 nmol of IH, 2.5 nmol ofsucrose, and 0.01 nmol of EDTA-Tris with 1.6 tumol of NaCI or ofKC1, pH 7.4, at 0 C. After 4 s, the reaction was terminated and theamount of phosphoenzyme was estimated by the method of Post andSen (32). The apparent affinity for the physiological ligands of(Na+,K+)-ATPase was estimated from the K0.5 for activation or inhi-bition of the K+-dependent p-nitrophenylphosphatase activity asdescribed above except that 0 to 128 mol of choline chloride wereadded to make the ionic strength constant. To estimate the apparentaffinity (K0.5) for ATP of high affinity or Mg 2+ or K+, the concentra-tions of ATP (0 to 100 pM) in the presence of 320 mM Na+ or Mg 2+

(0 to 20 mM) or K+ (0 to 40 mM) were changed and the activity wasmeasured. The concentration of half-maximum activation was esti-mated from plottings of these data. To estimate the Ko.5 for Na+ orATP of low affinity, the concentrations of ATP (0 to 1.4 m) or Na+

(0 to 320 mM) were changed and the half-maximum inhibition wasestimated. ATP (h) and ATP(1) means, Ko.5 for ATP-dependentactivation and inhibition, respectively.

K+-dependent p-ni- Ko.5(Na,K+)-ATPase trophenylphospha- Phosphoenzyme

tase Na+ K+ Mg2+ ATP(h) ATP(1)Imool mg' h-' pmol mg mM

Control 1105 ± 50 256 2 1859 ± 76 112 6 1.2 0.020 6BIPM-treated 522 ± 23 176 ± 3 1201 12 185 4 1.8 0.012 6

10660

by guest on Novem

ber 17, 2020http://w

ww

.jbc.org/D

ownloaded from

Conformational Difference between E1 P and E2 P in (Na +,K+)-ATPase

decreased to 60-70 nmol/mg of protein. The data showed thatone-third of the sulfhydryl groups of the (Na+,K+)-ATPasepreparations were modified with 0.2 mM BIPM treatment.

Preliminary experiments using Sephacryl S-200 columnchromatography suggested that BIPM was incorporated intoboth a and f, peptides (36). The more quantitative studies arenow in progress.

Relative Fluorescence Intensity Change Induced by Bind-ing of Physiological Ligands-To compare the effect of phys-iological ligands on fluorescence intensity, various ligandswere added to the enzyme. The addition of ADP or ATP orMg2+ with or without these nucleotides induced a slight neg-ative fluorescence change (Table II). Sixteen mM Na+ induceda -2.1% change which reflected the formation of NaE, be-cause 1.6 mM K+ reversed the change (not shown). Furtheraddition of ADP, ATP, or MgADP induced -3.2% changes,the value of which seemed to be nearly the same or slightly

FIG. 1. Excitation and emission spectra of (Na+,K+)-ATPasemodified with BIPM. Enzyme was modified with 0.2 mM BIPM for45 min as described in the text. Seventy pg of protein of the treatedenzyme (a, a') or the control (b, b') were suspended in 7 ml of asolution containing 25 mM IH, pH 7.4, 25 mM sucrose, 0.1 mM EDTA-Tris. The fluorescence measurement was performed at 25 C with asample volume of 3.2 mi. Excitation spectra (left, a, b, c) were run ata fixed emission wavelength of 365 nm, and for the emission spectra(right, a', b', c'), the fixed excitation wavelength was 308 nm for thetreated enzyme and 295 nm for the control. The dotted line showsthe excitation spectra of 6.1 M BIPM-fi-mercaptoethanol in thepresence of 25 mM IH, 25 mM sucrose, 0.1 mM EDTA-Tris, pH 7.4, ata fixed emission wavelength of 365 nm (c), and an emission spectrawith a fixed wavelength of 308 nm (c').

more than the summation of the decrease induced by eachligand. Addition of K+ to NaE, in the presence of Mg2+ andADP induced a 2% increase (-3.2 + 1.2). Thus, the Na +-

induced change was reversed by K+ . Addition of ATP in thepresence of Mg2+ to NaE 1 transiently increased the fluores-cence from -2.9 ± 0.4% to -0.6 + 0.5%; further addition ofouabain did not change the fluorescence as described previ-ously (18).

The addition of ADP or ATP to the enzyme in the presenceof 2 M Na+ induced a slight decrease in the fluorescence. Theaddition of ATP in the presence of Mg2+ and 2 M Na+ induceda -2.3% fluorescence change, and further addition of ouabainincreased the fluorescence to +4.7%.

The opposite change in fluorescence intensity induced bythe addition of ATP in the presence of Mg2 ' at high and lowconcentrations of Na + suggest that EIP and E2P are in differ-ent conformational states, because Na+ greatly affected theequilibrium between them (21, 23-25).

To compare the effect of K+ on fluorescence intensity withthat of Na+ , both cations were added to the sample andreference cells, respectively, to give the same final concentra-tions. The difference in the fluorescence intensity was shownto be almost constant in the presence of 16 mm to 2.8 M Na +

(not shown). The data indicate that the difference in fluores-cence intensity between NaE1 and KE 2 is maintained even inthe presence of very high concentrations of the respectivecations.

ATP-dependent Reversible Changes in Fluorescence In-tensity in the Presence of Low and High Concentrations ofNa+-Addition of ATP to the preparation in the presence of0.43 mM Mg 2+ with 16 mM Na+ transiently increased thefluorescence as shown (Fig. 2). The fluorescence intensitydecreased with time to the lowest level, which had been shown(18) to be the fluorescence of MgNaEiADP (see also TableII). Re-addition of ATP immediately increased the fluores-cence, and further addition of ouabain interrupted the dy-namic change and stabilized it at the maximum level.

Addition of ATP to the enzyme in the presence of 0.43 mMMg2+ with 2 M Na+ immediately decreased the fluorescenceintensity to the lowest level, and the intensity increased veryslowly with time (Fig. 3). Re-addition of ATP again decreasedthe fluorescence, but further addition of ouabain immediatelyincreased and then stabilized it at the maximum level.

Concentration Dependency of Na+ on ATP-induced Re-versible Fluorescence Change and the Amount of Phosphoen-zymes-The effect of Na+ on the relative fluorescence inten-

TABLE II

Fluorescence change induced by ligand(s) of (Na,K)-ATPaseThe protein, 70 pig, was suspended in 7 mil of a solution containing

25 m imidazole-HC1 (pH 7.4), 0.1 mM EDTA-Tris, 25 mM sucrosewith or without 2000 mM Na+. The sample and reference cells con-tained 3.2 ml of the suspension. When added, the ligands were 12.8td of 2 M Na+, 3 pl of 30.1 mM ATP-Tris or 3 pl of 30.1 mM ADP-Tris,2 pl of 686.4 mM MgCl 2, 4 1 of 1.6 M KCl, and 100 pl of 15 mM ouabainfor the sample cell. The same volume of water was added to thereference cell to keep the sample volume of both cells constant. Thedata shown are percentage values; 100% values of the fluorescenceintensity were taken from the difference between the fluorescenceintensity at 365 nm and that at 500 nm of the reference sample in the

absence of both Mg2+ and Na +, or in the presence of 2000 mM Na+(*).In the presence of Mg2+, Na +, and ATP, the fluorescence intensitychanged with time as described in the text. In such cases, the valuesobserved at 30 s after the addition of the third ligand are shown.When ouabain was added under the phosphorylation condition in thepresence of 2000 mM Na+ , the fluorescence intensity increased withtime to give a constant maximum level. The data show these values.The enzyme treated with 1 (Ref. 18) or 0.2 mM BIPM was used tomeasure the fluorescence intensity change in the absence and pres-ence of 16 mM Na+ or 2000 mM Na+ (*), respectively.

Na' 0 16 2000

Mg + 0 mM 0.43 mM 0 mM 0.43 nMi 0 mM 0.43 myM

Control 0.0 -0.2 ± 0.1 -2.1 ± 0.4 -2.9 ± 0.4 0.0* 0.0*+ADP -0.4 -0.6 -3.2 -3.2 ± 0.5 -0.3* -0.7*+ATP -0.5 - 0.2 -0.8 -3.2 -0.6 ± 0.5 -0.5* -2.3 ± 0.3*+ADP + K+ -1.2 ±- 0.2+ATP + ouabain -0.6 ± 0.5 +4.7 ± 0.1*

10661

by guest on Novem

ber 17, 2020http://w

ww

.jbc.org/D

ownloaded from

Conformational Difference between E1P and E2P in (Na +,K+)-A TPase

sity and the amount of phosphoenzymes formed under similarphosphorylation conditions are shown in Fig. 4, A and B. Thedirection and the extent of the fluorescence change inducedby ATP was dependent on the concentrations of Na+ present.Positive changes were observed in the presence of low concen-trations of Na+ . The extent of the positive change was reducedand the negative change increased with increase in the con-centration of Na+ . The extents of the positive and negativechanges were almost at saturation in the presence of 16 mMand 1.5 M Na+ , respectively. The Ko.5 for Na+ with respect toinduction of the fluorescence change by phosphorylation wasabout 0.4 M, which value was near that estimated from thesynthesis of ATP from K+-sensitive phosphoenzyme (21).

After exhaustion of ATP, the fluorescence intensity de-creased or increased to give the level of MgNaE 1ADP in thepresence of lower or higher concentrations of Na+ . The timerequired to attain this level increased with increasing Na+

concentration, and re-addition of ATP immediately changedthe fluorescence to give nearly the same levels as those ob-tained by the initial addition of ATP (data not shown but seeFigs. 2 and 3). Further addition of ouabain stabilized the

16mM Na

'St T TATP. 15 3% OUAB1.54WET-P' \ , ATP.15

"-20MIN -FIG. 2. ATP-dependent reversible conformational change in

the presence of 16 mM Na+. Seventy /tg of protein of the treatedenzyme were suspended in 7 ml of a solution containing 16 mM Na+,0.43 rmM Mg2+, 25 mM IH, 25 mM sucrose, 0.1 mM EDTA-Tris, pH 7.4.The fluorescence was excited at 308 nm, and emitted light wasdetected at 365 nm. The fluorescence measurement was performedwith an initial sample volume of 3.2 ml. The additions of ligand were5 ld of 30 mM ATP-Tris and 100 L1l of 15 mM ouabain (OUAB) to givethe final amount (micromoles) shown. The volume of sample andreference cells was kept constant by adding H2 0 to the reference cell.The upward direction indicates an increase in fluorescence. Thedifference of the fluorescence intensity between 365 and 500 nm ofthe initial solution was taken as a 100% value. The time course runsfrom left to right.

LLL

fluorescence change in the presence of low concentrations ofNa+; however, the addition remarkably increased the fluores-cence from the lowest to the highest level in the presence ofhigh concentrations of Na+ (Fig. 3 and 4A).

The amount of phosphoenzyme present immediately afterthe addition of ATP under the same ligand conditions (buthigher protein concentration) increased with the increase ofNa+ concentration and became saturated in the presence of32-2000 mM Na+ as shown (Fig. 4B).

The data indicate that phosphorylation induced the re-markable fluorescence intensity change and suggest that theEIP and E2P formations accompanied the negative and thepositive fluorescence changes, respectively, with respect to theNa+-form enzyme.

Time-dependent Changes in the Fluorescence Intensityand the Amount of Phosphoenzymes in the Presence of 8 or1500 mM Na+-To ascertain the reason for the transient risein the presence of low concentrations of Na+ , the changes inthe fluorescence intensity and the amount of phosphoenzymesafter addition of ATP were measured as shown in Fig. 5. Thetime course of the decrease in the fluorescence intensity wasthe same as that of the decrease in the amount of phosphoen-zymes. After exhaustion of ATP, addition of ouabain graduallyincreased the fluorescence, which was accompanied by a con-comitant increase in the amount of phosphoenzymes to nearlyhalf of the initial amount. Addition of K+ changed neither theamount of phosphoenzymes nor the fluorescence intensity.The phosphoenzyme formed immediately after ATP additionshowed sensitivity to K+ but not to ADP (not shown). Thesedata showed that the transient rise was due to the formation

2M Na

- 20MIN - AATP.15P.09 4% te

82MIN1 4o'N WOUAB1.5

.w OOA,%.0 1 20 M IN

FIG. 3. ATP-dependent reversible conformational change inthe presence of 2 M Na+. The experiments were performed asdescribed in Fig. 2 except that both concentrations of protein and Na+

were 20 /g/ml and 2 M, respectively. OUAB, ouabain. The two gapsin the trace represent a period of 120 and 82 min, respectively.

NaC[ ;M NaCI, M

FIG. 4. Concentration dependency of Na+ on the extent ofATP-induced transient change in the fluorescence intensityand the amount of phosphoenzyme. A, 100 pg of protein of thetreated enzyme were suspended as described in Fig. 2 except that theconcentrations of Na+ were varied from 8 to 2000 mM. Fluorescenceintensities (AF) shown are the levels obtained immediately afteraddition of ATP (0) and after exhaustion of ATP (A). Re-addition of27 LM ATP immediately changed the fluorescence intensity to theinitial level and further additions of ouabain stabilized the fluores-

cence (0). B, forty-five ,ag of protein of the treated enzyme weresuspended in 90 pl of a solution containing 2.5 /mol of IH (pH 7.4),0.01 pLmol of EDTA-Tris, 2.5 #mol of sucrose, and 0.125-200 pmol ofNaCI. Phosphorylation (25 C) was started by addition of 10 pl of asolution containing 0.03125 Lmol of MgCI and 4 nmol of [y-3 2P]ATP.After 4 s, the reaction was terminated and the amount of phosphoen-zyme (EP) was measured by the method of Post and Sen (32). One,and two-tenths nmol/mg of protein were taken as the value for 100%'of the phosphoenzyme.

10662

I

by guest on Novem

ber 17, 2020http://w

ww

.jbc.org/D

ownloaded from

Conformational Difference between E1 P and E2 P in (Na+,K+)-ATPase

rmM

10 20 30MIN,TIME AFTER ATP(-)or ATP32 (O)

40

known to be blocked by N-ethylmaleimide treatment (1-4,39). At this point, we thought it would be interesting toinvestigate the conformational change of BIPM-treated en-zyme which was further treated with N-ethylmaleimide. TheN-ethylmaleimide treatment of BIPM-treated enzyme in thepresence of K+ with ATP (39) reduced the amount of phos-phoenzyme formed from ATP and the (Na+,K+)-ATPase ac-tivity to 52% (630 ± 49 pmol/mg of protein) and 10% (74 ± 3

FIG. 5. Time-dependent changes in the fluorescence inten-sity and the amount of phosphoenzyme in the presence of 16mM Na+ . The fluorescence intensity (AF) was measured under thesame condition as that shown in Fig. 2 except that the concentrationof protein was increased to 112.5 Mg of protein/ml and the concentra-tions of Na + and ATP were 16 mm and 20 M, respectively. Themaximum fluorescence intensity was obtained immediately after ad-dition of ATP. The minimum fluorescence intensity was obtainedafter exhaustion of ATP. The difference between the intensity of themaximum and the minimum values was taken as a 100% value. Toestimate the amount of phosphoenzyme (EP), [y-32 P]ATP was addedto the reaction mixture, which was prepared exactly as describedabove. Four hundred p1 of the reaction mixture was withdrawn andthe reaction was terminated immediately by addition of 5 ml of 5%trichioroacetic acid containing 3 mM ATPNa 2 and 3 mm K2HPO4 atthe times indicated. The amount of phosphoenzyme was measured asdescribed in Fig. 4. One and two-tenths nmol/mg of protein weretaken as the value for 100% of the phosphoenzyme.

I?5

L0

FIG. 6. Time-dependent changes in the fluorescence inten-sity and the amount of phosphoenzyme in the presence of 1.5M Na +. The fluorescence intensity (AF) was measured under the samecondition as that described in Fig. 4 except that concentrations ofprotein, Na+, and ATP were 78.3 pg of protein/m, 1.5 M, and 26.6/M, respectively. The maximum negative fluorescence intensity wasobtained immediately after additions of ATP. After exhaustion ofATP, the fluorescence increased to the maximum value, which wasequal to MgNaE1ADP level. The difference between the intensities

of E2P and that the gradual increase in the amount of phos- was taken as a -100% value. Prelirphoenzymes after addition of ouabain was due to the forma- 3 2

p incorporation, which was insensition of ouabain-bound phosphoenzymes formed from Pi. Phos- with the increase in incubation tii

phosphoenzyme (EP), [-32P]ATPAphoenzyme formation from Pi has been shown to be strongly phosphoenzyme (EP), [y-32PATP inhibited by Na~~~~~~ (37) ~following the method described abe

inhi~bited by Na~ (37). ~reaction mixture were withdrawn aiTo investigate the reason for the transient fall in the pres- terminated at the times indicated b:

ence of high concentrations of Na+ , the changes both in the containing 3 ma ATPNa 2, 3 mm K2]fluorescence intensity and the amount of phosphoenzymes albumin, which was added as a csafter addition of ATP were measured (Fig. 6). Phosphoenzyme easily. The denatured enzyme suspwas formed immediately after addition of ATP, and it showed rpm for 20 min at 0 'C, and the pre

with 30 mm HCI containing I mMisensitivity to ADP but not to K+ (not shown). The decrease witae w tn n in the amount of phosphoenzymes preceded the increase in 8.1), and the suspension was incubthe fluorescence intensity. The data suggest that the initial centrifugation at 18,000 rpm for 20transient fall was due to the formation of EIP, because the withdrawn and 32Pi was extracted byenzyme was fully phosphorylated under the experimental (38) and counted. The line is the tconditions as shown above (Fig. 4B). The data also suggest induced by ATP. Open circles repr

zyme. Five hundred seventy pmol/that at least one other dephosphoenzyme, which showed value for 100% of the phosphoenyr

> ~~value for 100% of the phosphoenzymsimilar low fluorescence intensity to that of EIP, was present.

The Effect of Ionic Strength on Fluorescence Change In-duced by Phosphorylation-Concentrations of Na+ should be A 16mM Na+1M Chigh and low, respectively, to detect the conformational state 16mM Nof EiP and E2P at steady state because equilibrium between EP and E2P is shifted to the former in the presence of high Tconcentrations of Na+ (21, 23-25). To investigate the effect of 3% ATP093%ionic strength on fluorescence change, choline chloride was A ADPO9

AYP.09i ' . d Jadded to keep the ionic strength constant. ATP-dependent ADP.09 __increases or decreases in fluorescence were observed in the W \presence of 16 mM Na+ or 2016 mM Na+ (Fig. 7, A and B). 20MIN- DAddition of 1 or 2 M choline chloride in the presence of 16 mM 1M N Na+ did not affect the direction and the extent of ATP- Binduced fluorescence change (Fig. 7, C and D). However, the 2.016M N 3%rate of fluorescence decrease was considerably decreased in - IATP.09 OUAB.3 ATP.09 39the presence of high concentrations of choline chloride. ADP.09 OUAB3 ATP.093ADP.09 AP0

These data indicate that the large differences in fluores- -cence intensity of phosphoenzyme in the presence of high and -20MN -low concentrations of Na+ was due to the conformational Fic. 7. The effect of choline cdifference between EP and E2P and not to the difference in change induced by phosphorylatof the treated enzyme were suspendthe ionic strength used in the experiments. that the concentrations of Na were- ~~~~that the concentrations of Na + were

Effect of N-Ethylmaleimide Treatment on Fluorescence choline chloride or with 2 M NaCl. AChange of Phosphoenzyme-The conversion of E1P to E2P is a period of 34 min. OUAB, ouabain.

ninary experiments showed thattive to hydroxylamine, increasednme. To estimate the amount ofwas added to the reaction mixturewve. Two hundred sixty pl of thend the reaction was immediatelyy 10 ml of 5% trichloroacetic acidIHPO4, 20 mg/ml of bovine serumcarrier to precipitate the enzymeension was centrifuged at 18,000cipitates were washed two timesK2HPO 4, 1 m Na4P204. The

ith 2 hydroxylamine-Tris (pHated for 30 min at 37 C. Aftermin, 100 pl of supernatant werethe method of Martin and Doty

trace of the fluorescence changeesent the amount of phosphoen-mg of protein was taken as theie.

CHOLINE CHLORIDE

20MIN

#

+2M CHOLINE CHLORIDE

34MIN

- 20MIN

hloride on the fluorescenceion. One hundred jig of proteined as described in Fig. 2 except16 mM with or without 1 or 2 Mgap in the trace (D) represents

10663

)

by guest on Novem

ber 17, 2020http://w

ww

.jbc.org/D

ownloaded from

by guest on Novem

ber 17, 2020http://w

ww

.jbc.org/D

ownloaded from

by guest on Novem

ber 17, 2020http://w

ww

.jbc.org/D

ownloaded from

Conformational Difference between E1P and E2 P in (Na+,K+)-ATPase

fluorescence intensity of tryptophan residues of E1P is higherthan of NaEi. Skou and Esmann (43) reported that cholinehas a Na+ effect on tryptophan and eosin maleimide residuesin (Na+,K+)-ATPase. However, our data indicated that cho-line chloride does not replace Na+ and subsequently inducethe dynamic change in the fluorescence intensities of bothtryptophan and BIPM residues (Figs. 7 and 10).

The lag between the decrease in the amount of phosphoen-zyme and the increase in fluorescence after addition of ATPsuggests the presence of at least one other dephosphoenzymewhich has similar low fluorescence intensity to that of E1 P(Fig. 6). The dephosphoenzyme seemed to accumulate withthe increase in concentration ratio of [ADP]/[ATP]. Sincethe rate-limiting step of ATP hydrolysis is the conversion ofE1 P to E2P in the presence of ATP and high concentrationsof Na+ , it is highly possible that the increment of ADP due tohydrolysis of ATP caused the inhibition of EIP formation toaccumulate MgNaEATP, which has similar fluorescence in-tensity to that of E1P. The presence of such an intermediatehas been suggested (42, 44).

Karlish (19) and Hegyvary and Jorgensen (20) showed thedifference in the conformational states of E2 moieties usingfluorescein isothiocyanate-modified enzyme. Unfortunately,however, their treated preparations showed little (Na+,K+)-ATPase activity. We are presently conducting experiments todistinguish E2 moieties such as KE 2, E2P, and ouabain E2P bythe fluorescence intensities of tryptophan and BIPM residues.

Conformational Transition from E1P to E2P-Numerouskinetic data have been reported to show the presence of EIPand E2P during hydrolysis of ATP (1-8, 21). The presentexperiments show that the micro-environments of BIPM andtryptophan residues of EIP are different from those of E2Pand/or ouabain E2P. The difference of both the intrinsic andextrinsic fluorescence intensities was not due to the differencesof ionic strength used to detect E1P and E2P, respectively.This was because 1 or 2 M choline chloride did not change thedirections of fluorescence change induced by the phosphoryl-ation in the presence of low concentrations of Na+ , and be-cause ouabain-bound phosphoenzyme showed the same high-est fluorescence intensity irrespective of the concentration ofNa+ . N-Ethylmaleimide treatment partially abolished thetransient rise in the fluorescence of BIPM residues inducedby MgATP in the presence of 16 mM Na+ . The treatment didnot affect the transient decrease in fluorescence which wasinduced by formation of EIP. This result is consistent withthat of the assumed inhibition of conversion of EIP to E2P.From these results, we conclude that transition of EIP to E2Paccompanied the largest change in the fluorescence of BIPMresidues.

Conversion of EIP to E2P seems to be intimately related tothe energy coupling between hydrolysis of ATP and transportof Na+ across the membranes (21, 23-25). EIP and E2P are inequilibrium with ATP or Pi, respectively (45). Binding of Na+

is sufficient to convert E2P to EIP to synthesize ATP (21).These data and others suggest that the free energy whichcomes from the hydrolysis of ATP is conserved to form E1P(45) and that this free energy is used for the conformationalchanges which have been detected as fluorescence intensitychanges in the present experiments. If the high fluorescenceintensities of both intrinsic (Ref. 22, present study) and ex-trinsic probes are due to the increase in rigidity and/or hydro-phobicity of the micro-environment of these probes, and bothprobes reside on the same ATPase molecules, these data couldbe explained by using two simplified models of Na+ transport(Fig. 12). The enzyme which binds Na+ with high affinitycontains tryptophan residues which give the lowest fluores-cence intensity as well as BIPM residues which give inter-

Ar---- r----- -....... ' __ _ _fO -

B

NaE l

LB …/

ElP

ryfc

E2P

E2P

FIG. 12. Hypothetical models of Na + transport which weresuggested from the intrinsic and extrinsic fluorescence changeaccompanying E2P formation from NaE. T and B designate thetryptophan and BIPM residues involved in the fluorescence change.Open circles and dotted lines designate, respectively, Na+ and Na+

pump. The order of the relative fluorescence intensity of tryptophanresidues is as follows: NaE, < EIP < E2P, while that of BIPM residuesis as follows: EP < NaEI < E2P (see text).

mediate fluorescence intensity. Addition of MgATP to theenzyme caused formation of EjP, which presumably occludesNa + (6, 46), and the fluorescence intensity of the tryptophanresidues became intermediate, whereas that of the BIPMresidues become the lowest. Consequently, formation of E2Paccompanying the large decrease in the affinity for Na+ andboth the fluorescence intensities of tryptophan and BIPMresidues became the highest. In Fig. 12, model A, sodium ionsare transported by peristaltic movement, and a peptide whichcontains both fluorescent residues does not move across themembrane, i.e. the movement of the tryptophan and BIPMresidues is not as great as that of Na+ . On the other hand, inmodel B, sodium ions are transported by the movement of apeptide, and the amino acid residues which bind Na+ moveacross the membranes following the tryptophan and BIPMresidues in sequence. Studies of the sequence of changes inboth fluorescence intensities from E2P to EiNa via severalintermediates (47) and of the number of fluorescence residuesinvolved are now in progress to select one of the two models.

One may suggest that modified sulfhydryl groups may beunevenly distributed throughout the population of enzymemolecules and question whether the probe reports effects onthe whole population. The time course of 0.2 mM BIPMtreatment at 0 C in the presence of 5 mM KCI was studied.2

After 20 min, the amount of BIPM bound, the extent of thefluorescence change under various conditions (i.e. EIP toouabain E2P), and the residual (Na+,K+)-ATPase activitybecame almost constant values. Prolonged incubation up to120 min gave little change in these values. Those experimentssuggest that the probe reports effects on the whole population.

Acknowledgments-We are indebted to M. Sakuraya, M. Kudo, F.Kudo, and T. Tamaki for preparing the enzyme and K. Omori for hersecretarial assistance in preparing the manuscript.

REFERENCES

1. Post, R. L., Kume, S., Tobin, T., Orcutt, B., and Sen, A. K. (1969)J. Gen. Physiol. 54, 306S-326S

2. Glynn, I. M., and Karlish, S. J. D. (1975) Annu. Rev. Physiol. 37,13-55

3. Schwartz, A., Lindenmayer, G. E., and Allen, J. C. (1975) Phar-macol. Rev. 27, 3-134

4. Albers, W. (1976) in The Enzymes of Biological Membranes(Martonosi, A., ed) Vol. 3, pp. 283-301, Plenum Press, NewYork

5. Cavieres, J. D. (1977) in Membrane Transport in Red Cell(Ellory, J. C., and Lew, V. L., eds) pp. 1-37, Academic Press,London

2 M. Sakuraya, K. Suzuki, and K. Taniguchi, unpublished results.

10666

by guest on Novem

ber 17, 2020http://w

ww

.jbc.org/D

ownloaded from

Conformational Difference between EIP and E2P in (Na+,K+)-ATPase

6. Post, R. L. (1979) in Cation Flux across Biomembranes (Muko-hata, Y., and Packer, L., eds) pp. 3-19, Academic Press, NewYork

7. Robinson, J. D., and Flashner, M. S. (1979) Biochim. Biophys.Acta 549, 145-176

8. Cantley, L. C. (1981) Curr. Top. Bioenerg. 2, 201-2379. J0rgensen, P. L. (1975) Biochim. Biophys. Acta 401, 399-415

10. Lo, T. N., and Titus, E. 0. (1978) J. Biol. Chem. 253, 4432-443811. Koepsell, H. (1979) J. Membr. Biol. 48, 69-9412. Stahl, W. L., and Harris, W. E. (1979) in Na+,K+-ATPase Struc-

ture and Kinetics (Skou, J. C., and Norby, J. G., eds) pp.157-168, Academic Press, London

13. Karlish, S. J. D., Beauge, L. A., and Glynn, I. M. (1979) Nature282, 333-335

14. Gupte, S. S., Lane, L. K., Johnson, J. D., Wallick, E. T., andSchwartz, A. (1979) J. Biol. Chem. 254, 5099-5103

15. Karlish, S. J. D., and Yates, D. W. (1978) Biochim. Biophys. Acta527, 115-130

16. Forgac, M. D. (1980) J. Biol. Chem. 255, 1547-155317. Askari, A., Huang, W., and Antieau, M. (1980) Biochemistry 19,

1132-114018. Taniguchi, K., Suzuki, K., Shimizu, J., and Iida, S. (1980) J.

Biochem. (Tokyo) 88, 609-61219. Karlish, S. J. D. (1980) J. Bioenerg. Biomembr. 12, 111-13620. Hegyvary, C., and Jorgensen, P. L. (1981) J. Biol. Chem. 256,

6296-630321. Taniguchi, K., and Post, R. L. (1975) J. Biol. Chem. 250,

3010-301822. J0rgensen, P. L., and Petersen, J. (1979) in Na+,K+-ATPase

Structure and Kinetics (Skou, J. C., and Norby, J. G. eds) pp.143-155, Academic Press, London

23. Kuriki, Y., and Racker, E. (1976) Biochemistry 15, 4951-495624. Post, R. L. (1977) in Biochemistry of Membrane Transport,

FEBS Symposium No. 42 (Semenza, G., and Carafoli, E., eds)pp. 352-362, Springer Verlag, New York

25. Hara, Y., and Nakao, M. (1981) J. Biochem. (Tokyo) 90, 923-93126. Hayashi, Y., Kimimura, M., Homareda, H., and Matsui, H. (1977)

Biochim. Biophys. Acta 482, 185-19627. Nakao, T., Tashima, Y., Nagano, K., and Nakao, M. (1965)

Biochem. Biophys. Res. Commun. 19, 755-75828. Jrrgensen, P. L. (1974) Biochim. Biophys. Acta 356, 36-5229. Hayashi, Y., and Post, R. L. (1980) Fed. Proc. 39, 170430. Taniguchi, K., and lida, S. (1973) Mol. Pharmacol. 9, 350-35931. Taniguchi, K., Tazawa, H., and lida, S. (1979) in Cation Flux

across Biomembranes (Mukohata, Y., and Packer, L., eds) pp.41-47, Academic Press, New York

32. Post, R. L., and Sen, A. K. (1967) Methods Enzymol. 10, 773-77633. Bradford, M. M. (1976) Anal. Biochem. 72, 248-25434. Sekine, T., Ohyashiki, T., Machida, M., and Kanaoka, Y. (1974)

Biochim. Biophys. Acta 351, 205-21335. Riddles, P. W., Blakeley, R. L., and Zerner, B. (1979) Anal.

Biochem. 94, 75-8136. Peters, W. H. M., de Pont, J. J. H. H. M., Koppers, A., and

Bonting, S. L. (1981) Biochim. Biophys. Acta 641, 55-7037. Post, R. L., Toda, G., and Rogers, F. N. (1975) J. Biol. Chem.

250, 691-70138. Martin, J. B., and Doty, D. M. (1949) Anal. Chem. 21, 965-96739. Wallick, E. T., Anner, B. M., Ray, M. V., and Schwartz, A. (1978)

J. Biol. Chem. 253, 8778-878640. Giotta, G. J. (1975) J. Biol. Chem. 250, 5159-516441. Castro, J., and Farley, R. A. (1979) J. Biol. Chem. 254, 2221-222842. Mardh, S., and Post, R. L. (1977) J. Biol. Chem. 252, 633-63843. Skou, J. C., and Esmann, M. (1980) Biochim. Biophys. Acta 601,

386-40244. Kanazawa, T., Saito, M., and Tonomura, Y. (1970) J. Biochem.

(Tokyo) 67, 693-71145. Post, R. L., Kume, S., and Rogers, F. N. (1973) in Mechanism in

Bioenergetics (Azzone, G. F., Ernster, L., Papa, A., Quagli-ariello, E., and Siliprandi, N., eds) pp. 203-218, Academic Press,New York

46. Yamaguchi, M., and Tonomura, Y. (1980) J. Biochem. (Tokyo)88, 1387-1397

47. Karlish, S. J. D., Yates, D. W., and Glynn, I. M. (1978) Biochim.Biophys. Acta 525, 252-264

10667

by guest on Novem

ber 17, 2020http://w

ww

.jbc.org/D

ownloaded from

K Taniguchi, K Suzuki and S IidaN-[p-(2-benzimidazolyl)phenyl]maleimide.

potassium-sensitive phosphoenzyme of (Na+,K+)-ATPase modified with Conformational change accompanying transition of ADP-sensitive phosphoenzyme to

1982, 257:10659-10667.J. Biol. Chem. 

  http://www.jbc.org/content/257/18/10659Access the most updated version of this article at

 Alerts:

  When a correction for this article is posted• 

When this article is cited• 

to choose from all of JBC's e-mail alertsClick here

  http://www.jbc.org/content/257/18/10659.full.html#ref-list-1

This article cites 0 references, 0 of which can be accessed free at

by guest on Novem

ber 17, 2020http://w

ww

.jbc.org/D

ownloaded from