Mercury weakens membrane anchoring of Na-K-ATPase

E. IMESCH, M. MOOSMAYER, AND B. M. ANNEEDepartment of Pharmacology, Geneua University Médical Center, CH-1211 Geneva 4, Switzerland

Imesch, E., M. Moosmayer, and B. M. Anner. Mercuryweakens membrane anchoring of Na-K-ATPase. Am. J.Physiol. 262 (Rénal Fluid Electrolyte Physiol. 31): F837-F842,1992.—The présence of circulating inhibitors able to decreasethé rénal Na-K-adenosinetriphosphatase (ATPase) activity(natriuretic hormones) was postulated some 30 years ago. In théprésent work, thé natriuretic inhibitor HgCl2 was selected as amodel compound for thé structural characterization of a possi-ble natriuretic pathway for Na-K-ATPase modification. Thestructural effects of Na-K-ATPase inhibition by HgCl2 wereassessed by trypsinolysis of thé blocked enzyme in comparisonwith untreated préparations. The results show that inactîvationof Na-K-ATPase by HgCl2 leads to thé release of thé a-subunitfrom thé membrane preferentially in thé E2 conformation butalso in thé E! conformation. Apparently, HgCl2 weakens thémembrane anchoring of thé a-subunit, presumably by loosenîngthé a-|S-subunit interaction. By this mechanism, thé sensitivityof thé Na-K-ATPase to extracellular drugs, hormones, and anti-bodies, as well as to intracellular proteases and other regulatoryfactors, could be altered.

mercury(II)-modified sodium-potassium-adenosinetriphospha-tase; enhanced a-subunit trypsinolysis; reversai by dimer-captopropanesulfonic acid; weakened membrane anchoring;leak formation

THE SODIUM-POTASSIUM-TRANSPORTING adenosinet-riphosphatase (Na-K-ATPase, EC 3.6.1.37) is a well-known ubiquitous membrane System composed of acatalytic a-subunit of ~110 kDa and a /3-subunit glyco-protein of —50-60 kDa (21). The System performs elec-trogenic Na-K antiport (9) and ATP hydrolysis associ-ated with an autophosphorylation/dephosphoryla-tion step. The Na-K-ATPase molécule also carries théreceptor for carch'oactive steroids; thé binding of thèsecompounds to thé receptor results in thé inhibition of ailATPase and ion pump activity (27).

The molecular mechanism of thé Na-K antiportinvolves a crucial conformational E!/E2 change that canbe visualized directly by trypsinolysis (16). Various com-binations of pump ligands stabilize either thé Et or théE2 conformation (17). Distinct trypsin-sensitive bondsare then exposed according to whether thé enzyme is inthé E! or in thé E2 conformation (16). The action ofspécifie Na-K-ATPase inhibitors has its roots in ligandmimicry, i.e., copying thé action of pump ligands. Theonly spécifie inhibitors known so far, thé cardioactivesteroids, imitate thé action of K in several respects butremain bound to thé enzyme much longer and by thismechanism block thé turnover cycle (27). Similarly, thémolecular action mechanism of vanadate is shared byphosphate except for thé virtually irréversible binding ofvanadate, which leads to inhibition of active transport(6, 18). That thèse pump inhibitors act by mimickingpump ligands, but are poorly réversible, explains théinterruption of thé pump cycle by stabilization of a spé-cifie enzyme conformation.

Mercury compounds, on thé other hand, are unspe-cific Na-K-ATPase inhibitors because thèse are knownto interact generally with sulfhydryl groups of proteins

(22). Hence, thé molecular action mechanism of Hg(II)is expected to be entirely différent from cardioactivesteroids. Furthermore, their pharmacological effects aredistinct from cardioactive steroids, because in vivo theyare essentially powerful diuretics interacting with an asyet unknown rénal receptor protein (10, 23, 24, 29).

The experiments of thé présent work were designed toexamine thé structural changes associated with Na-K-ATPase inhibition by HgCl2. Comparative proteolysisrevealed greatly enhanced trypsin sensitivity of théa-subunit, leading to release of thé protein from thémembrane. Such a profound change in thé folding ormembrane embedding of thé Na-K-ATPase moléculeprésents an entirely new molecular mechanism for Na-K-ATPase modification, which to our knowledge hasnot yet been described elsewhere. The metal-inducedstructural modification of thé Na-K-ATPase could notonly alter its sensitivity to intra- or extracellular regu-latory compounds but could also contribute crucially toaccelerated internalization and digestion by cellularproteases.

EXPERIMENTAL PROCEDURES

Materials. EDTA (Titriplex II), acrylamide, ammonium per-sulfate, glycérine, tris(hydroxymethyl)aminomethane (Tris)and glycine were purchased from Merck; Tris-EDTA wasprepared by neutralization of EDTA by Tris. HgCl2, N,N'-methylene-bis-acrylamide, and 2-mercaptoethanol were fromFluka. 2,3-Dimercapto-l-propanesulfonic acid (DMPS); chy-motrypsin-free (N-tosyl-L-phenylalanine chloromethyl ketonetreated), dialyzed, lyophilized, and salt-free trypsin from bovinepancréas; and trypsin-chymotrypsin inhibitor (Bowman-Birk)were purchased from Sigma Chemical. Coomassie blue (brilliantblue R 250) and thé lithium dodecyl sulfate (LDS) solution(917.77 mM or 25% wt/vol) were obtained from Serva. Phos-phatidylcholine (grade I) and phosphatidylserine were fromLipid Products, Nutfield, UK. 8<;RbCl was from New EnglandNuclear. AWA^W-tetramethylethylenediamine (TEMED)and molecular mass protein standards (14.4 kDa, lysozyme; 21.5kDa, soybean trypsin inhibitor; 31 kDa, bovine carbonic anhy-drase; 45 kDa, hen egg white ovalbumin; 66.2 kDa, bovine sérumalbumin; and 97.4 kDa, rabbit muscle phosphorylase B) werefrom Bio-Rad. Ail chemicals were of thé highest purity avail-able; only bidistilled water was used. The HgCl2 solutions werefreshly prepared. The Pierce bicinchoninic acid assay (Rock-ford, IL) was used for protein détermination.

Purification of Na-K-ATPase. A slightly modified version ofthé dodecyl sulfate extraction procédure (8) was used forenzyme purification of thé rénal outer medulla of rabbit kidneysasfollows: 11.2 mg of microsomal protein were incubated in 7 mlof a solution containing (in mM) 3 disodium ATP, 25 imidazole,1 Tris-EDTA, and 2.5 LDS for 20 min at 25°C. The solutionwas then put on 4 ml of 15% sucrose, 16 ml 25% sucrose, 25 mMimidazole, and 1 mM Tris-EDTA (EDTA neutralized withTris), pH 7.5, 0°C, and centrifuged for 110 min at 250,000 g.The pellet was suspended in 0.5 ml of 1% sucrose, 25 mMimidazole, and 1 mM Tris-EDTA {pH 7.2) and stored at -70°C.The spécifie enzyme activities were between 25 and 35 U/min.

Na-K'ATPase activity. The linked enzyme assay was usedfor thé continuous détermination of Na-K-ATPase activity as

0363-6127/92 $2.00 Copyright © 1992 thé American Physiological Society F837

F838 MEMBRANE ANCHORING OF NA-K-ATPASE WEAKENED BY

follows: 0.5-2 ng Na-K-ATPase protein was added to 1 ml of asolution containing (in mM) 0.3 NADH, 2.5 phosphoerao/pyru-vate (cyclohexamine)-Tris, 30 imidazole, 1 Tris-EDTA, 2.5ATP, 5 MgCl2, 100 NaCl, and 10 KC1, as well as 8 ^1 pyruvatekinase/lactate dehydrogenase, pH 7.2, at 37°C, and thé oxîda-tion rate of NADH was recorded by thé absorbance decrease at340-nm wavelength.

Trypsinolysis of Na-K-ATPase. The conformation-spécifietrypsinolysis was performed according to a basic procédureestablished by J0rgensen (16). To 10 jil of solution containing 10fig Na-K-ATPase protein, 0.4 jxg trypsin was added to yield afinal trypsin/Na-K-ATPase ratio of 1:25 (wt/wt). The solutioncontained 5 mM MgCl2,1 mM Tris-EDTA, 30 mM imidazole orhistidine (pH 7.4), 20°C, and NaCl, KC1, or ATP as indicated inthé legends to Figs. 1-5. Trypsinolysis was stopped by thé addi-tion of 1.6 Mg trypsin inhibitor.

Gel electrophoresis, To 5 ^1 of solution containing 5 Mg Na-K-ATPase protein, 5 /J of 125 mM Tris (pH 6.8 with HC1), 3 M(20% vol/vol) glycerol, 147 mM (4% wt/vol) LDS, and 574 mM(4% vol/vol) 2-mercaptoethanol were added; thé solution wasincubated for 30 min at 60°C and then transferred to 80 X 70 X0.75-mm gels in a Hoefer SE 250 vertical slab unit. The gelswere formed in a solution of 30% polyacrylamide (29.2 g acry-lamide plus 0.8 g bis-acrylamide in 100 ml) diluted to 6 or 15%(14% sucrose was added to thé 15% polyacrylamide solution) ina solution of 400 mM Tris (pH 8.8), 3.4 mM (1% wt/vol) LDS,3.1 mM (0.05% vol/vol) TEMED, and 2.2 mM (0.05% wt/vol)ammonium persulfate; 6-15% gradients were formed in a gra-dient mixer. Electrophoresis was performed in 25 mM Tris(pH 8.3), 192 mM glycine, and 3.4 mM (1% wt/vol) LDS for75 min with ~20 mA/gel. The gels were then colored for 60 minin 0.6 mM Coomassie blue that had been dissolved inH20:methanol:acetic acid (45:45:10) and filtered. Décolorationwas performed overnight at room température inH20:methanol:acetic acid (87.5:5:7.5). Background colorationwas removed by incubation in methanol for 5 min. The gels werereincubated in destaining solution until they took their originaldimension, and then thèse were sealed into plastic sheets andscanned in a laser densitometer (LKB Ultrascan XL).

The apparent molecular masses of thé Na-K-ATPase a- and/3-subunits were estimated by comparing their migration dis-tance to molecular mass standards (14.1-200 kDa) and werefound to be between 94 and 98 kDa for thé a-subunit andbetween 50 and 60 kDa for thé 0-subunit. The préparationcontained at least 90% Na-K-ATPase protein.

Na-K-ATPase reconstitution. The purified Na-K-ATPase(180 Mg protein) was suspended in 60 n\f a solution containing(in mM) 30 histidine, 1 Tris-EDTA, 5 MgCl2,50 NaCl, 50 RbCl,and 23 cholic acid, pH 7.2, 0°C. The supernatant resulting froma 10-min centrifugation at 100,000 g was added to 50 //l ofsolution containing (mM) 5 MgCl2, 30 histidine, 1 Tris-EDTA,and 23 cholic acid, as well as 0.8 mg phosphatidylcholine and 0.2mg phosphatidylserine, pH 7.2, 0°C, according to previouslypublished procédures (5). Liposomes containing functional Na-K-ATPase were formed by 15-h dialysis at 0°C in 250 ml of asolution containing (in mM) 50 NaCl, 50 RbCl, 5 MgCl2, 1Tris-EDTA, and 30 histidine, pH 7.2. The liposomes contained—0.5 mg protein/ml. The passive 8fiRb uptake was measured bythé addition of external 86Rb and by détermination of théintraliposomal 86Rb by gel filtration on 1 x 15-cm Sephadexmédium columns at 0°C as previously described (5).

RESULTS

No cross-iink formation by Hg(II). Cross-link forma-tion between a- and /3-subunits was expected in thé prés-ence of high Hg(II) concentrations due to intersubunitHg(II)-disulfide bridges. Yet, incubation of Na-K-AT-

Pase in thé présence of HgCI2 at concentrations up to 5mM did not influence thé electrophoretic migration pat-tern of thé a- and /3-subunits and no a-a, a-fi, or /3-/3complexes appeared (Fig. 1). Thus HgCl2 does not cross-iink thé Na-K-ATPase subunits, at least not irreversibly,because thé 574 mM 2-mercaptoethanol added for proteinréduction before electrophoresis could hâve dissociatedputative cross -links.

For convenience of thé gel electrophoresis experiments,which required high protein concentrations, 0.1-1 mgNa-K-ATPase protein/ml was incubated with HgCl2.Because thé Hg(II) potency dépends on thé protein con-centration, dose-effect curves of Hg(II) inhibition wereperformed at thèse high protein concentrations (Fig. 2).Clearly, thé curves are shifted to thé right compared withthé 200 nM value of half-maximal inhibitory concentra-tion for HgCl2 inhibition observed at high Na-K-ATPaseprotein dilutions (3). Interestingly, a stimulatory phasepreceded thé inhibition in both conditions, thé mecha-nism of which was not further investigated.

Does mercury change Na-K-ATPase conformation? Toevaluate whether thé interaction of HgCl2 with Na-K-ATPase induced a structural change, thé enzyme waspretreated with increasing concentrations of mercury,and thé conformations were assessed by h'mited trypsintreatment in a condition that stabilizes either thé E:

(Fig. 3A) or thé E2 conformation (Fig. 3B). The resultsshow that Hg(II) pretreatment of Na-K-ATPase at con-centrations up to 10 AiM HgCl2 did not modify thé Ej or

Na,K- ATPase activity

(U/min)2 6 1 0 0 0

P

0 0.1 0.5 1 5

Fig. 1. Absence of cross-iink formation by HgCl2. Rabbit kidney Na-K-ATPase (2.5 U/min) was incubated at 10 Mg protein/10 M! for 30 min at37°C in a solution containing (in mM) 100 NaCl, 25 imidazole, 1 Tris-EDTA, pH 7.4, and 0.1-5 HgCl2. Enzyme activity was determined in a5-fil aliquot. Remaining 5-^tl samples were processed for electrophoresisas described in EXPERIMENTAL PROCEDURES, a and &, a- andQ- subunits.

MEMBRANE ANCHORING OF NA-K-ATPASE WEAKENED BY HcCi-, F839

140

O 120

u 100

Fig. 2. Mercury inhibition curves at high Na-K-ATPase proteinconcentrations. Increasing HgCl2 concentrations were added to 1 or 10Mg Na-K-ATPase protein/10 p\n containing (in mM) 100 NaCl,5 MgCla, 30 imidazole, and 1 EDTA, pH 7.4, and incubated for 30 minat 37°C. Suspension was then added to 1 ml of solution in a cuvettecontaining solution for Na-K-ATPase measurements by thé linkedenzyme assay as described in EXPERIMENTAL PROCEDURES.

thé E2 conformation; in thé présence of Na and ATP (acondition that stabilizes thé E! conformation), théE!-spécifie fragment migrating between thé a- and thé/3-subunit appeared independently of whether thé enzymehad been pretreated with HgCl2 (10 nM to 10 /iM). Like-wise, pretreatment of thé enzyme by HgCl2 in thé E2

conformation (in thé présence of K only) did also notmodify thé proteolysis pattern, because thé E2-specificfragments appeared unchanged on thé gels (Fig. 3S). Inthis condition (below 10 ^M Hg) thé enzyme turnoverappeared increased (Fig. 2). Although thé mechanism ofthé Hg(II)-stimulated ATPase activity was not furtherinvestigated, a trypsin-sensitive conformational changecan at least be excluded by thé results of Fig. 3.

Mercury weakens membrane anchoring of Na-K-ATPase. By contrast, if a fully inhibitory mercury con-centration of 100 AiM HgCl2 was used for Na-K-ATPasepretreatment, then thé a-subunit became highly trypsinsensitive and was totally digested in thé E: as well as inthé E2 conformation (Fig. 3, A and B). Control experi-ments hâve ruled out an effect of HgCl2 on thé trypsinactivity, because similar trypsinolysis results wereobtained when HgCl2 was removed in thé supernatantafter centrifugation or by chelating thé Hg(II) withDMPS before trypsin treatment, procédures that wereused for other experiments shown herein (Fig. 4). Thusthé results obtained at 100 /iM HgCl2 (Fig. 3, last lanes)clearly show that Hg(II) inhibition of Na-K-ATPase ren-ders thé a-subunit highly trypsin sensitive. Probably, théprotein folding is altered in a way that renders thétrypsin-sensitive régions more accessible to thé protease.Alternatively, thé Hg(II) treatment may weaken thémembrane embedding of thé a-protein and expose,thereby, more peptide bonds to trypsin.

Identicai structural effects at tow and high Hg(II). Asillustrated by thé dose-effect curves of Fig. 2, thé mercurypotency at 1 mg Na-K-ATPase protein was greatly

A

Tryps. - + - + + + +

^ , *- ••* • --

0 0 100 0.01 0.1 1 10 100

[HgCl2],

B

Tryps. - + -+ + + + +

P

0 0 100 0.01 0.1 1 10 100

[HgCl2].Fig. 3. Enhanced trypsinolysis of Na-K-ATPase a-subunit pretreatedby mercury at 1 mg protein/ml. Na-K-ATPase (10 wg protein/10 ^1) wasincubated either in a solution stabilizing thé E! conformation [A: con-taining (in mM) 50 NaCl, 50 KC1, 5 MgCl,, 5 ATP, 30 hîstidine, and 1Tris-EDTA, pH 7.2] or in a solution stabilizing thé E2 conformation (B:100 mM TA, pH 7.2, for 30 min at 37°C) with HgCI2 concentrationsranging from 10 nM to 100 ̂ M. Trypsinolysis (30 min at 20°C) and gelelectrophoresis were performed as described in EXPERIMENTALPROCEDURES.

reduced, and HgCl2 concentrations above 50 jiM wererequired to obtain full ATPase inhibition. The highHgCl2 concentration per se could hâve been responsiblefor thé trypsin hypersensltivity of thé a-subunit. To testwhether inhibition of Na-K-ATPase by lower HgCl2 con-centrations also enhanced thé trypsin sensitivity of théa-subunit so drastically, thé protein concentration wasreduced 10-fold to be able to block thé Na-K-ATPaseactivity by lower HgCl2 concentrations. The enzyme pro-tein (100 ng/ml) was incubated with 4 ^M HgCl2 in théprésence of only Na ions (Ej conformation) or only K

F840 MEMBRANE ANCHORING 0F NA-K-ATPASE WEAKENED BY HcCL,

0-6

Distance from front (cm)Fig. 4. Enhanced trypsinolysis of Na-K-ATPase a-subunit pretreatedby mercury at 0.1 mg protein/ml. Na-K-ATPase protein (1 jjg/10 n\)was incubated in a solution containing (in mM) 5 MgCla, 25 imidazole,1 Tris-EDTA, pH 7.4, and either 100 mM NaCl to stabilize thé E,conformation or 100 mM KCI to stabilize thé E2 conformation. To bothsolutions 4 pM HgCl2 was added, and thé incubation continued until théenzyme activity was entirely blocked (180 min in NaCl, 30 min in KC1).thé inactivated préparations were concentrated by centrifugation andresuspended at 10 ^g protein/10 jtl. Trypsinolysis (15 min at 20°C),electrophoresis, and laser densitometry were performed as described inEXPERIMENTAL PROCEDURES. The following densitometric scans areshown: no trypsin added in NaCl or KCI with or without HgCl2 (A);trypsinolysis in NaCl with or without HgCl2 (fi); and trypsinolysis inKCI with or without HgCl2 (C).

ions {E2 conformation) until total blockage of ATPaseactivity was reached.

The two enzyme préparations inhibited by HgCl2,either in thé Na or thé K médium, were concentrated, andthé free Hg(II) was removed by centrifugation. The prép-arations were then subjected to trypsin treatment, gelelectrophoresis, and comparative densitometric analysis.In thé absence of trypsin, thé densitometric scans showedthé normal migration pattern of thé a- and /3-subunits(Fig. 4A). Trypsinolysis of thé E1 conformation withoutHg(II) generated thé typical fragment appearing betweenthé a- and /3-subunit (Fig. 4B). However, when théenzyme had been treated by Hg(II) before trypsinolysis,thé a-subunit was totally absorbed; thé surface of thé0-subunit was only slightly decreased. New tryptic frag-ments appeared between thé front and thé /3-subunit,indicating that additional trypsin-sensitive bonds of théa-subunit were exposed by mercury (Fig. 45). The E2

conformation seemed to undergo even more profoundstructural changes in thé présence of HgCl2l because thé

a-subunit was entirely absorbed and thé /3-subunit wasalmost fully absorbed as well. Thus thé /3-subunit appearsto be more affected by mercury in thé E2 conformation.Only ~40% protein remained on thé gel after enzymeinactivation by Hg(II) in thé E2 conformation, indicatingthat numerous water-soluble low-molecular-weight frag-ments had been formed and released from thé gel.

In conclusion, inactivation of concentrated Na-K-ATPase protein (1 mg/ml) by a high HgCl2 concentration(100 t*M) produced thé same enhanced trypsin sensitivityof thé enzyme as inactivation of more diluted protein (0.1mg/ml) by 4 ^M HgCl2. Thus thé final form of Hg(II)-inactivated Na-K-ATPase is altered similarly regardlessof thé Hg(II) concentrations used for inactivation.

Normalization of enhanced trypsin sensitivity byDMPS. The inhibition of thé Na-K-ATPase activity bymercury can be reversed by treatment with (DMPS) (3).Is thé greatly enhanced trypsin sensitivity caused byHgCl2 pretreatment also reversed by DMPS, i.e., arestructural and functional changes related? If this were thécase, thé amount of trypsin-résistant a-subunit would beexpected to increase parallel with thé enzyme activity. Totest this prédiction experimentally, thé enzyme activitywas decreased to 21% of control by a 30-min incubationof 0.5 mg protein/1 ml with 10 nM HgCl2. The reactionwas stopped by thé addition of 5 mM DMPS, and théprotein was subjected to trypsin treatment. The amountof a-subunit résistant to trypsin was decreased to 47%compared with thé control experiment. The addition of 5mM DMPS for 120 min restored thé enzyme activity to87%, and thé trypsin sensitivity was back to normal.Thus thé réactivation of Hg(II)-inactivated Na-K-ATPase activity by DMPS was accompanied by restora-tion of thé trypsin sensitivity. When thé enzyme activityfell below 20%, DMPS was no longer able to reactivatethé enzyme activity, indicating that some irréversibleprotein altération had occurred (data not shown).

Leak formation. Is thé enhanced trypsin sensitivity ofNa-K-ATPase caused by mercury accompanied by achange in thé permeability of thé pump molécule? Tovisualize thé permeability of thé purified Na-K-ATPasemolécule, thé enzyme must be incorporated in artificielphospholipid vesicles or liposomes (4,13,15). In fact, thépurified, unreconstituted Na-K-ATPase consista of lipidbilayer fragments originating from thé basolateral kidneytubule membrane containing numerous aggregated Na-K-ATPase molécules; this préparation displays ouabain-sensitive Na-K-ATPase activity in thé présence of Mg,Na, K, and ATP but is not able to establish transmem-brane Na-K gradients, because there is no closed com-partment for ion accumulation. Therefore thé purifiedNa-K-ATPase molécules must be incorporated into anartificial vesicular membrane or liposomes.

It has been shown previously that thé pure lipid bilayeris virtually imperméable to 86Rb ions and that thé intactNa-K-ATPase molécule créâtes a 86Rb-selective perme-ability pathway (2). In thé présent work, thé 86Rb per-meability of thé intact Na-K-ATPase molécule was deter-mined by adding external 86Rb to thé liposomes andmeasuring its uptake kinetics. The typical Na-K-ATPase-induced passive 86Rb uptake was seen (Fig. 5).

MEMBRANE ANCHORING OF NA-K-ATPASE WEAKENED BY HcCL, F841

By contrast, when 100 ^M HgCl2 was présent, théuptake by thé liposomes was greatly reduced and reachedonly —20% of control (Fig. 5). To confirm that thé miss-ing 86Rb uptake was due to an 80% leak component (lead-ing to loss of 86Rb during gel filtration of thé liposomesfor external 86Rb removal) and not to turnover arrest ofNa-K-ATPase, HgCl2 was also added when 86Rb hasalready entered thé liposomes; —60% of thé entrapped86Rb leaked out in this condition (Fig. 5, inset), indicatingthat mercury did indeed open a leak pathway across théNa-K-ATPase molécule. When 1 mM ouabain was addedinstead of mercury in thé same condition, no leakappeared within 60 min (data not shown). The création ofa leak pathway by high concentrations of mercury, butnot by ouabain, represents another useful test for thédifferentiation of ouabain-like and mercury-like Na-K-ATPase inhibitors.

DISCUSSION

Mercury releases a-subunit from its membrane anchor.The présent report shows that thé inhibition of thé Na-K-ATPase activity by HgCl2 is associated with greatlyenhanced trypsin sensitivity of thé a-subunit. TheHg(II)-treated a-subunit is rapidly degraded to low-mo-lecular-weight peptides or amino acids no longer visibleon thé gels in conditions that leave a large fraction of théuntreated a-subunit intact. Conversely, thé trypsin sen-sitivity of thé /3-subunit is less drastically increased bymercury treatment. The extensive digestion of thé a-pro-tein by trypsin suggests that Hg(II) releases thé a-sub-units from their membrane anchor. The /3-subunit seems

15-r1

Time , h

Fig. 5. Leak formation by mercury inhibition of Na-K-ATPase activity.Na-K-ATPase purified from rabbit kidney outer medulla was reconsti-tuted into liposomes; 100 jtM HgCl2 was added to a part of thé lipo-somes, and Na-K-ATPase inhibition was ascertained. External 8fiRbwas added to both control and Hg(II)-treated liposomes, and 8(JRbuptake by liposomes was measured in parallel and expressed as a frac-tion of total ^Rb added (cpmlol ). HgCl2 was added also 5 h after 86Rbaddition, to verify that decreased uptake was indeed due to a leak(inset). Techniques used for liposome préparation and radioflux mea-surements are described in EXPERIMENTAL PROCEDURES. Liposomepréparation contained —0.3 mg protein/ml; epin™, radioactivity withinliposomes in counts/min.

to be thé membrane anchor of thé a-subunit (1, 25).Hence, Hg(II) could weaken thé interaction between théa-subunit and thé /3-subunit and thereby facilitate thérelease of Na-K-ATPase protein from thé lipid mem-brane with a préférence for thé E2 conformation.

Mercury-inactivated Na-K-ATPase forms a leakpathway. The structural altération caused by mercury isassociated with thé blockage of thé Na-K-ATPaseactivity. Loosening of a- and /3-subunit interaction is amechanism postulated in thé présent work for thé struc-tural Hg(II) effects. In conséquence, thé leak pathwaycreated by Hg(II) might be associated with thé weakeneda- and /3-subunit interaction. Apparently, a strong sub-unit interaction contributes to thé tightness of thé ionpassage across thé pump molécule, i.e., prévention ofbackleak.

Possible nature of metal-binding interface. Analogousto other Hg(II)-binding proteins (22, 26), it is likely thatHg(II) interacts with sulfhydryl groups of thé Na-K-AT-Pase molécule. Analysis of thé primary structure of Na-K-ATPase reveals ~23 cysteine residues on thé a-sub-unit and 7 on thé /3-subunit; titration experiments showthat only 1-4 sulfhydryl groups per a-j3 complex are freelyaccessible (12). The profound structural altérationinduced by Hg(II) binding may be due to perturbation ofthiol or disulfide groups that normally stabilize thé ter-tiary or quaternary enzyme conformation. Three disulfidebonds réside in thé /3-subunit (20), and one of thoseappears to be essential for enzyme activity, an interestingfinding, which for thé first time indicates direct func-tional involvement of thé /3-subunit in thé turnover cycle(19).

The characteristics of thé mercury-Na-K-ATPaseinteraction are reminiscent of thé mercury-resistancemetalloregulatory protein, which is, for instance, sensi-tive to HgCl2 concentrations of 1 X 10~B M despite théprésence of 1 mM dithiothreitol and is résistant to Cd(II)and Zn(II) (26). The mercury-resistance metalloregula-tory protein binds mercury as a rare tricoordinate com-plex, which can form metal-bridged dimers (14). Thusmetal-binding interfaces of macromolecules can promotethé dimerization of proteins (11), thé interaction betweenligands and receptors (28), and thé interaction betweenproteins and nucleic acids (7). Whether thé metal-bind-ing interface of Na-K-ATPase fulfills such objectives isnot yet known.

We thank Frank West and Jacqueline West for assistance with thémanuscript and Fred Pillonel for thé artwork.

This work was supported by Swiss National Science FoundationGrant 31-25666.88.

Address reprint requests to B. M. Anner.

Received 31 May 1991; accepted in final form 10 December 1991.

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F842 MEMBRANE ANCHORING OF NA-K-ATPASE WEAKENED BY HcCL2

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