Plant Seienc~ 82 (1992) 135-144 135 Elsefier S~enfific Pubfishe~ briand Ltd.

Characteristics of vanadate-sen tive ATPase activities migrating to different den ties on a sucrose gradient in microsomes

Acer pseudoplatanus cells

V i ~ o f i a Pat t i , G i o v a n n a Cur t i and Piera L a d o

Universi~ deg~ Stu~ ~ Milano, D~artim~w ~ BiologY, Centto ~ Studio del C ~ R ~lla Biolog~ Cellulare e Molecola~ ~lle P ~ n ~ Via Ce~r~ 26, 20133 Milan (Ba~)

(Rec~ved March 13~, 1991; ~ o n rec~ved Novemb~ 22nd, 1991; ~ e d Novemb~ 22nd, 1991 )

The tonoplaH verde p ~ p ~ i o n s obt~ned from ~ d ~ e d v a c u ~ or from ffa~iona~d m~rosom~ are often co~amina~d by v a n a d ~ n f i f i ~ ATPa~ a~Ni~, ~ ~ ~me a~ho~ s u g g ~ d ~ t~s activi~ w~ a~ooa~d wi~ ~e ~ n o ~ t and n~ due m a ~ m a membrane contamination. We i n ~ g ~ e d ~e ~ f i b u f i o n of v a n a d ~ n ~ f i ~ ATPa~ ~ f i ~ from A~r p m ~ t mi~mom~ on a t w o ~ p ~ e g a ~ e ~ (1 ~ 1.13 and 1.1 ~ 1.19 g ml-~). A bo~ 30-40% of ~e v a n a d ~ n ~ f i ~ ATPa~ ~f i~- ~ ~ways mig~es to the 1 ~ ~ f i o n together with the bulk (70-80%) of the ~ n ~ t N e ATPa~ activity and K ~ i m d ~ e d PPa~ activity ( ty~ol of ~e mno~ast). The ~ f i n g effects ~ ~trate and vanadate on the light ff~fion ATPa~ actifi~ a~ ~d- ' ~ f i~ . The ~ U ~ e speoficity of the vanadate-sensitive ATPase actifities ~ t h e two f f~ f io~ c ~ o n ~ ~ ~ ~ n e r ~ reposed ~ r ~asma membrane A T P ~ ml~g out the possibili~ that the v a n a d ~ n ~ t N e ATPa~ activity present ~ the ~g~ ff~fion depends on an am~o~a~ envdope comaminafion ~ on an ~pedfic A D P ~ O~er m~k~s of ~ m a membrane, ufi~ne d~hosph~e ~ u c o ~ r d - ~ u c o s ~ a n s ~ r a ~ (UDPG-ST) and ~ucan synthetase I1, are d~tfibuted fike v a n a d ~ n ~ f i v e ATPa~ b~ween the two fractions ind~ating ~ the vanadat~senskive ATPa~ activity ~ h b r a t i n g ~ the light fraction ~ associated with the ~ m a membrane and nm with the tonoplast. The two vanadate-se nfitive ATPa~ ~t ivi t i~ which eq~libra~ ~ &fferent den~ies ~ffer ~ ~ dependen~ ofpH, s e n s i t i ~ ~ he~ and ~ ~m~at ion by ~sophosphafidy~hd~e (LPC). The~ ~suks a~ ~ ageement wi~ ~ f in~n~ ~ ~e ~ m a mem~ane ATPa~ ~ ~ant calls is n~ a homo~no~ activity.

Key w ~ : Acer pseudop~tanus; mno~a~ ATPa~ activity; ~ m a memb~re ATPa~ activity

In~oducfion g e n e r ~ con~dered representative of plasma membrane, or by other plasma membrane

Many data ind~a~ that tonoplast preparations, marker~ depending on the matefi~ and/or the obt~ned ~om ~ o h ~ d vacuous or ~om ~ac- expefiment~ conditions of ~actionation. The tionated microsomes, are more or ~ss con- presence of a vanadate-senfitive H÷-ATPase aco taminated by some vanadat~senfitNe ATPase, tifity has been particularly fignificant in prepara-

tions ~om call cuRures [1-3], so much so that the autho~ have sugges~d that this vanada~- sensili~e ATPase actifity was a ~ o o a ~ d with lhe

Cor~sponden~ to: P~ra Lado, Dipa~men~ ~ ~ o ~ a C3, tonopla~, fince thor tonopla~ preparation was

Un~Adibb~v~t~n~Mi~nB~SA ' bo~nVi e a CdOfise a~m Mbumine2 ; ~ 20133 BTM p~an~s~I s.taly. ~ee ~om other plasma membrane markers. In our (hydroxymethy~)methylamin~propane; DTT, ~thim~eito~; hboratory, a tonopla~ preparation obt~ned from EGTA, ~ h ~ e n ~ ~&amyno~h~her)-N,N'4~r~ vacuous ~olated from Acer pseudop~tanus calls a~fic a~d; LPC, lysopho~hatid~choline; MES, 2-(N- by osmotic shock, flee from endoplasm~ mo~hdino~thane-sul~nic acid; PMSF, phen~ m e ~ s ~ n ~ r e t i cu lum and mitochondfi~ membrane marker~ flu°fidM e;ES~ buffer m a d P e Pas~ pyr°ph°spham~by ; m i ~ n g solutionPtsP~equ~ c o n c ~ t r a t i o P r ~ m°~a~s; TRIS~ cont~ned not only a ~rge amount of vanadate- of TRIS and MES ~ ~ de~de~d pH vMue~ UDPG~T, sen~tive ATPase, but ~so a ~milar amount of the uriSte ~ph~ph~te ~uc~e-s~rol.glucmgUansfe~. plasma membrane marker ~ucan synthetase II,

01~452D~$05.00 © 1992 ~ f i ~ Soenfific P u ~ h ~ s briand Ltd. Pfin~d and Punished ~ ~dand

136

sugge~ing rather a contamination by ~asma homogen~ation and the fi~t centrifugation ~ the membrane [4]. pre~nce of 0.5 M m a n ~ t d ~med at ~mo~ng

It has ~cently been reposed that the ATPa~ amy~plasts, w~ch are abundant ~ Acer cultu~d acti~ty of plasma membrane, p a r t i ~ solu~hzed cells. Homogen~e was centrifuged ~ 1000 × g ~r ~om microsom~ of various m a t e r i ~ di~fibuted 8000 x g for 10 rain; supernatant was ~ t e d I:1 on a d e n ~ gra~ent ~ two ~st in~ peak~ show- with buffer D (wilhout m a n , toO and centrifuged ing ~ffe~nt characteristics [5-~. ~ a 50.2 Ti Beckman rotor at 108 000 x g (rm~0

In t~s work we fu~her ~vestigated the ~ r i b u - for 60 min. All the pell~s were resuspended ~ fion of vanada~-~nfit~e ATPa~ acti~ff of Acer buffer C and ~ored at -80°C. proto~a~ microsomes on a continuous and on a Su~ose denM~ gradients. ~) Continuous gr~ two-~ep sucrose gra~ent and we compared ~ent: 2.5 ml ~ u o ~ of 8000 × g/108 000 × g some charac~ristics of t~s ATPa~ activity pellet ~suspenfion, diluted to correspond to about equilibrating at ~f fe~nt denfitie~ We found that 150 × 106 protoplasts, were layered onto a 36oml the vanadate~enfitive ATPa~ activity migrating continuous gra~ent of 12-50% sucrose (w/w) ~ with the tono~a~ verities has characteristics of me~um E. T~s was centrifuged at 27 000 ~v./min pH, ~ m p e r ~ u ~ and phospholi~d ~nfitivity ~ ~ a SW 28 Beckman rotor (130 000 x g for rma O ~rent ~om those g e n e r ~ reposed for ~asma for 15 h. The gra~ent was ~ d e d into 16 ~ac- membrane ATPa~. tions of 2.4 ml ufing an ISCO ~actionato~ the

~actions were ~ lu~d 1:5 with me~um F and ~en- Matefia~ and Methods trifuged ~ a 50.2 Ti Beckman rotor at 108 000 g

for 60 min; pallets were resuspended ~ me .u rn C P~nt mater~! and stored at -80°C. (b) T w o ~ p gradient: ali-

Suspenfion cell cdtures of Acer pseudop~tanus quots equM to those used for the continuous gra- L. (Bligny call 1 ~ , ~ n ~ y prodded by P~o~ J. ~ent we~ hyered onto a t w o ~ p gradient Guern ( G i ~ s u ~ Y v ~ , FrancO, we~ grown ~ the confisting of 13 ml 43% sucrose (d = 1.19 g ml -~) fiq~d me.urn described by Bligny [~ at 25°C ~ ~ me.urn E, 13 ml 30% sucrose (d = 1.13 g con~ant fight (1800 lu~ and m~nt~ned by sub- m1-1) ~ me .urn E, and 10 ml me .u rn C ~% cdturing wee~y. S~-day-dd subcultures at the sucros~ d = 1.03 g ml-~). A~er centfifugation at end of exponenfi~ phase ~bout 1.5 × 106 c~ls 27 000 ~v./min in a SW 28 Beckman rotor for 3 ml -~) we~ used for the experiments, or 16 h, membrane ~actions were ~moved ~om

the 8°/d30% and the 30°/d43% interfaces ufing a ~o~t~n of protoplas~ and preparat~n of mem- Pa~eur ~pette. The bw density and ~gh density brahe fract~ns ~actions we~ ~ h ~ d 1:10 with me .u rn C and G,

Proto~a~s we~ prepared by enzymatic ~ge~ ~spe~Ndy, and centrifuged ~ a 50.2 Ti Beckman fion. Cells ~bout 10 g ~esh wright) we~ washed rotor at 108 000 × g for 60 min; pallets we~ re- with me .urn A, then incubated under bw a~ta- suspended and ~ored as for the continuous gra- tion ~0 ~ / m i n ) at 25°C ~ 20 ml of me .urn A ~ent. All operations were performed at 0-4°C. c o n m ~ g 1% (w/v) cdlulase RS and 0.1% pec- tolyase Y 23. A~er 120 m ~ of dige~ion the su~ Media composition penfion was f i l ~ d through 10~#m mesh n~on Me.urn A 0yfis of p r o t o ~ a ~ : 25mM TRISw cloth, diluted with me .urn B and centrifuged ~ MESL (pH 5.5) c o n t ~ n g 0.5 M man~tol. Me~- 180 × g for 5 min ~ 4°C. The pell~ed proto~asts um B (homoge~zation): 30 mM TRISmMESL we~ diluted with 3 vdumes of me.urn B (pH 7.5), 5 mM EGTA, 3 mM DTT, 1 mM PMSF, (homoge~zation medium with 0.5 M man~tol) 3 mM MgSO~ 0.5% (w/v) BSA, 0.5 M man~td . and counted ~ a Nageo~e chamber. Me.urn C (membrane ~suspenfion): 5 mM

The proto~a~ suspension (5-7 x 106 proto- TRISwMESL (pH 7.5), 1 mM DTT, 0.5 mM ~a~s ml -t) was homoge~zed with a hand oper- PMSF, 0.1% BSA, ~25 M sucrose. Me.urn D (d~ ~ed ~ a ~ Po~e~Elve~em homoge~er . The ~tion of 8000 × g supern~anO: same as me .urn

137

B but without mannitol. Medium E ~ucrose gra- MgSO~ 0.1 mM ammonium molybdate, 4 × 10 -4

dient): 5 mM TRISe-MESL (pH 7.5), 1 mM DTT, mM nigerian, 5 ~g/ml oligomy~n, 0.3 mM Na- 0.5 mM PMSF. Medium F: same as medium C but pyrophosphate, 0.03% Triton X-10~ ± 100 mM without sucrose. Medium G (dilution of heavy KNO3. The reaction was run for 30 min at 30°C. gradient fraction): same as medium C but w~h a The v~ues are expressed as the difference between sucrose concentration suitable to reach a fin~ con- the v~ues obt~ned in the presence and in the cen~ation of 0.25 M sucrose when added to the absence of K +. heavy fraction.

P r o ~ assay Enzyme assays Membrane protons were de~rmined according

ATPase acti~ty was assayed by measuring the to the method of Lowry modified by Markwdl et Pi rdeased according to the method described by ~. [13] on membrane ~ iquo~ diluted 50-fold with De M~helis and Spanswick [9]. The reaction me- 1 mM MgSO~ centrifuged at 100 000 × g for 60 dium (1 ml) cont~ned: 40 mM TRIS~MESL min and resuspended in d~ti l~d water to avoid in- buffer at the indicated pH, 100 mM KCI, 3 mM ~r~rence by BSA and other components of the MgSO~ 0.1 mM ammonium mo~bdate , 4 × 10 -4 resuspen~on medium. mM nigerian, 3 mM Na-ATP.

The reaction was ~a~ed by adding an appropri- Chemica~ ate aliquot of membrane suspen~on (co~espon- LPC was ~om Sigma C h e m ~ Co., type I con- ding to about 1-10 ~g membrane pro tons and taining primari~ p~mitic and ~earic aods. BrO 58 allowed to proceed for 60 min at 30°C. Variations R was ~om Aldrich Chemic~ Company. The of these standard conditions are spe~fied in the ~ock solution of BrO was 20 mg/ml in ethanol 48% Tables. In all conditions substrate consumption (w~) according to P~mgren et al. [14]. did not exceed 10% and the Pi re~ased was linear over con~dered time. Sta t~cs

A~d~senfit ive ATPase activity was c~culated The assays of each experiment were run in as the difference in the ATPase activities assayed tfipl~ate and ~1 the experimen~ were repeated at in the absence and in the presence of 5 mM NAN3. lea~ three times. The behaviour of each experi- NRra~ens i t i ve ATPase and v a n a d a ~ e n ~ t i v e ment is ful~ reproduob~, even if the actNity ATPase acti~ties were c~culated as the difference v~ues were ~omewhat variable in different mem- in the ATPase actifities a~ayed in the absence of brane preparations. Therefore, the reposed r e s d ~ the inhibito~ (50 mM KNO 3 and 100 ~M are from representative exper iment . Standard NaaVO4 respect~dy) with 5 mM a~de included de~aton o fa~ays was less than !% for ATPase a~ in the reaction medium. The ~ze of uninhibi~d tivity measurements. background activity in the presence of a~de, ni~ate and vanadate together is very low (in the Resu~s range of 5-10% of the actifity in the absence of in- hibitor~. LPC or BrO 58 were added when in- Preparat~n of membrane veMc&s dicated in the Tables. The microsom~ ~a~ion was obt~ned ~om

Glucan synth~ase II was assayed according to proto~asts isolated ~om Acer cells using differen- Ray [1~; UDP-~ucose-sterol-~ucosyltransferase fial centrifugation. Table I shcws the ATP- according to Hartmann-BouiHon and Benven~te hydr~yfing act i~t~s of microsom~ membranes I11]; cytochrome c oxidase was a~ayed after 3 min sedimented at 108 000 × g ~om a supernatant ob- of proncubation of membranes with 0.03% t~ned a~er centrifugation of lhe homogenate at digitonin according to Hodges and Leonard [1~. 1000 x g or 8000 × g. Differenti~ sen~ti~ty to

PPase activity was measured as described for the g e n e r ~ con~dered speofic inhib~ors was used ATPase assay. The reaction medium (1 ml) con- to ~st ing~sh the ATPase activities of plasma t~ned: 25 mM BTP-MES buffer (pH 7.5), 3 mM membrane, m~ochondri~ and vacuolar mere-

138

TaMe L AT~hydr~ing~fififi~ ~ 108 000 x g ~acfion evant shoulder in the low denfity re#on co~ obtained aft~ prelimina~ ~mfi~gation ~ 1000 x g or 8000 responding to the peak of niWate senfitive ATPase X g. VMu~ ~ brakes ~ p ~ m ~e peseta of ~e ~tM AT- activit~

Pa~ a ~ i ~ . The percentage of vanadate-senMtive ATPase F~cfion ATPa~ activity ~ m ~ ~ x 60 min -~ acti~ty found at low den~ty re#on of grad~nt

x lO -6 proto#a~ was greater than that commonly reposed for the vanada~ sen~five ATPase of the '~ght fraction'

A~d~ Ni~m~ Vanada~- obtMned ~om other materiM~ but ~m~ar to that sensffive sen~f ive sens~ive

found by Montfichard et M. [2] and by Henry and ~H 8.~ ~H 7.~ ~H 6.5) PiMt [1] in tonopla~ preparations obtained ~om

1000 × g 750 (l~ 140 ~ 166 (16) ~ola~d vacuoles or from density gradient- 108 000 x g 3387 ~ 400 (7~ 873 ~ ffactionated m~rosomes from call cultures. To

8000 x g 4 7 4 2 ~ 4 8 3 ~ 1054(69) verify whether the vanadate-senfitive ATPase 108 000 x g 335 (7) 193 ~ 474 ( 3 1 ) equilibrating at low denfity is associated with the

tonoplast, as suggested by these authors [1,~, we compared the d~tribufion of ATPase activities of vacuolar and plasma membranes with that of other marker enzyme activities (K+-stimulated pyrophosphatase for the tonopM~; glucan syn-

brane. Each ATPase acti~ty was measured at i~ thetase II for plasma membrane). Figure 1B shows optimum pH (vanadate-sensitive at pH 6.5, afide- that the peak of K+-dependent PPase activity was senfifive at pH 8.5 and ni~ate-senfifive at pH 7.8, fully coinddent with that of the ni~ate senfitive respectivdy). ATPase acti~ty and the #ucan synthetase H acfiv-

The data of Table I show that centrifugation at fly had the same di~fibufion as the vanadate- 8000 x g resulted in the dimination of the bulk of senfifive ATPase activity. These data suggested mitochondrial activity (93%) although it Mso that the vanada~ senfifive ATPase activity found brought about a confiderable reduction (about in the low denfity r e#on belongs to the plasma 70%) cf tonopla~ and plasma membrane activi- membrane rather than to the tonopla~. ties. The fractionation of the 1000-108 000 x g In order to see whether the vanadate-senfitive pellet on discontinuous sucrose gradient (data not ATPase activities equilibrating at different den- shown) wh~h would have provided a be~er yidd, fities have different characteristics in the following did not Mlow a sat~factory separation of the three experiments a t w o 6 ~ p sucrose gradient was ATPase activities, due to the large amount of mi- defigned to separate membranes enriched in ~ther tochondfiM membrane present in this pallet. Thus nffrate~en~tive or vanadate~en~five ATPase ac- we loaded the 8000-108 000 pellet on a confinu- tivity (see Materials and Methods), and we col- ous sucrose gradienL ~cted a 'light' fraction at the 8%/30% interface and

a 'heavy' ~acfion at the 30%/43% interface. D ~ i b u t i o n o f the A TPase a c t ~ s on a cont~u- ous sucrose gradient A TPase acH~t~s associa~d with. low and high den-

Figure IA shows the d~tfibution on a continu- Mty membranes ous sucrose gradient of the nkrate-senfitive (pH The di~ribution of each of the ATPase activities 7.8) and vanadate-senfitive (pH 6.5) ATPase activ- between, the two ~acfions obt~ned from the ities. There ~ a sharp peak of nkrate senfitive AT- 8000-108 000 x g microsom~ preparation load- Pase activity at 27-30% sucrose (corresponding to ed on the two-~ep sucrose gradient is shown in a density of 1.1 l -1 .13 g ml -~) The main peak of Table II. The bulk of the ni~ate-senfifive ATPase v a n a d a ~ e n f i f i v e ATPase actifity is at 33-40% activity (77%) equilibrated in the light ~act ion and sucrose ~orresponding to a denfity of 1.14-1.17 g the greater pa~ of the vanadate-sens~ive ATPase ml-~), as generaHy reposed. HoweveL the profile actifity (65%) and of the mkochondr i~ ATPase of vanadate-senfitive ATPase activity shows a r d - activity (79%) were present in the heavy fraction.

139

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R ~ 1. Confinuo~ s ~ # a ~ t ( 1 ~ 5 ~ w ~ o f t ~ ~ m M ~ ~ l ~ ~ X ~ ~ m ~ ~ ~ m ~ & ~ m d ~ d ~ r 15 h ~ ~ 0 ~ ~ n ~ a SW28 B ~ k ~ n ~ . A: ~ - - ~ ~ d ~ m n f i ~ e ~ d ~ - - ~ ~ m R N e A ~ P~e ~fif if i~, ~ d (~) ~ e m ~ o ~ . B: ~ - - ~ # ~ ~ n ~ a ~ H and ~ - - ~ K ~ m ~ m ~ PP~e a ~ , ~ d (*) ~ ~ n ~ n ~ m . T ~ f ~ f i o m c ~ d from t ~ ~ a ~ e m ~ d ~ e ~ o m M ~ ~ - l ~ ~ x D we~ ~ m f i ~ d m 108 ~ x g ~ t ~ en~me ~ . The e n ~ m ~ ~f i f i f i~ me~u~d ~ ~e ~ o m M ~ f i ~ ~e: 1~ nm~e ~ x l0 ~ W ~ M m x ~ ~ n -~ ~ r n~r~e~enfifive ATPa~; 268 nmM~ ~ x ~ ~ n -I x l0 -6 protop~s~ ~ r vanada~6enfifive ATP~e; 122 nmdes ~ x ~ ~ n -~ x l0 ~ p ~ o # ~ s ~ r K ~ P P ~ 1~ pm~ UDPG x 15 ~ n -1 x l0 ~ ~ o ~ a ~ ~ r # ~ a n ~ n ~ e II. T ~ m ~ ~ o ~ n ~ n ~ m w ~ ~ ~ 1 0 6 ~ o # ~ By ~mpaf in~ ~ r e ~ h ~ a~f i f f , ~e ~ m ~ ~e ~ s ~ ~ M ~ me~u~d ~ ~ t ~ ~ s ~ ~ m ~ ~ ~ ~ ~ m m M ~ , ~ ~ ~ ~ ~ : 82% ~ r ~ a ~ r e n f i f i v e A T P ~ 85% ~ r K+-PPa~, 65% ~ r vanadatemenfifive ATPa~, 65% ~ r # ~ a n synthetase H ~ d 9 ~ ~ o r ~ n contenL

Table H. A~d~, nit~te- and v a n a d ~ n f i t ~ e ATPa~ a~ In all experiments, the presence in the fight ~ac- ~fifi~fmc infion~adedth onefig~an:two.stepsucro~adh ient.eavy~fi°nsf°maS000-1D 08atacome~00om 0 x t ion of a confiderable percentage of vanadate- a rep~ntat ive expefimem and ~ e ~xpres~d ~ n m ~ ~ × sensitive ATPase activity was confirmed. Prolong- 60rain -~ x 10 ~ p r ~ o ~ V~u~ ~ brakes ~ p ~ m ~e ed centrifugation time (from 3 to 16 h) did not p e ~ e ~ ~ d b u f i o n of e ~ h actifi~ betw~n ~ e two fractions, bring a b o u t significant changes in this d i~dbu-

tion. Sim~ar resulB were obtained when eRher ~- - - ~ ~gtt ht~3/1.1~aacfi°, n - - ~ ( 1 . 1 ~ l . l H oeavy~acfi°, n ~esh or previou~y ~ o z e n (stored at -80°C) ml-~) mi-~) microsomes were loaded on a sucrose grad~nt. An

atmmpt to ,~act ionate the same m ~ r o s o m ~ A~d~nfifive ATPa~ preparation on a dexffan grad~nt [15] fai~d to

~H 8.~ 43 ~1) 159 ~ improve the separation of these two acfivR~s (data N ~ e n s i t ~ e ATPa~ not shown). Vanad~n~fiv(p e H 7.8) ATP~e 83 ~ 25 (2~ Table III shows that the effects o f ni~ate and

~ n &~ 69 (3~ ~28 ~ vanadate on the ATPase activ~y found in the ~ght ~act ion were additive, confirming that the

140

Ta~ellL Eff~tsof~trateandvanadateon ~eATPa~ ~- and 108 000 X g microsomes (Table IV, A) and f i ~ ~ d ~ the hght fraction ofa tw~ep ~ c ~ g ~ d ~ . between the two gradient ~actions (Table IV, B) in

V~eru S n ~ p n H m ~ C°pime~.86~°inmmin-th ~[ p~ntY ~ ~cx ~10~ pr°t°~asteX s'Pefimencf t 5 mM NaNa 3. n~l~e ~ YsP~S~e~ inhib~o~ensifivt e h e same proportioA nTPasea~ctivities . t h e c°rresp°ndinT g h e s e results

confirm that the vanadate-senfitive ATPase activi- ATPa~ ~tivity ty associated with the fight ~action bdongs to the

plasma membrane rather than to the tonoplast. -Vanada~ (l+00Vanada# teM) AVanadate In the discontinuous gradient the recovery of

the activities (Table IV) is lower than in the contin- - N i ~ e 85.5 6~6 24.9 UOUS gradient (about 80%, see ~gend of Fi~ 1). +Ni~e (50 mM) 3~8 15.7 2~1 HoweveL as ind~a~d in the ~gend of Fig. 1, the

Miquot of the microsomal ~action used in this ex- ANiline 45.7 44.9 pefiment for the assays of total activitie~ was

recentrifuged with a ~eatment corresponding to that of the ~actions a~er collection ~o m the gra- dient ~ee Materials and Methods), while the acfiv-

vanadate-sensRive and the hi,ate-sensitive AT- Ry v~ues for 108 000 × g microsomes of Table IV Pase activities are due to two different emymes, refer to a membrane ~action pelleted only once at

Data of Table IV show the d~tfibution of other 108 000 x g. A control experiment showed that membrane marker activities: cytochrome c o x - about 35% of the vanadate~enfitive ATPase activ- idase, K÷-stimulated PPas~ ~ucan synthetase II Ry, and about 20% of the other activities (data not and UDPG-ST. These membrane marker activities shown) are lost in a succe~ive centrifugation of di~fibuted betwedn 8000 x g membrane ~action microsomes at 108 000 x g. Fu~hermore the low

TaMe IV. Acfi~fi~ of ~ide-, ~tm~- and vanada~-~nfifi~ ATPa~ of ~h~-mark~ en~mes and ~otein conm~ ~ 8000 x g pellet, ~ 8000 × g-108 000 x g mi~omes (A) and ~ the two ~acfions of a two-~ep sucrose ~a&cm (B). Data come from one ~ p ~ m a t i ~ ¢xpefimenL V~ues ~ ~ k e ~ ~present the per~m ~fibufion of each activity between 8000 x g pellet and microsom¢~ ~ A, and between Hight and heavy ~act~ns, ~ B.

A B

8000 x g pellet M~m~m~ ~g~ fraction H~vy ~ f i o n ~000-I08 000 x ~

Azide-~nsitiv¢ ATPa~ a 4468 ~i) 414 (8) 43 ~1) 159 ~ C~oc~ome c ofida~ b 91 629 ~ 4100 (4) 126 (~ 1731 ~3)

Nitmtc-~nfitive ATPa~ a 404 ~ 237 (3D 83 ~D 25 ~D K~sfim~a~d PPase c 445 ~ 258 ~ 94 (7~ 37 ~

Vanada~-~nfifive ATPa~ a 916 ~ 482 ~ 70 ~ 127 ~ Glucan ~ m h ~ e II ~ 615 ~ 331 ~ 80 ~1) 115 (5~ UDPG~T e 1842 ~ 1083 ~ 269 ~ 322 (5~

P r ~ f 63 23.9 13

~md~ ~ x 60 min -~ x 10 -6 pr~ofla~s. ~pm~ cytoc~ome c x min -~ x 106 p rmo~t . ~ m ~ ~ x ~0 min -l × 10 ~ prmoflags. dpmd~ UDPG x 15 min -~ x 10 -6 proto~a~s. epm~es UDPG x 30 rain -~ x 10 ~ pro~asts. f#g x 10 ~ Votoflasts.

141

yidds of enzymat~ a c t i v ~ s in the d~continuous l y in g activity in question is not attributable to an gradient can be aafibuted to some loss during the aspe~fic ADPase [17], inasmuch as, in the collection of the ~action~ as supposed by the fact presence of ADP, activity was 15% of that with that ~so the p ro ton recovery was reduced. ATP in each of the two ~actions.

The percent di~ribution of the different enzyme Dependence on pH. The effect ~f pH on the a c , v ~ ' s " " in the two ~acfions was fully reprodu~- vanadate~en~five ATPase activity asso~a~d w~h ble in the ~x two-~ep performed gradients and it lhe two ~ c t i o n s is shown in Fig. 2. The activity is the same as the percent di~ribufion c~culated in found in the heavy ~action showed a sharp pH op- the two '~actions' obt~ned by adding the activi- timum of 6.5, in good agreement with what is ties of the ~actions 1 to 9 and 10 to 16, ~om Ihe generally observed for plasma membrane ATPase. continuous gradient. The shape of the vanadate-sen~five ATPase in the

fight ~action was different, as i~ pH optimum was Characterizat~n of the vanada~ensit~e A TPase ~ sharp and shifted to a more ~kaline pH. ~ the light and heavy fracHons Heat-stability of the vanada~ensit~e ATPase

Subswa~ specifi~ty. We inve~igated the sub- activ~ies. We invesfiga~d the effect of tempera- ~rate speof io ty by measuring the phosphohy- ture on the ~abi~ty of vanadate-sen~tive ATPase droly~ng activity of the light and heavy ~actions activities of the two membrane ~actions by pr~n- in the presence of different nuc~otides. The te~uRs cubating lhe membranes in the assay medium reposed in Table V show that in both ~actions without ATP for 30 min at 25 °, 33 ° and 40 °. AT- ATP was the most effective sub, ra te , whi~ GTP Pase activity was then assayed at 30°C for all and UTP were scarcdy hydrolyzed. Pi rdeased in samples. the presence of ADP was around 15% of that rdeased in the presence of ATP, in both ~actions.

The sub , ra te spedfioty of the enzyme is the same in the two ~actions and corresponds to that generally reported for plasma membrane ATPase .E ~om plant materials [16]. Moreover, these data ~ 2 o o -

C) show that the vanada~-sen~five phosphohydro- ~o

~ ~50 c).

.qo

~. Table V. Substrate specificity of the vanadate-sensifive ~ phosphohydrolyfing acfivR~s of m~rosomes and of fight and ~ 100 heavy ~acfions ~ o m a two-step sucrose ~adient . The activities ~ were assayed at pH 6.5 in the p re~nce of 3 mM GTP, UTP or I~_- ADP, and are expressed as percentage of the corresponding ac- ~ fivity with 3 mM ATP as a substrate. The rates of hydrolysis ~ with ATP were 579, 94 and 196 nmol Pi × 60 min -~ × 10 -6 ~ 50 protoplasts, in microsome~ ~ght ~acfion and heavy ~acfion, ~spect ivdy.

M ~ m ~ ~ g ~ Heavy 0 - I 1 I I I ~000-108 000 x ~ f f~ f ion f f~ t ion

6.0 6.5 ZO Z5 8.0

AT~ 100 100 100 pH GTP 9 13 7 Fig. ~ Effe~ ~ pH on the vanadate-~ns i f i~ ATPases ~ d UTP 5 9 5 ~ ~ e hgM ~nd heavy ~ t i o ~ of a t w ~ e p ~ o m ~ a ~ e m . ADP 13 15 14 Valu~ come from a ~p~mnta t ive ~pef imem. ~ - - ~ , ~ght frac-

tion; O - - O, heavy ~acfiom

142

Table VL Effe~ of preincubation m ~c~as~g ~mp~mu~s Table ¥1L Effect of Br~ 58 and of LPC on the vanadate- on ~e x a n a d ~ n f i f i v e ATPa~ activi~ ~ ~e lwo ~acfions ~nfifive ATPa~ acfififi~ ~ ~e tig~ and heavy ~actions of a o f ~ t w ~ e p sucrose grad~nt. Membran~ we~ pr~incuba~d t w ~ e p sucro~ grad~nt. V~ues come ~om one ~ p ~ fer 30 min ~ ~ y medium ~H ~ withom ATP ~ ~e ~dicated tive expefimenL and am exp~ssed as n m ~ ~ x ~0 min -~ x ~mper~u~s; then the ATP~e ~acfion w~ s ~ e d by ad~fion 10 -6 pr~o~a~s. of ATP and run ~ 30°C for 60 min. ATPa~ actifi~ ~ non- p~incubated membranes was assayed ~ parallel. Data come from T~mmem ATPa~ a ~ i ~ one ~ p ~ n ~ f i v e experiment and a~ e x p ~ e d as nmdes ~ x 60 min -~ x 10 ~ p r o t o ~ t s . V~ues in bracke~ ~ p ~ n t Lig~ ~acfion Heavy ~acfion the pe~ent effect of p~ incub~n .

% % Temper~u~ of ATP~e activity p ~ c u b ~ n Effea of Br~

Lig~ ~acfion Heavy ~acfion Nil 76 203 Br~ 58 (150 #g x ml -~) 123 +62 309 +52

No preincubation 73 169 ~00 #g x m1-1) 117 +54 304 +50 25°C 69 ( - ~ 155 (-8) 33°C 69 (-5) 136 ( -2~ Effea of LPC 40°C 66 ( -1~ 123 ( -2~ LPC ~0 ~g x m1-1) 129 +70 485 +139

Bfij (150 ~g x ml -~) + LPC 150 +22 515 +67

Bdj O00 #g x ml -~) + LPC 151 +29 505 +66

TaMe VI shows tha t the responses to heat o f the two A T P a s e activit ies were different . Pre incuba- t ion at increaf ing tempera tures p rogres~ve ly s t imula t ing effect o f Br~ (150 #g ml -~) was a b o u t reduced the A T P a s e act ivi ty in bo th fractions. 50°,6, d id no t change at a 300 ~g mg -~ concent ra - However , the inact iva t ing effect was more m a r k e d fion, and was the same in the two fract ions. The e~ in the heavy f rac t ion (see ReL 5). fect o f LPC was greater and the s t imula t ion o f the

Effect of lysophophatidylcholin~ Several recent ATPase greater in the heavy f rac t ion than in the da t a show that the A T P a s e act ivi ty in the p l a sma Hght. membrane ~f ic t ly depends on i ~ ~pid environ- These results suggested that the na tu ra l ment (phosphol ip ids , s t e ro id , though the mecha- phospho l ip id may act no t only by unmask ing the nism involved in this regu la tory process remains la tent active sites, but also in some more specific unknown [18,19]. We invest igated the effect o f two manner . To conf i rm this poss ibi l i ty we l e ~ e d the

de te rgen~ known to act ivate the A T P a s e act ivi ty effect o f LPC in the absence and in the presence o f in p lasma m e m b r a n e veficles by modi fy ing its l ipid lhe detergent Btij 58. The da ta o f TaMe VII show envi ronment and by abol i sh ing the la tency o f the tha t LPC fu~he r s t imulates A T P a s e act ivi ty in the enzyme (TaMe VII). presence o f Brij and tha t also in these condi t ions

We used (i) a de tergent o f the BrO series 0 o n g the ~ imu la t i ng effect o f LPC is 2 - 3 t imes greater chain po lyoxyethylene acyl e t h e r ~ which perme- in the heavy than in the Hght fract ion. abil izes the membranes to subst ra tes even when given at low concent ra t ions and does no t inhibi t Discussion ATPase act ivi ty even at high concen t ra t ions [14], and (ii) LPC, a na tu ra l de tergent which has been The resu l~ presented in this pape r indicate that , r epor ted to act ivate ATPase act ivi ty also in a when a microsomal p r e p a r a t i o n ob ta ined f rom latency-free system [14,20]. LPC was used at 50 ~g Acer cell p r o t o p l a s ~ is f rac t iona ted on a cont inu- m1-1 fince ffs effect was the same even at double ous or a d i scont inuous sucrose grad ienL a b o u t concent ra t ion (da ta not shown). 35 -40% o f the vanadate-senf i t ive A T P a s e act ivi ty

The da ta o f Table VII show that bo th BrO 58 always migrates to low denf i ty toge ther with the

and LPC s t imula ted the vanada t e~en f i t i ve A T P - bu lk o f the ni t ra te-senf i t ive A T P a s e and ase activi ty o f the Hght and heavy fract ions. The K÷-s t imula ted PPase t o n o p l a ~ markers .

143

The finding that the inhibiting effects of nkrate put in evidence in our research, could correspond and vanadate on the ATPase activity of the fight to two distinct enzymes of the plasma membrane fac t ion were completdy additive suggests that the or to different conformations of the same enzyme two inhibitors do not act on the same enzym~ The determined by different characteristics of the sub , ra te specific~y of the vanadate~en~tive AT- membrane or of regions of the same membrane Pase activities present in the two fractions cot- (~erol and/or fipid compo~fion, fipid/protein responds to that of the plasma membrane ATPas~ ratio). thus ruling out also the possibility that the From a phy~ological point of view, these di~ vanadate-sen~tive phosphohydrolyzing activity in ferent ATPase activitie~ whether they reflect the the fight fraction ~ due to amyloplast envdope expres~on of different genes or a different corn- contamination [21], or to an aspecific ADPase por t ion of the membran~ could be finked to di~ [17]. UDPG-ST and glucan synthetase II acfivi- ferent phy~ological condition~ In our case the tie~ markers of the plasma membran~ have the differences could be finked to the presence of cells same di~fibufion between the two fractions as the of different age in the non~ynchronous Acer vanadate-sensitive ATPase activity. These data in- culture employed. This view is in agreement with dicate that the vanadale~en~tive ATPase of the the data of Kasamo and Nouchi [26], showing that low density fraction belongs to the plasma mem- the plasma membrane ATPase ~olated from di~ brahe and does not seem to be associated w~h the ferent tissues of mung bean had a different sen- tonoplast, as sugge~ed by Montrichard et al. [2] ~tivity to phospholipids, and wkh the finding of and Henry et al. [1]. Poo~ et al. [27] of a difference in the apparent

The most interesting aspect of our data ~ that den~ty of the beet plasma membran~ depending the two vanadate~en~tive ATPase~ which on whether ~ ~ ~olated f o m growing or from migrate to different d e n ~ t ~ differ as to depen- dormant material. dence on pH, sen~tivity to heat inactivation and stimulation by LPC, wh~e thry have the same Acknowkdgements substrate speoficity and are both inhibited by Ca ++ (data not shownL Th~ finding ~ in agree- The authors wish to thank M.I. De M~hefi~ E. ment with previous data obtained in different con- Marr~ and F. Rasi-Caldogno for valuable sugges- ditions and material~ ind~ating that the plasma tions and for critical reading of the manuscript. membrane ATPase of plant c d ~ ~ not a Research suppo~edbyNat iona lResearchCounci l homogeneous activity. Different vanadate- of Italy, Special Project RAISA, Sub-project N.2, sen~tive ATPases have been distinguished on the Paper No. 27~ ba~s of different responses to inactivation by Mg and to stimulation by FC On rad~h membrane References veeries [22]) or on the grounds that th~ ATPase, partially solubilized from m~rosomes, di~ributed 1 ~no~aH ~ . HenrYHa÷n~mn~ocating A T P a P ~ . E . ~ L in~NtiOno~ubu s c d ~ V .anada~ O~ant the on a den~ty grad~nt in two d~tinct peak~ show- s~., 56 (198~ 149-15& ing different characteristics as to pH optimum and 2 F. Momdch~& A. Pu~n and Y. Gaudem~, InaCtion sen~tivity tO heat inactivation ([5], in radishL sen- of the vacu~ar ATPa~ of Acer pseudop~tanus cd~ by ~tivity tO photoinactivation ([6] in Rosa vanada~. ~oc~mie, 71 (1989) 813-81~

damascena), stimulation by K÷and by LPC ([7] in 3 Ch~ac~sti~ of ATP~e f r o M m " Thom, J. W~nbfinksugarcana e n d Ap~o~a ~ a n M dar~z~, mung beanL In some cases the hypothes~ of the vacu~e memb~n~. Phy~. ~am., 58 (198~ 497-504. exi~ence of two d~tinct ATPases on the plasma 4 R. C~ombo and V. Pa~, T o n o ~ t ATPa~ activity ~ membrane has been supposed by immunological proto~ast and vacuO~ memb~ne p~par~s ~om ~udies [23] and espedally by the identification of Acerpseudop~tanus cel~ Giorn. B~. R~., 123 (198~ 1~ at ~ast three isoforms of the plasma membrane 5 ~rM m.C. Cocuco N f Nasm~ndmemb~nE e . Mar~, H+_ATPAsC e~mematiOa nnd oOff ont ehe

ATPase gene [24,25]. ~ c o ~ ~ o r ~om ~d~h m~rosom~. ~ant S~., 73 The two vanadate-sen~tive ATPase activit~s (1991) 45-5(

1 ~

6 C.W. Imbrie and T.M. Murphy, Photoinactivation of 18 D.T. Cooke and R.S. Burden, Li~d mod~ation of de~ rgen t~u~f i zed ~asma membrane ATPa~ from ~ m a membran~bound ATP~se~ Phyriol. Ham., 78 Rosa damascen~ Plant Phyri~., 74 (198~ 617-621. (199~ 153-159.

7 N. Hito, T. Kimura and T. Asahi, Parti~ purification and 19 M.C. Cocucci and E. Mar~, L y s o p h o s p h ~ y ~ h ~ i n ~ characterization of an ATPa~ ~ mung bean hypocot~ acfiv~ed, vanada te -~b i t ed , Mg2+-ATPa~ from ra~sh ~asma membran~ suggestion for a new ~pe of ~gher microsomes. Bioc~m. Biophys. A~a, 771 (1984) 42-5Z ~an t ~asma membrane ATPa~. Plant Call PhyriC., 29 20 M.G. P f l m g ~ M. Sommarin, P. Ulvskov and ~L. (1988) 875-882. Jggensen, Mod~afion of ~asma membrane H%ATPa~

8 R. Blign~ Growth of s u s p c n s ~ n ~ t u ~ d Acer from o ~ roots by ~sophosphatid~choline, flee fa t~ pseudoplatanus cells ~ a u t o m ~ u ~ of h~ge v~um~ a~ds and p h o s p h ~ a ~ A 2. Phyri~. Plant., ~4 (198~ Hant Phyri~., 59 (197~ 502-505. l l - l ~

9 M.I. De M~helis and R.M. Spanswick, H ~ p u m ~ n g 21 E HarinasuL T. Takabe. T. Akazaw~ M. Tagaya and F. dfixen by lbe vanad~e ~nriti~e ATPa~ in membrane Fuk~, Charac~f izaf ionofanATPa~ a ~ o ~ a ~ d w i l h the ver id~ from corn root~ Plant Phyri~., 81 (198~ ~ner envflope membrane of a m ~ o ~ a ~ s from suspen- 542-547. r ion-c~tu~d ce~s of sycamo~ (Acer pseudoplatanus L.).

l0 EM. Ray, A u ~ n - ~ n ~ n g rites of m~ze co~optiles are Hant Phyri~., 88 (198~ !19-12~ localized on membran~ of the endo~asmic ~t ic~um. 22 M.1. De Micheli~ C. OINari, M.C. Pugliarel~ and F. Plant Phyfi~., 59 (197~ 594-599. Rasi-C~dogno, Effect of Mg 2÷, Triton X-100 and

I I M.A. Hartmann-Bo~flon and P. Benve~s~, Ster~ ~mperatu~ on basil and FC~fim~ated ~asma mem- biosynthetic capa~fi~ of purified membrane fractions brane ATPa~ activity. Plant S~., 54 (198~ liT-12& from m~ze c~eoptil~. Ph~ochemistry, 17 (1978) 23 T. Kimur~ M. Meesh~a and T. Asahi, l m m u n ~ o ~ c ~ 1037-104Z ~u~es on ATPases ~ mung bean hypocot~ ~asma mem-

12 T.K. Hodges and R.T. Leonar~ Purification of a ~asma brahe: propos~ of ~ e p ~ n c e of two mo~c~ar specks membran~bound adenoride tfiphosphatase from corn of ATPa~. Hant Cell Phyri~., 29 (1988) 883-88& root~ M~hods Enym~., 32 (1974) 392-40~ 24 J.M. Pardo and R. Serran~ S t ru~u~ of a ~asma mem-

13 M.A.K. Markwdl, S.M. Haas, L.L. Bieber and N.E. brane H÷-ATPa~ gene from the ~an t Arabidopsis T ~ b ~ L A m o r t i c i a n of the Lowry procedu~ to ~aliana. J. Bid. Chem., 264 (198~ 8557-856Z rimpli~ protein de~rm~ation in membrane and l~opro- 25 B. Mich~et, C. Per~z, A. Goffe~u ~nd M. Boutry, The tein sample~ An~. Biochem., 87 (1978) 206-210. ~asma membrane H÷-ATPa~ of Nicot~na plura-

l4 M.G. P f l m g ~ M. Sommarin, E Ulvskov and C. bag~olia, ~: J. D~nty, M.I. De M~heli~ E. Mar~ and Lar~o~ Effe~ of d~ergen~ on the H+-ATPa~ a ~ N i ~ E Rari-Cfldogno ( E d ~ Plant Membrane Transpo~: of inride-out and righ~rid~out ~an t ~asma membrane The Cur~nt Positio~ El~fier S~ence Pubfishers B.V., veric~s. Bioc~m. Biophys. Act~ 1021 (199~ 133-14ff 198~ p~ 455-460.

15 K.A. Chu~hill and H. Sze, A~on-~nri f iv~ H ~ p u m ~ n g 26 K. Kasamo and I. Nouchi, The r~e of phospholi~ds in ATPase ~ membrane verities from o ~ ro~t~ Plant ~asma membrane ATPa~ activity in Vigna radiam L. Phyri~., 71 (198~ 610-61% (mung bean) roo~ and h y p o c o ~ . Plant PhysiC., 83

16 H. Sze, H+-translocating ATPases: advances uring mere- 0987) 323-328. brahe vefide~ Annu. Rev. Phyfi~., 36 0985) 175-208. 27 R.J. Po~e, D.E Bfis~n, Z. Kr~ky and R.M. John~on~

17 L. Tognoli and E. Marrb, Purification and chara~eriza- Density gradient localization of ~asma membrane and tion of a div~ent c ~ n - a c t i v ~ e d ATP-ADPase ~om pea tonopla~ from ~orage ti~ue of growing and dormant ~ d ~em microsom~. Bioc~m. Biophys. Act~ 642 (1981) beet. Plant Phyfi~., 74 (198~ 549-55~ l - l ~