Biochem. J. (1967) 102, 675

The Sodium-plus-Potassium Ion-Activated AdenosineTriphosphatase of Cerebral Microsomal Fractions: Treatment with

Disrupting AgentsBy J. R. COOPER* AD H. McILWAIN

Department of Biochemi8try, Institute ofPsychiatry(British Postgraduate Medical Federation, University of London),

Maudeley Hospital, London, S.E. 5

(Received 10 October 1966)

1. The Na+-plus-K+-stimulated adenosine triphosphatase [(Na+,K+)-ATPase]ofmicrosomal preparations from ox brain was inactivated or diminished in activityby exposure to 2-8M-urea. Similar concentrations ofurea diminished the turbidityofthe suspensions. 2. Low concentrations (about 2.5mM) ofNaATP with the ureagave partial or complete protection oftheATPase, without altering the concomitantchange in turbidity. Some protection ofthe (Na+,K+)-ATPase was afforded by trisATP, but the greatest protection was found with NaATP and in its presence thechange in (Na+,K+)-ATPase with 3M-urea included a phase in which activity wasenhanced by 40%. 3. The protective effect was specific to NaATP: KATP,NaADP, NaAMP and sodium pyrophosphate were without protective effect and insome cases they augmented the action of urea. 4. The turbidity of cerebral micro-somal suspensions was diminished also by ultrasonic irradiation; NaATP did notalter this change. After ultrasonic treatment up to 55% of the protein and of theATPase activity were no longer deposited by centrifugal forces of 4*5 x 106g-min.5. Ultrasonic treatment and centrifugation could be carried out with little or noloss of ATPase and ammonium sulphate flocculation of the supernatant thenafforded in the first material precipitated a three- to five-fold enrichment of(Na+,K+)-ATPase activity. 6. Sodium borohydride and dimethyl sulphoxide alsodiminished the turbidity of the microsomal fraction but enrichment ofthe ATPasewas not effected by these reagents; ten other compounds were without action onthe ATPase. 7. Acetyl phosphate was hydrolysed by the microsomal preparationandthis activity was increased by added K+. Acetyl-phosphatase activity persistedin the ultrasonically treated and ammonium sulphate-fractionated preparations,which were more exacting in their requirements for K+. 8. The findings are

discussed in relation to the mechanism ofthe (Na+,K+)-ATPase.

Microsomalfractions howing (Na+,K+)-ATPasetactivity, though often referred to as an enzyme (seeSkou, 1964), are more correctly to be regarded asmorphological entities. Processes of disruption orextraction are thus a necessary part of the bio-chemicalstudyof the (Na+,K+)-ATPase. A previousinvestigation (Stahl, Sattin & McIlwain, 1966)showed that simple washing procedures couldextract from ox-brain microsomal preparations anADP-ATP exchange system, which earlier in-vestigators had regarded as an essential part of the

* Present address: Department of Pharmacology, YaleUniversity, New Haven, Conn., U.S.A.t Abbreviations: ATPase, adenosine triphosphatase;

(Na+,K+)-ATPase, Na+-plus-K+-stimulated ATPase.

(Na+,K+)-ATPase; yet its extraction did notdiminish the (Na+,K+)-ATPase activity of thepreparations.In the present study, cerebral microsomal

fractions have been subjected to more extensivetreatments, including ultrasonic irradiation andexposure to high concentrations of urea, with theobject of differential extraction and of learningmore about the (Na+,K+)-ATPase and its relation-ship to other microsomal constituents. Suchrelationships are inherently likely to be importantto the participation of the (Na+,K+)-ATPase inactive movements of Na+ and K+ across cellboundaries (see Bonting, 1964; Mcflwain, 1963;Skou, 1964). While the present investigations were

675

J. R. COOPER AND H. McILWAINin progress Skou & Hilberg (1965) reported the useof urea in examining a differently prepared micro-somal ATPase and this receives comment below.

EXPERIMENTAL

Micro8omal 8u8pen8ion8. Ox brains were placed in aplastic bag in ice, within 15min. of the animals' slaughter.About 45min. later, grey matter was snipped from thecerebral hemispheres and about 70g. of cortex from twobrains was blended with 9vol. of 0-32M-sucrose, adjusted topH7-4 with KOH. The centrifuging sequence of Stahl et al.(1966) was followed, except that for washing the mito-chondrial fraction was resuspended in a test-tube homo-genizer and that the combined supernatant yielded amicrosomal pellet that was suspended in 0-32M-sucrose at aconcentration corresponding to 10-15mg. of protein/ml.This was either used immediately, or was kept for up to3days at 60 or at -20° for up to 3weeks.

Expo8ure to urea. This was normally carried out at roomtemperature (16-20°) with mixtures containing microsomalsuspensions equivalent to 1-2mg. of protein/ml., and theconcentrations of urea and other reagents that are specifiedin individual experiments. When extinction values weremeasured the mixtures were made in cells of a UnicamSP.600 spectrophotometer with which readings were takenperiodically at450mu (Fig. 1). After chosen times, measuredportions of the mixtures were pipetted to tubes for enzymeassays; this involved at least tenfold dilution ofthe urea.

Ultra8onic treatment. Microsomal suspensions containing7-18mg. of protein/ml. of 0-32M-sucrose (2ml.) were placedin a 10ml. cup, cooled in ice-water, into which dipped alm.-diam. probe of a 60w MSE ultrasonic unit. Theultrasonic irradiation was carried out intermittently, forperiods of30sec. each followed by a 30sec. interval, until theirradiation periods totalled 3min. After treatment thesamples were centrifuged (Spinco preparative machine L2)at 1-5 x 105g for 30min. or 60min. and the supernatants anddeposits collected. When specified (Table 2) the depositswere resuspended in 2ml. of 0-32M-sucrose and the ultra-sonic treatment was repeated.Ammonium 8ulphate fractionation. Saturated aqueous

(NH4)2SO4 neutralized with aq. NH3 was added to thesupernatants from ultrasonic treatment, to give the con-centrations specified in Table 3. The resulting suspensions,turbid when the (NH4)2SO4 was 30% saturated or above,were centrifuged at 1-5 x 105g for 30min. and the super-natants removed. The solid that separated formed aplaque-like deposit with the higher (NH4)2S04 concentra-tions that adhered to a side ofthe tubes. It was resuspendedin water for assay.ATPa8e as8ay. Activity was determined by the method of

Schwartz, Bachelard & Mcllwain (1962) except that normalconcentrations of reagents for (Na+,K+)-ATPase were50mM-tris-HCl, pH7-6, 3mM-tris ATP, 3mM-MgCl2,100mM-NaCl and 30mM-KCl. For ATPase as quoted inTables 1-4, the NaCl and KCI were omitted. Also, afterincubation the reaction was terminated by the addition oflml. of ice-cold 7-2% (w/v) HC104. The extract was thenneutralized with 1 ml. of 1-3 N-KOH, refrigerated for 10min.and centrifuged. A 2ml. portion of the supernatant wastaken for phosphate determination. Since pyrithiamine andPersantin interfered with the determination, the followingmodification was employed when these drugs were tested.

After incubation the reaction was stopped by the addition of0- 1 ml. of N-HCI, and the samples were placed in a boiling-water bath for 1 min., cooled and neutralized with 0-5M-NaHCO3. The tubes were centrifuged and 0-5ml. of thesupernatant was placed on a column (1 cm. x 7cm.) of thecation-exchange resin Amberlite IRC-50 (Na+ form). Theeffluent and subsequent water wash were collected and madeto 3ml., and a portion was taken for phosphate determina-tion.

Acetyl-phzo8phatame a8ay. Incubation media contained50mM-tris-HCl, pH 7-6, 3mM-MgCl2 and 30mM-KCl, and to0-5ml. of the media was added the sample to be assayed, in0-3 ml. After preincubation at 370 for 5min. the reaction wasinitiated by adding 0-2ml. of 50mM-acetyl phosphate aseither the lithium or tris salt. The sample was incubated for20min., and the reaction stopped by the addition of 1 ml. ofneutralized hydroxylamine. Control samples without theenzyme were carried through the procedure, and wherespecified (Table 4) mixtures lacking K+ ions were included.The acetyl phosphate remaining was then determinedphotometrically as described by Lipmann & Tuttle (1945).Examination of reaction mixtures buffered between pH 7-2and 8-2 showed greatest activity at pH7-6; the reactionprogressed linearly with time for 30min.

Other reagent8 and determinations. NaATP was obtainedas the crystalline hydrated disodium salt of99-100% purityfrom the Sigma Chemical Co., London, S.W.6. The tris orpotassium salts when required were prepared from thesodium salt by passing concentrated solutions through a1 cm. x 7cm. column of Amberlite resin IR-120 (H+ form),and immediately neutralizing with either tris base orKHCO3. Tris base and hydrochloride and lithium acetylphosphate were also obtained from the Sigma Chemical Co.Urea (ultra pure) was obtained from Mann ResearchLaboratories, New York, N.Y., U.S.A. Diethylamino-ethyldiphenylpropyl acetate hydrochloride, SKF525A, wasa gift from Smith, Kline and French Laboratories, Phila-delphia, Pa., U.S.A. Persantin (dipyridamole) was suppliedby Geigy Pharmaceuticals, Ardsley, N.Y., U.S.A. Proteinwas determined by the method of Lowry, Rosebrough,Farr & Randall (1951).

RESULTS

The microsomal fractions that formed the startingmaterial of the present investigations were similarin preparation and properties to those of Swanson,Bradford & McIlwain (1964) and Stahl et at. (1966),including their requirements for Na+ and K+ ions.Their rate of hydrolysis of ATP in solutions con-taining magnesium salts was 0-3-0-5 that obtainedwith further addition of Na+ and K+ (see Tables 1and 2). These Tables indicate also the variationstypically seen between different preparations, inenzyme activity and in degree of Na+- and K+-activation.

Treatment with urea

Optical effects. The actions of urea in unfoldingand dissociating protein chains (see Koshland, 1958;Nozaki & Tanford, 1963) were applied to myosinATPase by Stracher (1961), with 2-10M-urea. In

676 1967

(Na+,K+)-ATP-ASE AND DISRUPTING AGENTS

4 6 8 0 5 10

Concn. of urea (M) Time (min.)

677

15 20

Fig. 1. Urea and the extinction value ofmicrosomal suspensions. (a) The differently marked points refer to differentmicrosomal preparations. In each case the suspensions (12mg. of protein/ml., 0.Iml.) were added to 0f05ml. of5Omm-NaATP, 10M-urea ofvolume needed for the final concentrations quoted, and water to lml. Readings weretaken at450m, 20min. after mixing. In parallel experiments (not quoted) NaATP was omitted and the extinctionvalues were found to be the same. (b) Microsomal suspensions were exposed as just described, with the followingadditions: 2, none; 0, 4M-urea; *, 4m-urea+2 5mM-NaATP.

the present study a similar range of concentrationswas used, and these brought about physicalchanges in the microsomal suspensions, as well as

alterations in their ATPase activity (Fig. 1).Pronounced clearing of the originally opalescentmicrosomal suspension took place within a fewseconds of admixture with urea. The changeprogressed for several minutes and required the highconcentrations of urea shown in Fig. 1(a). It wasnot altered by the presence of2*5nM-NaATP.ATPa8e inactivation and protection. At 3M-urea,

when change in the extinction of the suspensionshad undergone about half its maximal change, thesuspensions had also changed greatly in ATPase.Their activity, when tested either in the presence or

absence of Na+ and K+, had fallen by 50-80%;examination was carried out in reaction mixtures inwhich the urea was diluted 30-fold, and thus theeffect of urea was an inactivation of the ATPaseratherthan an inhibition. This situationwas greatlyaltered when ATP also was present during theexposure to urea (Fig. 2). Thus after exposing themicrosomal suspension at room temperature to2'5mM-NaATP plus 3mM-urea for 20min., andsubsequently diluting it 30-fold for assay in the

standard reaction mixture, the (Na+,K+)-ATPaseactivity exhibited had increased by 40%. Thisincrease in the Na+- and K+-activation was accom-

panied by diminution in the ATPase activity in theabsence of Na+ and K+ (Table 1), though thisactivity remained at a higher level than afterexposure to urea only. The augmentation in(Na+,K+)-ATPase could occur as a result of thepresence of urea and ATP independently of Na+,but the greatest activities were obtained when bothNa+ and ATP were present with urea (Fig. 2). Theprotective effects of Na+ and ATP were showntowards all concentrations of urea examined, i.e.up to 6M, though only at the lower concentrationswas (Na+,K+)-ATPase activity enhanced.

Specificity of protection by 8odium ATP. OnlyNaATP among several related compounds pro-tected the microsomal ATPase from inactivation byurea (Table 1). The KATP and tris ATP were

ineffective; the protective effect of NaATP wasshown at a range of concentrations (Table 1, Expt.C) and was maximal at 2-5mM with urea at 4M.NaAMP and NaADP at 2-5mM did not protectagainst urea, though sodium pyrophosphate aug-mented the loss of activity (Table 1, Expt. B).

Vol. 102

100c

5

0

dS

o Ioo.-0-

S0@4

W

J. R. COOPER AND H. McILWAIN

30 \

04C-5-p0

0 3 4 5 6

Concn. of urea (M)

Fig. 2. ATPase of microsomal suspensions, previously ex-posed to urea inthe presence (0) and absence (0) ofNaATP.Mixtures were prepared containing the different amounts ofurea and with or without 2-5mm-NaATP. The microsomalsuspension was then added and the mixtures were kept at20° for 10min.; portions were then taken for assay ofATPase (A) and (Na+,K+)-ATPase (B) under the standardconditions stated in the Experimental section.

In Table 1 are quoted ATPase estimations in thepresence and absence ofNa+ andK+ (independentlyof the presence or absence of Na+ during exposureto urea). The protective effects described wereexhibited inbothcircumstances: i.e. the 'MgATPase'as well as the '(Na+,K+,Mg2+)-ATPase' wasprotected, though to a smaller degree. Certain ofthe added substances, it will be noted, augmentedthe action of urea under one or both of thesecircumstances rather than opposing it. Sodiumpyrophosphate and KATP were most potent in thisrespect. After treatment ofmicrosomal suspensionswith urea in the presence or absence of NaATP, cen-trifugation under the conditions used for originallypreparing the microsomes gave a deposit that againcarried all the (Na+,K+)-ATPase activity of thesuspensions.

Ultrasonic irradiation

Exposure of cerebral microsomal suspensions toultrasonic irradiation was noted by Bradford,Swanson & Gammack (1964) to be deleterious totheir ATPase activity. However, about 12% of theATPase or (Na+,K+)-ATPase of the suspensionswas no longer deposited by the centrifugal forcesused in preparing the microsomes. By choosingthe conditions described in the Experimentalsection, involving intermittent treatment of well-cooled suspensions, the procedure has now beencarried out with minimal loss; Table 2 indicates that

Table. 1. Protection of (Na+,K+)-ATPase against inactivation by urea

In all experiments microsomes were exposed to urea at4M; the nucleotide orphosphate salts were added at 2-5mMexcept in Expt. C. For each exposure, reagents were added to 0-1 ml. of microsomal preparation (10-15mg. ofprotein/ml.) with 0-05ml. of adenine nucleotide or pyrophosphate (50mm), 0-4ml. of urea (10M) and 0-45ml. ofwater. The suspensions were kept for 10min. at room temperature and then 0-1 ml. portions were taken for ATPaseassay.

Addedcompounds

ATPase activity (,umoles of Pi/mg. of protein/hr.)

(a) No Na+ orR+

A NoneUreaUrea, NaATPUrea, KATPUrea, tris ATP

B NoneUreaUrea, NaATPUrea, NaADPUrea, NaAMPUrea, sodium pyrophosphate

C NoneUreaUrea, 0-5mm-NaATPUrea, 1-25mm-NaATPUrea, 2-5mM-NaATPUrea, 5mm-NaATP

11-81-65-80-32-23-90-91-41-30-30

13-01-02-99.11-74-8

(b) Na+ (100mma)+K+ (30ma)

22-85-1

20-72-32-5

12-64.9

10-95.41-20

29-63-1

16-514.820-212-4

(b)- (a)

11-03.5

14-92-00-38-74-09.54-10-90

16-62-1

13-612-718-57-6

Expt.

678 1967

(Na+,K+)-ATP-ASE AND DISRUPTING AGENTS

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Vol. 102 679

J. R. COOPER AND H. McILWAINrecoveries of the (Na+,K+)-ATPase averaged 72%and reached 98%.

Solubilized material and 8altfractionation

The ultrasonic treatment caused major changes inthe microsomal suspensions shown in Tables 2 and3; their extinction values diminished, e.g. in thesuspensions of Table 2 by about 40%. Moreover,much of the (Na+,K+)-ATPase originally present inthe suspensions was now no longer deposited bycentrifugal forces greater than those used in pre-paring the microsomes. Some 40-50% of the ori-ginal activity now remained supernatant aftercentrifuging at 4-5 x 106g-min. (Table 2). After theultrasonic treatment, a similar proportion of thetotal microsomal protein was also no longerdeposited and thus the specific activity of theATPases was little changed, in supernatant or inresidual fractions (Table 2). Opportunity was,however, offered by the presence ofATPase activityin the supernatant for application of differentmethods of fractionation.Enrichment of the enzyme was first afforded by

the ammonium sulphate fractionation of Table 3.Addition ofincreasing concentrations ofammoniumsulphate could precipitate almost all the ATPaseand 80% of the protein of the supernatant fromultrasonic treatment. Selectivity was, however,shown in the ATPase being more readily brought outof solution than were admixed proteins; increase inspecific activity or ATPase per unit of protein, was

then obtained. With the first separation of quitesmall amounts of protein with 20% saturatedsolution of ammonium sulphate, some 40% of theactivity was associated. Almost all the activity wasflocculated with 30%-saturated ammonium sul-phate, at similarly high specific activity. Higherconcentrations of anumonium sulphate gave pre-cipitates that carried increasing proportions ofprotein inactive in the ATPase assay.

Treatment of microsomal suspensions with asodium iodide reagent has been investigated byNakao, Yashima, Nagamo & Nakao (1965). Ultra-sonic treatment and centrifugation of this prepara-tion also has now been found to yield a supernatantwith ATPase activity (Table 4).

Acetyl-pho8phatase activity. Microsomal fractionsfrom kidney cortex carry a K+-activated acetyl-phosphatase activity, which is considered to arisefrom their (Na+,K+)-ATPase (Bader & Sen, 1966),though fractionation methods were not employed inreaching this conclusion. As the procedures justdescribed had afforded a separation of (Na+-K+)-ATPase from admixed protein, the resultingpreparations were examined for acetyl-phosphataseactivity.Such activity was found (Table 4); the original

microsomes liberated phosphate from ATP andacetyl phosphate at about the same speed. Ultra-sonic treatment of the microsomal suspensionsincreased their acetyl-phosphatase activity andgave preparations in which acetyl phosphatase wasactivated by Mg2+ and K+ and inhibited by Na+

Table 3. Enrichment of (Na+,K+)-ATPace activity by ammonium 8uIphate treatment

Microsomes were ultrasonically treated and centrifuged as described in the Experimental section. To separateportions of the supernatant, neutralized saturated (NH4)2SO4 was added at 0° to give the final saturation valueshown in the Table. After the addition of (NH4)2S04, the samples were centrifuged at 150000g for 30min. Thesupernatant fraction was aspirated and discarded; the (NH4)2S04 precipitates were suspended in water and theirprotein and activities determined.

(a) Protein(mg./ml.)

PreparationA: Supernatant from ultrasonic treatment

Precipitates with (NH4)2SO4 at:20% satn.40% satn.60% satn.80% satn.

B: Original microsomesSupernatant from ultrasonic treatmentPrecipitates with (NH4)2SO4 at:30% satn.35% satn.40% satn.

8*17

0-601-806-06-8

19*35.92-23-03.5

(b) Phosphate liberated(,umoles of Pijmg. of

protein/hr.)

No WithNa+ or K+ Na+ and K+

1-64 10.9

3-831-900-740.594-44-8

594815*513-815*014-0

7.7 50-55-8 28*85-2 26-2

(a) x (b)Enzyme units

No WithNa+ or K+ Na+ andK+

13-4 89

2-3 353*4 8750 934-0 94

84-328-4

16-917*318-1

29083

1118792

680 1967

(Na+,K+)-ATP-ASE AND DISRUPTING AGENTSTable 4. Acetyl'phosphatase activity of different ATPase preparations

Preparations (i), (ii), (iv) and (vi) were made as described in Table 3, with (NH4)2S04 at 30% saturation toobtain (iv). Preparation (iii) was the material deposited by the centrifugation, which yielded (ii), and correspondedto the 'residue' ofTable 2. We are indebted to Mr I. Pull for the NaI-treated microsomal suspension thatwas madeaccording to Nakao et al. (1965) and that, after ultrasonic treatment and centrifugation as described in Table 2,yielded the supernatant (v). When specified, Na+ was present at lOOmM and K+ at 30mM.

Preparation(i) Microsomal suspension

(ii) Supernatant from ultrasonic treatment(iii) Residue from ultrasonic treatment(iv) (NH4)2S04 precipitate from supernatant(v) Supernatantfrom ultrasonic treatment ofNaI-treated

microsomes

ATPase(,umoles of Pi/mg. of

protein/hr.)

No WithNa+ or K+ Na+ and K+

7.7 24*79.4 24-1

10*2 25*57.8 22-61.0 28.g

Acetyl phosphatase(,umoles of Pi/mg. of

protein/hr.)-

No WithK+ K+16X8 23-831-1 40*129*9 34.82-8 27-65.5 53-8

(vi) As preparation (ii)As preparation (ii)As preparation (ii)

Additions to acetyl phosphatase reaction mixtureNone3mM-MgSO43mM-MgSO4, l0Omm-NaCl

ions. On centrifugation the acetyl phosphatase wasdistributed between supernatant and deposit inabout the same proportion as was the (Na+,K+)-ATPase. Moreover, the acetyl phosphatase of thesupernatant was deposited by ammonium sulphatetogether with the ATPase; its activation by K+ions greatly increased after this treatment. Thepreparation obtained by ultrasonic treatment andcentrifugation of microsomes treated with sodiumiodide reagent (see above and Table 4) also showedincreased activation by added K+ ions.

Miwellaneous agents

Of other disruptive or solubilizing agents,detergents have been examined previously(Schwartz et al. 1962; Swanson et al. 1964). Amonga number of reagents tested in the present study(Table 5) dimethyl sulphoxide and sodium boro-hydride caused pronounced diminution in theextinction value ofcerebralmicrosomal suspensions,but on centrifuging the treated solutions all

(Na+,K+)-ATPase was again deposited. Furtheroptical clearing with higher concentrations of thesulphoxide or borohydride than quoted in Table 5caused loss of (Na+,K+)-ATPase. The otherreagents of Table 5 were examined in exploringpossible cofactor requirements of the system.Bruchhausen (1964) reported actions of pteridinederivatives on an erythrocyte ATPase, but actionwas not found in the amethopterin examined in thepresent study. The nicotinamide nucleotides were

Table 5. Agents examined for effect on

(Na+,K+)-ATPase

Potassium borohydride was added to microsomalsuspensions in 50mM-tris, pH8, and kept at 370 for 10min.beforetakingaportionofthe suspensionforassay. Dimethyl.sulphoxide was added to the microsomal suspensionandkeptat 180 for 20min. before taking samples for assay. The otheragents were added to ATPase reaction mixtures before themicrosomal suspensions were added.

Agent

KBH4KBH4KBH4NAD+,NADH,NADP+or NADPH

Thiamine pyrophosphatePyrithiamineGSHYeast extract (lOmg./ml.)Amethopterino-PhenanthrolinePersantinSKF525ADimethyl sulphoxideDimethyl sulphoxideDimethyl sulphoxide

Concentra-tions

examined(mM)

2060

2400-66

10-5f 5

1

0-310-50-3

42014004200

Inhibition ofof(Na+,K+)-ATPase(%)

0

46*77*0

0

0

0

0

0

0

0

0

0

21*37*

* In these instances the extinction value of the micro.somal suspensions was diminished: by60mx-KBH4 to 81%and by 24Omm-KBH4 to49% ofits original value.

11x318-614-5

11*349.529-3

Vol. 102 681

J. R. COOPER AND H. McILWAINincluded in view ofobservations of Siekevitz (1965),thiamine pyrophosphate and pyrithiamine in viewof those of Armett & Cooper (1965), SKF525Abecause of its effects on other microsomal systems(Cooper, Axelrod & Brodie, 1954), and Persantinbecause of similarities in its action to that ofouabain.

DISCUSSION

Conceivably an agent ideal for the presentinvestigation would separate the (Na+,K+)-ATPaseunchanged from its microsomal matrix. Thoseexamined, urea and ultrasonic irradiation, as wellas the detergents of a previous study (Swanson etat. 1964), have modified ATPase activity during itsseparation. This was the result also of a distincttype of enrichment employing sodium iodide andcysteine, which extracted associated material,leaving an enriched (Na+,K+)-ATPase (Nakao et al.1965). It is encouraging that the alterations in(Na+,K+)-ATPase encountered after each of thesetreatments have made the enzyme more exacting inits requirement for Na+ and K+forATPase activity,or have included an increase in rate of hydrolysis.This makes it more likely that the Na+ and K+ re-quirements are properties of the enzyme system it-self rather than being imposed by independentmicrosomal systems that, for example, functionedby allowing access of ions to the enzyme.

Features in the attion of urea. (i) Comparison ofFigs. 1 and 2 shows that the concentrations of ureathat caused optical clearing of microsomal sus-pensions were similar to those that (in the absence ofNaATP) diminished the suspensions' (Na+,K+)-ATPase. Both of these actions of urea are pre-siimably to be ascribed to its property of formingnew linkages of the hydrogen-bond type withpeptide chains, while dissociating those native tothe microsomal components (see Kauzmann, 1959;Nozaki & Tanford, 1963). That the loss of activitycaused by moderate concentrations of urea can beopposed by NaATP, whereas that of greater con-centrations cannot, suggests the affinities involvedbetween substrate and enzyme, and enzyme andurea, are similar in magnitude. (ii) Further,NaATP not only protects the microsomal ATPasefrom inactivation by urea: joint action of NaATPand 3-4m-urea can enhance the (Na+,K+)-ATPase.This comes about not by an increase in the totalATPase activity observed (Table 1) but by diminish-ing the contribution made by the component notrequiring added Na+ and K+. This result is com-patible with the suggestion that these two enzymeactivities are interconvertible, a view supported bySkou & Hfilberg (1965), but as a result of investiga-ting microsomes exposed to deoxycholate, EDTAand histidine during their preparation. The

properties of this material differed from those ofthemicrosomal preparation now examined, for deoxy-cholate itself modifies the (Na+,K+)-ATPase(Schwartz et al. 1962; Skou & Hilberg, 1965). Withthe deoxycholate-EDTA-histidine-treated pre-paration, in contrast with the present results, aprotective role for Na+ was not found: Na+ in-creasedaninactivationcausedbyN-ethylmaleimideboth in the presence and absence ofATP. Effects ofNa+ in the presence of ureawere stated to be similarto those in the presence of the imide, but with-out data. Na+ did, however, protect a similarcerebral microsomal ATPase from inactivationbrought about by incubation with chlorpromazine(Squires, 1965).The potency of the (Na+,K+)-ATPase makes its

control mandatory in vivo. In the derived micro-somes, relevance to control mechanisms may thusbe seen in the finding that the extent ofactivationbyNa+ and K+, and also the resulting (Na+,K+)-ATPase activity, can both be increased by urea.Comparable features were previously reported in theaction of detergents and of ouabain and theirrelevance to control of the enzyme's functioningwas appraised (Swanson, et al. 1964; Swanson &Mcflwain, 1965).

(iii) Properties of metal salts of ATP in purelychemical systems appear relevant to the specificityshown by NaATP as protective agent. Combina-tion ofATP with Na+ in ATPase reaction mixturesindependently of the enzyme was noted earlier(McIlwain, 1963; Schwartz et al. 1962). Melchior(1954) emphasized that firm complexes existed inaqueous solutions between ATP and Mg2+, Na+ orK+, and that the preferred configuration taken upbyATP differed in the different salts. In the presentsystem, Na+ is without protective action unlessATP is present; therefore, although the two couldstill be acting separately, the simplest assumptionwould regard NaATP itself as the protective agent.This view is supported by the greater protectiveaction shown by NaATP in comparison with thesodium salts of the other phosphates examined. Indilute aqueous solution, orthophosphate, AMP andADP had less ability to bind Na+ than had ATP,which could attach 2Na+ ions/mol. (Hickling &White, 1965). This parallels the protective effectsshown in Table 1.

(iv) The chemical properties ofKATP often differlittle from those quoted above for NaATP, but inprotecting the microsomal ATPase from urea thetwo salts differ greatly. The diminution of ATPaseinduced by urea was augmented by KATP. Injudging aspects of the ATPase related to thesephenomena, proposed intermediates and stericfactors appearrelevant. AnalagousATPasesystemsof giant axons and erythrocytes required Na+ andATP to be together on the inside of the cell mem-

682 1967

Vol. 102 (Na+,K+)-ATP-ASE AND DISRUPTING AGENTS 683

brane (Glynn, 1962; Whittam, 1962); K+ acted atthe other side and catalysed breakdown of a labilephosphate derivative that was formed when Na+,ATP and Mg2+ were present (Post, Sen & Rosenthal,1965; Hems & Rodnight, 1965). In the presentexperiments protection is afforded by the simul-taneous presence oftwo reactants that areneeded onthe same side of the cell membrane but that them-selves are inadequate for enzymic change. The ureaexperiments thus support formulations of (Na+,K+)-ATPase action that permit substrate-additioncompounds of the type Enzyme .NaATP.

Acetylphosphaase and utrasonic treatment. It is afeature of the (Na+,K+)-ATPase mechanisms thatpropose successive Na+- and K+-activated steps(see above) that the phosphorylated intermediatehas the properties ofan acylphosphate. The second,K+-activated, stage is then an acyl phosphatase forwhich acetyl phosphate is concluded to bearelevantsubstrate (Bader & Sen, 1966; Hokin, Sastry,Galsworthy & Yoda, 1966). The present investiga-tions give additional support to this, for themicrosomal suspensions that formed the startingmaterial carried acetyl-phosphatase activityapproximately equal to their (Na+,K+)-ATPaseactivity, and the two activities remained associatedafter ultrasonic treatment and armonium sulphatefractionation (Table 4). It is especially noteworthythat activation ofthe acetyl phosphatase by K+ wasshown throughout, and increased after thefractionation. The inhibition caused in the acetylphosphatase by Na+ may also be relevant to thefunctioning of the enzyme in its normal cellularenvironment.The extent to which ultrasonic treatment may

have given solution of the ATPase is uncertain.Centrifugation demonstrated diminution in particlesize to a degree often associated with solution andarnmonium sulphate indicated that over 75% ofmicrosomal protein was not deposited on centri-fuging solutions that were 40% saturated withammonium sulphate. The (Na+,K+)-ATPase was,however, flocculated by relatively low armoniumsulphate concentrations. The enrichment achievedby removing in this way most of the microsomalprotein indicates that the process merits furtherstudy, which is in progress.

We are indebted to Mr I. Pull for providing several of themicrosomal preparations used in this study, and to Mr W.

Chivers for technical assistance. J. R. C. is a Special Fellowof the National Institute of Neurological Diseases andBlindness, U.S. Public Health Service.

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