Journal of Biochemical and Biophysical Methods, 5 < 1981) 299-306 299Elsevier/North-Holland Biomédical Press

Préparation of Na,K-ATPase-containing liposomeswith predictable transport properties by a procédurerelating thé Na,K-transport capacity to thé ATPase

activity

Béatrice M. Anner and Marlis Moosmayer

Deparmettt of Pharmacotogy, Centre Médical Universitaire, Cl 1-12H (ieneva 4, Switzet-land

(Accepted 7 Scptemher 1981)

SUMMARY

A mieroprocedure for thé préparation of Na.K-ATPase-containing, liposomes with a minimal start ingmaterial (200 /ig) of purificd Na,K-ATPa,se is présentée!. Phosphatidylcholine is addcd gradually tocholate-solubilizcd Na.K-ATPase of various concentrations and thé lipid-induced decrcase in enzymeactivity is monitorcd. After removal of thé détergent hy dialysis, thc Iransport paramciers of thé resultingNa.K-ATPasc-liposomes are established by a microassay. By relating thé transport properties to théNa,K-ATPase activity présent beforc dialysis, a procédure is devclopcd which allows to prépare standar-dizcd Na.K-ATPase-liposomcs with predictable transport properties.

Key words: Na.K-ATPase-containing liposomes; microprocedure; prcdictahle transport properties;standardization.

Incorporation of active soluble Na,K-ATPase (EC 3.6.3.1) in liposome mem-branes restores thé transport function of thé isolated enzyme. Such reconstitutedNa,K-ATPase-liposomes offer a new tool for studying thé mechanism of thé puretransport System and thé properties of 90 nm [1] high Na, low K vesicles.

Although several investigators hâve been workîng with thé reconstituted enzyme[2-6], a rigorous method to prépare Na,K-ATPase-liposomes has not yet beendeveloped. The présent report fills in this gap with a step-hy-step procédure forpréparation of standardized N a, K-ATPase-liposomes. The method makes possible tochoose a suitable transport capacity by adjusting thé soluble Na,K-ATPase activitybefore thé vesicles are formed. Importantly, thé procédure allows to perform anextensive transport analysis with a minimal quanti ty of 200 jug purified Na,K-ATPase.

OI65-022X/8I/0000-0000/S02.50 tf> 1981 Elsevier/North-Holland Biomédical Press

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MATERIAL AND METHODS

Sodium cholate, salts and buffers were analytical grade products from Merck;phosphatidylcholine type V-E was purchased from Sigma, and NADH, phos-phoenolpyruvate and pyruvate kinase/lactate dehydrogenase were from Boehringer.Carrier-free 22NaCl and 86RbCl were from thé Radiochemical Center, Amersham.Double-distilled water was used for ail solutions. Purified membrane-bound Na,K-ATPase of a spécifie activity of 1500 to 1700 jumol/mg/h was prepared from rabbitkidneys according to J^rgensen [7] and was stored at — 25°C for several weeks at aconcentration of 2.5 mg/ml in 25 mM imidazole/29 mM sucrose/1 mM Tris-EDTA,pH 7.50.

Step}; Cholate-solubilizaiion of Na,K-A TPase: Soluble active Na,K-ATPase wasprepared by suspending 200-1200 jug of Na,K-ATPase protein ( l O O O O O X g pellets)in 200 jtl of ice-cold 'transport' solution (0-100 mM NaCl/0-100 mM KC1/50 mMcholine chloride/30 mM imidazole/5 mM MgCl.,/1 mM EDTA/1 mM cysteinechloride, pH 7.10) and 24 mM sodium cholate. The suspension was transferred to a1 ml glass homogenizer fitted with a Teflon pestle and was homogenized at 150rev./min for 15 s. The suspension was then centrifuged for 15 min at 100000 X g (30lb/inch2) in a Beckman Aîrfuge installed in a 4°C coldroom. The clear supernatantwas removed and tested for Na,K-ATPase activity by adding 2-5 /il to a 1 mlcuvette filled with 1 ml of a solution containing 100 mM NaCl, 10 mM KO, 5 mMMgCl2, 2.5 mM Tris-ATP, 2.0 mM phosphoenolpyruvate. 25 mM histidine, 8julpyruvate kinase/lactate dehydrogenase, pH 7.2, and 4ju l of a 60 mg NADH/mlsolution to yield an initial absorbance of 1.8, and thé ADP production wascontinuously measured [8] at 37°C. A 10-20 \i\e of thé supernatant was usedfor determinaton of protein by thé method of Lowry et al. [9] and to establish théprotein profile of thé soluble Na,K.-ATPase by gel electrophoresis in thé présence ofsodium dodecyl sulfate [10].

Step 2: Graded addition of phosphatidylcholine: A phosphatidylcholine/cholatesolution was prepared by drying 100 mg lipid under a stream of ultrapure N, in arotavapor as previously described [6] and dissolving thé thin film formed in 5 ml of'transport' solution containing 24 mM sodium cholate. The resulting solution wasdîvided in convenient portions and was stored under N2 at — 25°C for several weeks.To 5-/nl aliquots of supernatant, 1-10 /LI! of thé phosphatidylcholine/cholate solutionwere added and thé decrease in Na,K-ATPase activity was measured. A lipidconcentration producing a 50-70% decrease in activity was selected and thé corre-sponding quantity was added to thé whole supernatant. Generally a final phos-phatidylcholine concentration of 10 mg/ml gave optimal results.

Step 3: Formation of vesicles: The Na,K-ATPase/phosphatidylchohne/cholatesolution was homogenized for 60 s and was transferred to 7 mm dialysis tubing. TheNa,K-ATPase-liposomes formed while thé cholate was removed slowly by dialysisfor 60 to 90 h at 4°C in a 1000-fold excess of cholate-free 'transport' solution. The

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liposomes were quantitatîvely removed from thé dialysis tubing and thé final volumewas exactly determined in order to introduce a correction factor for possible dilutionof thé suspension. The liposomes were stable for 1-2 weeks at 4°C. Excess freeenzyme was removed by centrifuging thé préparation at 100000 X g for 10 min in aBeckman Airfuge and discarding thé pellet.

Step4: Transport analysis: The principle of thé transport analysis is described inprevious publications [2,3,5,11-13]. Essentially, 22Na is used as a tracer for Na-fluxesand 86Rb as a tracer for K-fluxes, which are measured in thé absence and in théprésence of ATP. In highly active préparations thé ATP concentration was main-tained constant with an ATP-regenerating System containing 2 mM phosphoenol-pyruvate and 0.1 mg/ml pyruvate kinase. The variation in intravesicular labeledcations was measured by a microversion [13] of thé Sephadex G-50 fil tration method[2,3,5], Routinely 5jul liposomes were eluted per column. The transport-associatedATP-hydrolysis was measured in thé présence of 1 mM external ouabain either bydetermining thé inorganic phosphate by a highly sensitive colorimetric method [14]or by measuring thé 32Pi released from [y-32P]ATP [15]. For transport experimentswith différent internai and external ion concentrations, thé external solution wasremoved by centrifuging thé liposomes for 30 min at 100000 X # (30 lb/inch2) in aBeckman Airfuge and resuspendîng thé liposome pellet in thé desired solution. Noloss of transport activity was caused by thé centrifugation step.

RESULTS AND DISCUSSION

In order to standardize thé préparation of thé Na,K-ATPase-liposomes, a rela-tionship between thé Na,K-ATPase activity of thé soluble enzyme and thé transportactivity of thé final liposomes was sought. For this purpose, încreasing amounts ofNa,K-ATPase protein of a spécifie activity of 1600-1700 ,umol/mg/h were cholate-solubilized and thé extracted protein and Na,K-ATPase activity were measured.Approximately 50% of thé initial protein were soluble in a 24 mM cholate solution(Fîg. la). The solubilized enzyme activities augmented from 145 to 745 ^rnol/ml/hat initial protein concentrations of 1-3 mg/ml and were 1700 and 3200 ^mol/ml/hat 4 and 6 mg/ml initial protein, respectively (Fig. Ib). The addition of 10 mgphosphatidylcholine per ml supernatant resulted in a 60-90% decrease in enzymeactivity (Figs. Ib and 2). This inhibition is not due to thé additional cholate addedwith thé soluble lipid, for addition of thé same amount of pure lipid produced asimilar inhibition (data not shown). Note that thé lipid-induced inhibition of théenzyme activity is necessary to obtain transport function. A freeze-thaw-sonicationstep does not improve thé reconstitution by thé présent procédure where solubleenzyme and phospholipid are used.

For optimal reconstitution it was necessary to remove thé détergent for at least60 h. When thé dialysis was interrupted after already 15 h, and thé formed vesicleswere sedimented and resuspended in a cholate-free soutien, thé transport capacitywas about 2fold lower than after a 60 h dialysis, in agreement with thé observation

302

0 2 4TOTAL NKA PROTEIN (mg/ml }

3200 r

2400

f= 1600

800-

0 5000 10000TOTAL NKA ACTIVITY (jjmoles/ml/h)

5000

1000

500-

100

50

102 4 6 8

PHOSPHATIDVLCHOLINE(mg/ml)

10

Fig. I. Cholalc-solubîlizalion of Na.K-ATPase. Supcrnatanls containmg increasing amounls of solubleNa.K-ATPase wcre prepared by adding 200, 400, 600, 800 and 1200 pg of Na.K-ATPase protein of 1670jiiniol/mg/h spécifie activity to 200 ,11! of 'transport' solution (for composition sec undcr Materials andMcthods) containing 24 mM sodium cholatc and sedimenting ihc undissolved enzyme for 15 min atlOOOOOXg. The hiipernatant was analysed for protein (•) in a. and for Na.K-ATPase (NKA) activity(O). Na,K-ATPase aclivity in thé presenee of 10 mg phosphalidylcholine per ml before dialysis (O) andin thé final liposomes suspension aflcr dialysis (•) in b. The activities in presenee of lipid were correctedfor thé dilution duc to thé addition of thé lipid solution.

Fig. 2, Inhibition of Na.K-ATPase activity by phosphatidyleholine. To supernatants containing 166 (O).438 (3), 716 (•). 1836 ( D ) and 3274 (•) junol/ml/h of solublc Na.K-ATPase, 2-10 mg of phosphati-dyleholine were added and thé Na.K-ATPase (NKA) activity was determined as dcseribed undcrMaterials and Mcthods.

that approximately 50% of thé Na,K-ATPase was still in soluble form at this stage(data not shown).

The dialysis increased thé enzyme activity in thé préparation containing thélowest amount of protein and reduced thé activity by 8-30% in thé high proteinpréparation (Fig. Ib). The reconstituted enzyme keeps its activity for at least 2 weeksat 4°C, indicating that thé phospholipid may protect thé enzyme from denaturation.

Generally, 150-400 fil Na,K-ATPase-liposomes were prepared. To allow a com-plète transport analysis of a 150 ,ttl préparation, thé transport assay was refined to a

303

200 r

16

300 600 900

NKA ACTIVITY BEFORE DIALYSIS

)

2 A 6 8 10

>ji LIPOSOMES /COLUMN

Fig. 3, Détermination of internai isotope in microsamples of Na,K-ATPase-liposomes. The liposomeswere prepared as described under Materials and Mcthods and were loaded wiih Rb for 24 h. l - I O /niwerc pipeited with a Pipelman 20 /il syringe (Gilson) on iee-cooled I emX 10 cm Scphadex G-50 columnsand were eluted with 'transport' solution. The liposomes elutcd in thé void volume wi th in 2-3 min and théexternal isotope within 20 min. The entrapped K f >Rb (•) was detcrmîned by scintillation counting inInstagcl (Packard).

Fig. 4. Relationship between thé soluble Na,K-ATPase activily présent before dialysis and thé transportcapaeity présent after dialysis. The soluble Na,K.-ATPase (NKA) activîty was dctcrmined by thétechnique described under Materials and Melhods in thé présence of 10 mg phosphalidylcholine (PC) perml. The total ATP-aclîvaled Na-transport (•) was determined as follows: 20 jiil of liposomes were addedto 0.4 jiCi of dried 22Na and I fi\f 60 mM Tris-ATP. The 22Na uptake reached cquilibrium within 30min at 25°C. The ATP-free Na-uptake (O) was measured after a 30 min incubation in thé présence of2 2Na and in thé absence of ATP. The ATP-free [M(lRb]K. uptukc (D) was established by rneasuring théinternai [H6Rb]K after a 24 h incubation (at isotopic equilibrium) of 40 /il liposomes in ihe présence ofI iid ""Rb and thé total ATP-activated [*fiRb]K efflux (• ) after adding 20 ^1 of ^Rb-loaded liposomesto tubes conlaîning 1 jul of 60 mM Tris-ATP and rneasuring thé inlcrnal [H f >Rb]K remaîning in thé vesiclesafter a 30 mîn incubation at 25°C (at equilibrium). The internai isotope content was measured asde.seribed for Fig. 3 and duplicates were made by eluting two 5-^1 samplcs in parallcl. The total (internaiplus exlernal) isotope was measured in 2-,iil samples of thé whole liposome suspension. The transport wascalculated in nmoles cations transported per mg phosphatidylcholine (PC).

micromethod. Fig. 3 illustrâtes thaï thé internai isotope content of only 1 fil can bedetermined quantitatively by thé gel-filtration method. Sînce thé elution was per-formed at 2-4°C, no ions escaped from thé internai médium during thé 2-3 minelution time. In fact, some samples were left for 15 min in thé column before elution

304

and their internai labeled cation content was not différent from rapidly elutedcontrols.

In thé reconstituted System, only thé inside-out Na.K-ATPase molécules areactivated by thé externally added ATP so that Na ions are moved from thé externalto thé internai space and K ions from thé internai to thé external space. Therefore,thé most convenient way to test thé transport capacîty of thé Na,K-ATPase-liposomesis to add 22Na to thé external médium and to measure thé 2 2Na uptake oecurringwithin 15-30 min in thé présence or absence of ATP. Within this time period, astable plateau for thé ATP-activated Na-uptake is attained [11],

The results shown in Fig. 4 illustrate that thé total ATP-activated Na-transport isa function of thé soluble Na,K-ATPase activity measured in présence of phosphati-dylcholine before dialysis. When thé soluble enzyme activity was for instance 126jumol/ml/h before dialysis, thé total Na-transport measured after dialysis was 136nmol Na/mg phosphatîdylcholine (Fig. 4). When thé soluble Na.K-ATPase activityreached 300 jumol/ml/h, thé total ATP-activated Na-transport was about 180 nmolNa/mg phosphatidylcholine (Fig.4). This was thé maximal transport capacity: anincrease of thé soluble enzyme activity to 900 jU mol/ml/h did not further augmentthé total cation transport. The maximal transport capacity (thé plateau phase inFig. 4) may indicate that ail available vesicles contaîn transport molécules.

Thus thé method described herein allows to détermine thé optimal concentrationof soluble Na,K-ATPase required to saturate a given amount of phosphatidylcholinewîth transport molécules. A 2-3-fold excess of soluble enzyme with respect to lipidsaturation can be obtained (Fig. 4). Only such saturated Na,K-ATPase-liposomes areoptîmally reconstituted with regard to their lipid content. Perhaps thé transportactivity of reconstituted liposomes should generally be expressed per lipid ratherthan per protein. Indeed, a liposome suspension may contain a small amount ofactive protein which is entirely reconstituted—yielding high transport rates perprotein—with only a fraction of thé liposomes containing transport molécules. Onthé other hand, an optimally reconstituted (saturated) liposome suspension maycontain excess protein and thus thé transport rates seem low when expressed perprotein. In addition, ail préparations are expected to contain a certain amount ofprotein which was inactivated durîng solubilization and hence does not contribute tothé cation transport. In thé Methods section of thé présent report it is indicated thatthé free protein can be removed by differential centrifugation. Note that théNa,K-ATPase-liposomes are so stable that they can be sedimented and resuspendedwithout loss of transport activity. This is a simple method to change thé ioniccomposition of thé external ('intracellular') solution.

In a preceeding publication it was reported that thé ATP-free Na,K-ATPasetransports K ions [13]. Along this line it is shown in Fig. 4 that thé total ATP-freeK-uptake, measured at isotope equilibrium, i.e., after a 24 h incubation in théprésence of 86Rb, displays thé same dependence on thé soluble Na,K-ATPaseactivity measured before dialysis as thé ATP-activated Na-transport. Therefore, théATP-free K-transport can be equally well used as an indicator of thé transportcapacity of thé liposomes.

The total ATP-activated 22Na uptake is twice as high than thé total ATP-activated

305

[s6Rb]K extrusion (Fig. 4). This is due to thé présence of Na:K plus Na:Naexchange activities as explained in détail elsewhere [6].

The initial transport velocities, measured within 40s after thé ATP additionincreased in proportion with thé total transport (data not shown). However, as noend point is reached when initial velocities are established, thé degree of reconstitu-tion of thé liposome population cannot be detected by this method.

Taken together, thé results described in thé présent work show that thé totalcation transport measured in thé absence and présence of ATP is a reliable indicatorof thé degree of reconstitution, and that thé degree of reconstitution can be selectedby adjusting thé soluble Na,K-ATPase activity measured before thé dialysis in théprésence of phosphatidylcholine. Thus, thé four-step microprocedure describedherein offers for thé first time thé possibility to prépare standardized Na,K-ATPase-liposomes with predlctable transport properties using minimal quantities of purifiedNa,K-ATPase.

SIMPLIFIED DESCRIPTION OF THE METHOD AND ITS APPLICATIONS

A method for thé préparation of standardized Na,K-ATPase-containing liposomes is described. Firsl,increasing aniounts of purified Na,K-ATPase are solubilized by cholate in well defined conditions. Then,thé soluble Na.K-ATPasc activity is reduced by 50-70% by thé addilion of exogenous phosphalidylcho-line. The liposomes are formed by détergent removal (dialysis) and their Na,K-transport capacity isdetermined by a microassay. A relationship betwccn their transport properties and thc ATPase activityprésent before dialysis is established. This corrélation allows to sélect and adjust thé transport propertiesof thé N a, K-ATPase-liposomes before they are formed. Another importani advantagc of thé method is ilsréduction to microscale which allows to perform a large number of expcriments with a minimal quantityof purified enzyme. The method is applicable to prépare Na,K-ATPase-liposomes for studying thémcchanism and thé kinetics of thé Na,K-transport catalyzed by thé reconstituted Na,K-ATPase inreproducible conditions, or to form stable Na-gradicnt vesicles which can be used as a tool to investigatcthé mcchanism of secondary (Na-dependcnt) transport System.

ACKNOWLEDGMENTS

This work was supported by grant No. 3.575-0.79 of thé Swiss National ScienceFoundation.

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