Eur. J. Biochem. 261, 84±88 (1999) q FEBS 1999

A 75-kDa Na+,K+-ATPase competitive inhibitor protein isolated from ratbrain cytosol binds to a site different from the ouabain-binding site

Koushik Roy, Atin K. Mandal and Parimal C. Sen

Department of Chemistry, Bose Institute, Calcutta, India

A Na+,K+-ATPase inhibitor protein has been purified to homogeneity from rat brain cytosol by ammonium sulphate

precipitation, DEAE anion-exchange chromatography and hydroxyapatite adsorption column chromatography. The

purified protein migrates as a single polypeptide band of 75 kDa on 7.5% SDS/PAGE. Amino acid composition data

shows the presence of a high number of acidic amino acids in the molecule in relation to the pI value of 4.6. The

inhibitor binds Na+,K+-ATPase reversibly and blocks ATP binding sites at micromolar concentrations with an I50 of

<700 nm. As a result, formation of the phosphorylated intermediate of Na+,K+-ATPase is hindered in the presence

of the inhibitor. It does not affect p-nitrophenylphosphatase activity. Tryptophan fluorescence studies and CD

analysis suggest conformational changes of Na+,K+-ATPase on binding to the inhibitor.

Keywords: inhibitor protein; Na+, K+-ATPase; rat brain.

Because Na+,K+-ATPase (EC 3.6.1.3) regulates several essentialcellular functions ± such as intracellular homeostasis of sodiumand potassium ions, pH, membrane potential, cell volume,cellular uptake of amino acids and sugars in different tissues ±its activity must be finely controlled [1]. Numerous endogenouspeptidic [2,3] and nonpeptidic [4±6] factors have been reportedto influence the activity of this pump. Arnaiz et al. [7] reportedthat brain extract from rat can either stimulate or inhibit Na+,K+-ATPase. Recently, our laboratory has reported some lowmolecular mass (12±13 kDa) modulator proteins of ATPasesfrom rat brain [8,9]. Previously, we predicted the existence of ahigh molecular mass (<80 kDa) Na+,K+-ATPase inhibitorprotein in rat brain cytosol [10]. In the present study, wedescribe the purification (to homogeneity) and characterizationof a 75-kDa inhibitor protein of Na+,K+-ATPase from rat braincytosol. The inhibitor binds to Na+,K+-ATPase at a site differentfrom the binding site of ouabain.

MATERIALS AND METHODS

Materials

DEAE-cellulose, EGTA, ouabain, phenylmethanesulfonyl fluor-ide (PhCH2SO2F), N-a-p-tosyl l-lysine chloromethane(TosLysCH2Cl) and N-tosyl-l-phenylalanine chloromethane(TosPheCH2Cl) were purchased from Sigma Chemical Co.[3H]-Ouabain (15 Ci´mmol21) and [g-32P]ATP (3000 Ci´mmol21)were obtained from NEN, USA and Bhaba Atomic ResearchCentre, India, respectively. Hydroxyapatite was from Bio-Rad.(NH4)2SO4, Tris, ATP-disodium salt, EDTA and sucrose were

from Sisco Research Laboratory, India. All other chemicals usedwere of analytical grade. Membrane filter was obtained fromMillipore Corporation.

Methods

Preparation of microsomal membranes and cytosol. Malealbino rats, Charles foster strain, weighing about 100 g werekilled after anaesthetization with chloroform; whole brains wereremoved and washed with isotonic saline. Microsomal mem-branes were isolated as described previously [11,12] and werethe source of Na+,K+-ATPase. The supernatant from the post100 000 g centrifugation (cytosol) was collected and used as thesource of the inhibitor protein.

Purification of the inhibitor protein. The supernatant was kepton ice and solid (NH4)2SO4 was added slowly with constantstirring to a saturation of 35%. It was then centrifuged at15 000 g for 1 h, the pellet was suspended in 20 mm Tris/HClcontaining 1 mm EDTA, 1 mm EGTA, 1 mm PhCH2SO2F,0.1 mm TosLysCH2Cl, 0.1 mm TosPheCH2Cl, 1 mm b-mercap-toethanol (pH-7.5) and dialysed against 50 vols of the samebuffer for 24 h with two changes. The dialysed fraction wasloaded on to a 20-mL DEAE±cellulose column equilibratedwith 20 mm Tris/HCl (pH-7.5) containing 0.5 mm EDTA and1 mm b-mercaptoethanol. The column was washed with5 £ column volume of the same buffer containing 50 mmNaCl. The inhibitor protein was eluted with a 0.05±0.3 m NaClgradient, peak fractions were pooled together and subjected tohydroxyapatite chromatography. The column was pre-equili-brated with 10 mm sodium phosphate buffer (pH 6.8) andpurified protein was eluted by washing the column with thesame buffer. It was dialysed against 10 mm Tris/HCl buffer (pH7.5) containing 0.5 mm b-mercaptoethanol for 48 h with fourchanges and used for subsequent studies.

Purification of Na+,K +-ATPase. Na+,K+-ATPase was purifiedfrom rat brain microsomal membranes as described byBhattacharyya and Sen [8].

Correspondence to P. C. Sen, Department of Chemistry, Bose Institute, 93/1,

A.P.C. Road, Calcutta 700009, India. Fax: + 91 33 350 6790,

Tel: + 91 33 350 2402, E-mail: parimal@boseinst.ernet.in

Abbreviations: PhCH2SO2F, phenylmethanesulfonyl fluoride; TosLysCH2Cl,

N-a-p-tosyl l-lysine chloromethane; TosPheCH2Cl, N-tosyl-l-phenylalanine

chloromethane; pNPPase, p-nitrophenyl phosphatase.

Enzymes: Na+,K+-ATPase (EC 3.6.1.3).

(Received 27 October 1998, revised 17 December 1998, accepted

6 January 1999)

q FEBS 1999 Binding of Na+,K+-ATPase inhibitor from rat brain (Eur. J. Biochem. 261) 85

Protein assay and gel electrophoresis. Protein was estimated bythe Bradford method [13] using BSA as a standard. SDS/PAGEwas performed according to the method of Laemmli [14] andsilver staining was carried out according to Morrissey [15].

Na+,K +-ATPase activity assay. Na+,K+-ATPase activity wasassayed as described by Robinson [16] and Sen et al. [17].Briefly, the reaction mixture consisted of 20 mg membraneprotein and 30 mm histidine, 25 mm sucrose, 1 mm EDTA (pH7.5), 3 mm MgCl2, with or without 130 mm NaCl and 20 mmKCl in a volume of 0.4 mL; it was incubated at 37 8C for 5 min.The reaction was initiated by the addition of 1 mm ATP,incubated at 37 8C for 30 min and then terminated with 0.08 mLof 30% ice-cold trichloroacetic acid. The liberated Pi wasestimated colorimetrically as described by Sen et al. [17].Na+,K+-ATPase activity was measured as the difference inactivity between Mg2+/Na+/K+ and Mg2+ alone and was <90%sensitive to ouabain. The enzyme activity was measured inpresence of purified inhibitor protein from rat brain cytosolunder different conditions.

p-Nitrophenylphosphatase activity assay. The activity wasassayed according to Robinson [16] with slight modifications

as described by Sen et al. [17]. The assay mixture, in a volumeof 0.4 mL, contained 50 mm Tris/HCl (pH 7.5), 3 mm MgCl2,3 mm p-nitrophenyl phosphate with or without 20 mm KCl. Thereaction was initiated by the addition of the enzyme, incubatedfor 30 min and terminated with 0.4 mL of 1.5 m NaOH. Theabsorbance of liberated p-nitrophenol was measured spectro-photometrically at 410 nm. p-nitrophenyl phosphatase ( pNPPase)activity was calculated from the difference in activity betweenMg2+/K+ and Mg2+ alone. The enzyme activity was measuredunder different conditions in the presence of ouabain and theinhibitor.

I50 of the inhibitor

The concentration of inhibitor that caused 50% inhibition ofenzyme activity (I50 value) was determined by assaying theactivity of a fixed amount of enzyme (20 mg, specific activity230 ^ 37 mmol Pi´mg protein21´h21) with variable amounts ofthe inhibitor.

Nature of inhibition

Na+,K+-ATPase activity was measured in the presence ofdifferent concentrations of ATP at a fixed concentration(700 nm) of the inhibitor. The nature of inhibition wasdetermined from the Lineweaver±Burk plot of the velocityversus the substrate concentration.

In order to determine whether the inhibition was reversible orirreversible, enzyme-enriched membrane was incubated with theinhibitor protein for 30 min at 37 8C, diluted, centrifuged andassayed for enzyme activity according to Chandra et al. [10].

[3H]-Ouabain binding study

[3H]-Ouabain binding to Na+,K+-ATPase was measured accord-ing to Van Winkle et al. [18] and as described by Sen et al. [17].The standard medium contained 30 mm histidine/HCl/Tris(pH 7.5), 2 mm MgCl2, 50 mm NaCl, 1 mm EDTA and 1 mm[3H]-ouabain (1 mCi) with 2 mm ATP and 50 mg membraneprotein. Different concentrations of inhibitor protein were addedafter 15 min and aliquots were taken out at 5-min intervals.Aliquots were filtered through 0.45-mm membrane filters andwashed four times with 1 mL ice-cold water. The radioactivityretained on the filter was measured by liquid scintillationcounting.

Phosphorylated intermediate formation of Na+,K+ATPase

The EÄ P intermediate was produced using [g32P]ATP as substratefollowing the method of Post and Sen [19] in the absence of andin the presence of different concentrations of inhibitor protein.Radioactivity was determined by scintillation counting.

Tryptophan fluorescence study

Tryptophan fluorescence of Na+,K+-ATPase was recorded in aPerkin-Elmer spectrofluorimeter (model MPS 44B) between 290and 370 nm at an excitation wavelength of 280 nm in thepresence of inhibitor protein and ouabain.

Cd

The CD spectra of Na+,K+-ATPase and inhibitor protein weretaken either alone or in combination in a Jasco spectro-polarimeter (model 600) between 200 and 250 nm.

Fig. 1. Different stages of purification of inhibitor protein from rat brain

cytosol. Samples were resolved by 7.5% SDS/PAGE according to Laemmli

[14]. Lane 1, molecular mass standard (kDa); lane 2, crude cytosolic protein

(15 mg); lane 3, ammonium sulphate precipitate (10 mg); lane 4, DEAE

fraction (5 mg); lane 5, hydroxyapatite wash (0.5 mg). The gel was silver

stained according to Morrissey [15].

Table 1. Summary of purification of inhibitor protein from rat brain

cytosol. The crude cytosolic fraction was precipitated with 35% ammonium

sulphate and centrifuged at 15 000 g for 1 h. The fraction obtained from

35% ammonium sulphate precipitation was dialysed, loaded on to a DEAE±

cellulose column and the 0.2±0.24 m NaCl eluent further purified on a

hydroxyapatite column. The active fraction was eluted in buffer wash and

tested for inhibitory activity. The percentage inhibition of Na+,K+-ATPase

activity was calculated by taking the activity of the control membrane

without inhibitor as 100% (specific activity 225 ^ 30 mmol Pi´mg

protein21´h21). Enzyme protein (20 mg) was used in each case. Results

are shown as mean ^ SEM of three independent experiments.

Steps Total protein (mg)

Amount of protein

causing 50%

inhibition (mg)

Crude cytosol 110.0� 80 �^ 12

0±35% (NH4)2SO4 precipitation 18.5� ^ 4.4 102 �^ 14

DEAE±cellulose 3.9� ^ 1.2 53 �^ 7

Hydroxyapatite 0.056� ^ 0.011 21 �^ 3

86 K. Roy et al. (Eur. J. Biochem. 261) q FEBS 1999

Amino acid analysis

About 20 mg protein free from all ions was digested with 6 mHCl in a nitrogen atmosphere followed by derivatization withphenylisothiocyanate and run through a PICO TAG HPLCcolumn (amino acid analysis system, Millipore Corp., WatersChromatographic division). Peaks on the chromatogram wereidentified by comparing the retention time with known standardsobtained from Sigma.

IEF

The isoelectric pH of the inhibitor was determined on a 5%polyacrylamide gel using an Atto-IEF apparatus (model AE-3230) according to the manufacturer's instructions.

RESULTS AND DISCUSSION

The inhibitor protein was purified by ammonium sulphateprecipitation, DEAE anion-exchange chromatography andhydroxyapatite adsorption chromatography. The 35%(NH4)2SO4 precipitate showed decreased specific inhibitoryactivity on Na+,K+-ATPase (Table 1); this could be because thisstep excluded the low molecular weight (12±13 kDa) potentcytosolic ATPase inhibitor proteins reported previously [8]. The0.2±0.24 m NaCl eluent of DEAE±cellulose comprised the peakfractions of the inhibitor protein suggesting that it is acidic in

Fig. 2. Inhibition of Na+,K+-ATPase activity by different concentrations

of inhibitor protein. Enzyme activity was measured in the presence of

increasing concentrations of inhibitor protein. Activity in the absence of

inhibitor was taken as 100% (specific activity 230 ^ 37 mmol Pi´mg

protein21´h21; n = 4).

Fig. 3. Binding of ouabain vs. inhibitor. Ouabain-binding sites of Na+,K+-

ATPase were saturated for 15 min. Two concentrations of inhibitor protein,

0.25 mm (X), 1.0 mm (w) were then added at 15 min (arrow). The [3H]-

ouabain incorporated by the enzyme was measured by liquid scintillation

counting at 5 min intervals.

Table 2. Effect of ouabain and inhibitor on Na+,K+-ATPase and p-

nitrophenyl phosphatase activity. Na+,K+-ATPase and pNPPase activities

of rat brain microsomal membrane were determined in the presence of either

ouabain or inhibitor protein or both. Enzyme activities in the absence of

ouabain or inhibitor protein were taken as 100% (specific activity

232 ^ 30 mmol Pi´mg protein21´h21). Results are shown as mean ^ SEM

of three independent experiments.

Condition Activity (%)

Control (Na+,K+-ATPase) 100�

+ Ouabain (0.5 mm) 49� ^ 7.2

+ Inhibitor (0.7 mm) 52� ^ 10.3

+ Ouabain + inhibitor 5� ^ 2.4

Control ( pNPPase) 100�

+ Ouabain (0.25 mm) 47� ^ 10

+ Inhibitor (0.7 mm) 94�

+ Ouabain + inhibitor 49� ^ 8.1

Fig. 4. Formation of phosphorylated intermediate (E,P) of Na+,K+-

ATPase in the presence of inhibitor protein. Rat brain microsomal

membrane protein (50 mg) rich in Na+,K+-ATPase activity, was phosphory-

lated according to Post and Sen [19] in the absence and the presence of

different concentrations of inhibitor protein. Phosphorylation in the absence

of the inhibitor was taken as 100% (14 ^ 3 nmol´mg membrane protein21;

n = 3).

q FEBS 1999 Binding of Na+,K+-ATPase inhibitor from rat brain (Eur. J. Biochem. 261) 87

nature. The protein did not bind to hydroxyapatite (calciumphosphate) material at low ionic strength (10 mm sodiumphosphate buffer, pH 6.8) and was eluted with buffer washfractions (Table 1). The fraction showed a single protein band ofmolecular mass <75 kDa when subjected to SDS/PAGE on a7.5% gel (Fig. 1).

Na+,K+-ATPase activity measured in presence of differentconcentrations of inhibitor protein showed that 1.5 mm ofinhibitor led to <90% inhibition of enzyme activity with an I50

of 700 nm (Fig. 2). The high sensitivity of Na+,K+-ATPase tothe inhibitor is comparable to some of our recently reported data[8,10].

A Lineweaver±Burk plot of the ATPase activity in theabsence and presence of the inhibitor protein at differentconcentrations of ATP suggested that the inhibitor is competitivein nature with respect to ATP (data not shown), i.e. the inhibitorbinds either at the same site or at a position close to the ATPbinding site of the enzyme. Repeated dilution, centrifugationand washing of Na+,K+-ATPase-enriched membrane preincu-bated with the inhibitor, retained the ATPase activity (data notshown), suggesting that the binding of the inhibitor to theenzyme is reversible; this may be of physiological significance.

Measurement of the [3H]-ouabain binding of Na+,K+-ATPaseshowed that after 10 min incubation, the reaction reached aplateau and the affinity of ouabain towards ATPase remained

Fig. 5. Tryptophan fluorescence of Na+,K+-ATPase in the presence of inhibitor protein and ouabain. (A) Fluorescence of Na+,K+-ATPase in the presence of

inhibitor protein: 1, Enzyme only (15 mg); 2 and 3, enzyme in the presence of 20 mg and 40 mg inhibitor, respectively. (B) Fluorescence quenching of Na+,K+-

ATPase (25 mg) with increasing concentrations of ouabain: 1±6, 0, 1, 2, 3, 4 and 4.5 mm ouabain, respectively; 7, 4.5 mm ouabain and 40 mg inhibitor protein.

Table 3. Amino acid composition of the inhibitor protein. Protein

(<20 mg), free from all ions, was digested with 6 m HCl in a nitrogen

atmosphere followed by derivatization with phenylisothiocyanate and run

through a PICO TAG HPLC column. The experiment was repeated twice.

Composition (%) represents the approximate number of respective amino

acid per 100 amino acid residues of the protein molecule.

Amino acid Composition (%)

Asp 4�.71

Glu 13�.59

Ser 3�.91

Gly 7�.82

His 3�.35

Arg ND

Thr 2�.60

Ala 10�.42

Pro 9�.68

Tyr 2�.60

Val 7�.82

Met 1�.30

Cys 0�.74

Ile 1�.86

Leu 14�.15

Phe 5�.21

Lys 10�.24

Trp ND

ND, Not detected.

88 K. Roy et al. (Eur. J. Biochem. 261) q FEBS 1999

unchanged even after the addition of varying concentrations ofinhibitor (Fig. 3). This suggests that the inhibitor probably bindsto a site distinct from the ouabain binding site. This is supportedfurther by the experiment that showed an additive effect ofouabain and the inhibitor towards ATPase inhibition (Table 2).

Again, ouabain is believed to bind to an E2 state of theenzyme and inhibits both Na+,K+-ATPase and p-nitrophenylpho-sphatase ( pNPPase) activities [20,21]. The inhibitor proteinhowever, was unable to block pNPPase activity (Table 2)predicting that the inhibitor binds to a different state of theenzyme. Formation of the phosphorylated intermediate (E1,P)of Na+,K+-ATPase is inhibited with increasing concentrations ofthe inhibitor (Fig. 4). This observation clearly suggests thatinhibition at the E1 state of the enzyme molecule is responsiblefor the decreased enzyme activity in the presence of the inhibitorprotein.

Conformational changes often lead to enzyme inactivation.Thus, trytophan fluorescence of Na+,K+-ATPase was measured,either alone or in combination with the inhibitor, to detect anystructural change of the enzyme molecule. The results of thisstudy suggested that the inhibitor protein is devoid of Trpresidues, but that Na+,K+-ATPase shows enhanced fluorescenceafter binding the inhibitor (Fig. 5A) probably due to exposure ofsome hidden Trp moieties of the enzyme molecule. Ouabaincauses quenching (Fig. 5B). CD spectra suggested a slight lossof a-helix and random coil in the enzyme±inhibitor complexwhich is probably compensated for by other secondarystructures such as b-sheet or turn, or a combination of both(data not shown).

Amino acid analysis data (Table 3) showed that in the protein,aspartate and glutamate residues constitute 18%, lysine accountsfor 10%, and alanine, proline and leucine also represent aconsiderable amount (<10±15%). The 75-kDa protein moleculeis rich in acidic amino acid residues as substantiated by theisoelectric pH of <4.6 (calculated by IEF).

The data presented here suggest the presence of anendogenous inhibitor of Na+,K+-ATPase; this may be ofphysiological significance, especially in the light of thereversible nature of the binding process.

Amino acid sequence analysis and antibody experiments forfurther characterization of the inhibitor protein are in progress inour laboratory.

ACKNOWLEDGEMENTS

We are thankful to P. K. Ray (Director, Bose Institute) for his active interest

and providing facilities for this work. Thanks to K. P. Das of our Department

for fruitful discussion in interpreting the CD data. This work is supported in

part by grants from Council of Scientific and Industrial Research,

Government of India (37/0845/94-EMR-II and 37/0942/97-EMR-II) and

financial help from Bose Institute.

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