Further evidence for an ATP-driven sodium pump in the marine alga Tetraselmis (Platymonas) viridis

! Pl4nt PhysioL w,J. 150. pp. 264-270 (1997)

Further Evidence for an ATP-driven Sodium Pump in the Marine Alga Tetraselmis (Platymonas) viridis Yu.

BALNOKINI,

L.

POPOVAl,

and H.

GIMMLERh

I

Institute of Plant Physiology, Russian Academy of Sciences, Moscow, Russia

2

Julius-von-Sachs-Institut fur Biowissenschaften, Universitli.t Wiirzburg, Germany

Received May 17, 1996· Accepted August 2, 1996

Summary

The ATP-dependent 22Na + accumulation by inside/out plasma membrane vesicles isolated from the marine green microalga Tetraseimis (Platymonas) viridis Rouch. has been studied. The 22Na + uptake was observed only within a narrow region of weakly alkaline pH with a maxium at pH 7.8-8.0. When permeant anions such as N0 3-, were absent from the reaction medium, ATP-dependent 22Na + uptake was low but could be stimulated by 6 ~mollL CICCP. In the presence of N0 3-, however, the rate of ATPdependent 22Na + uptake was much higher than in the absence of nitrate and not affected by the uncoupler CICCP. The pH optimum of ATP-driven 22Na + uptake differed from that of ATP-dependent L\pH formation across the vesicle membranes (optimum pH 6.0-7.0). These data indicate that a L\~H+­ dependent Na +/H+ antiporter does not contribute to the observed uptake under the experimental conditions; 100 ~mollL orthovanadate completely inhibited ATP-dependent 22Na + uptake, whereas the uptake was not affected by 50 ~mollL amiloride and only slightly reduced by 200 ~mol/L of this inhibitor. It is concluded that the ATP-supported 22Na + uptake by Tetraseimis (Platymonas) viridis plasma membrane vesicles is carried out by a mechanism independent of the proton-motive force. For example it could be catalysed by a primary Na +-pump. It is thought that this pump is an orthovanadate-sensitive electrogenic Na +-ATPase.

Key words: Ir -ATPase, ionic homeostasis, plasma membrane, Platymonas, sodium-ATPase, sodium uptake, salt stress, Tetraseimis. Abbreviations: BTP = 1,3-bis[tris(Hydroxymethyl)methylamino]propane; CICCP = carbonyl cyanide m-chlorophenylhydrazone; OTT = dithiothreitol; L\~H+ = electrochemical H+ potential difference; L\

The maintenance of low cytoplasmic Na + concentrations appears to be a general property of all plant organisms. Na + is expelled from the cells against the gradient of its electrochem* Correspondence: Julius-von-Sachs-Institut fur Biowissenschaften, Universitlit Wiirzburg, Minlerer Oallenbergweg 64, 0-97080 Wiirzburg, Germany. © 1997 by Gustav Fischer Verlag. Jena

ical potential. It is generally accepted that a Na +/H+ antiporter in the plasma membrane (PM) is one of the mechanisms responsible for the extrusion of cytosolic sodium in exchange against H+ (Padan and Schuldiner, 1993) and that this ion exchange is secondarily energized by the proton-motive force (L\~H+) generated by the H+ -ATPase of the PM. Evidence for the existence of a Na +/H +-antiporter in the PM of halotolerant green microalgae was presented by Kaplan and Schreiber (1977, 1981), Katz et al. (1986, 1989, 1991, 1992)

ATP-driven sodium pump in Platymonas and Popova and Balnokin (1992). However, the question arises whether a Na +IH+ antiporter is an effective regulator of Na + concentrations in the cytoplasm under conditions of high salinity in combination with alkaline pH in the medium. If the Na +IH+ antiporter exchanges Na + for H+ with a stoichiometry of 1 : 1 (an electroneutral mechanism), the electrical potential (L1q» does not contribute to the driving force of sodium transport. Only the energy of the concentration component of L1IlH+, that is L1pH, is utilised in this case (Padan et al., 1981; Skulachev, 1989). Due to the presence of a pH-stat mechanism in the cytoplasm of microalgae, only slight changes in the cytoplasmic pH, mostly restricted to values between pH 7 and 8, do occur while the external pH varies in a broad range (Raven, 1985; Kugel et al., 1987; Gimmler et al., 1988; Madshus, 1988). Thus, the L1pH. across the PM of microalgae has the direction and magnitude required for Na + export by means of an electroneutral Na +IH+ antiporter only at acidic pH values of the medium. However, such situations do not always occur in nature. Many marine and halotolerant algae live in an environment the pH values of which are more alkaline than the cell cytoplasm. Under such conditions Na +-extrusion from the cytoplasm to the exterior could proceed only if the anti porter is electrogenic (an exchange of two or more protons against one Na + ion) (Padan et al., 1981; Skulachev, 1989). However, data on electrogenicity of the PM Na +IH +-antiporter of halotolerant algae and bacteria are at variance (Katz et al., 1989; Pick, 1992). Another possible mechanism for Na + export from the cytoplasm of halotolerant microalgae at alkaline pH into the medium is a primary Na+-pump (Na+-ATPase). The existence of a primary Na + pump has already been postulated to operate in the PM of the marine alga Platymonas viridis (Balnokin and Popova, 1994), because this alga is able to maintain low intracellular Na + levels in spite of proton gradients directed from inside to outside (Galkina and Balnokin, 1992) and PM vesicles isolated from this alga accumulate 22Na + when incubated with ATP and Mg (Balnokin and Popova, 1994). In this paper we present further evidence for the existence of such an ATP-driven Na +-pump in the PM of Platymonas viridis and describe some characteristics of this enzyme. Material and Methods

Algal culture The marine unicellular alga Tetraselmis (Platymonas) viridis Rouch. was grown under axenic conditions in artificial sea water with the following composition: 450 mmollL NaCl, 50 mmollL MgS04, 10 mmollL KN0 3, 0.5 mmollL NaH 2P0 4, 5 mmollL Ca(N0 3h. 1 mmollL KBr, 20 J.lmollL MnCh, 25 J.lmollL LiCl, 4 J.lmollL Na2Mo04, 6 J.lmollL AlCI3, 0.4 J.lmollL CoCh, 1.3 J.lmoll L RbCl, 0.04 J.lmollL CUS04, 0.3 J.lmollL ZnS04, 0.4 J.lmollL K], 0.4 J.lmollL BaCI2, 0.1 mmollL K1B407' 6 J.lmollL Nal Si03, 90 J.lmollL SrCh, 60 J.lmollL NaF, 1.5 J.lmollL FeCI3, and 6 J.lmollL NalEDTA. The pH of the medium was adjusted to 8.0 with NaOH; 50 mL flasks were used as culture vessels (axenic maintenance culture). For mass production cells were grown under non axenic conditions in 6-L rectangular Perspex vessels (50 X 20 X 6 cm) at 25-26 ·C, bubbled with a continuous stream of air plus 1.5 %

265

Cal' The suspension (light path 6 cm) was illuminated with 43 W m- 2 of fluorescent light (14: 10 h light: dark regime). Cells were harvested after the 7th day of culture (late logarithmic growth period) by centrifugation.

Determination ofintracellular sodium content Cells of Platymonas viridis of the standard culture were transferred to media of the same composition with the exception that the pH was buffered with 20 mmollL TRIS/MES to pH 5.5, 7.2, or 9.0. Cell concentrations of about 5 X 106 cells mL -1 were used. Cells were illuminated at room temperature with 40 W m -1 of fluorescent light and bubbled with air for 4 h. Then, salts of the medium were removed by layering the cell suspension onto a washing solution (0.8 mollL mannitol, 20 mmollL Ca(N03h. Balnokin and Mazel, 1985) and subsequent centrifugation. The resulting pellet was suspended in distilled water, and cells were destroyed by ultrasonification treatment using an ultrasonic desintegrator. Sodium content in the homogenate was determined with a flame photometer. The total cell volume, necessary for the calculation of sodium concentrations, was measured according to Okamoto and Suzuki (1964) from the difference between electrical conductivity of the cell suspension and that of the medium using an OK-10211 conductometer (Radelkis). Determined volumes very likely reflect slight overestimations, since Tetraselmis possesses a theka giving a considerable «free space» (Kirst, 1977 a). However, such overestimations do not change the principal conclusions of this paper.

Isolation ofplasma membrane vesicles Highly purified plasma membrane vesicles were prepared according to the method described by Balnokin et al. (1993) with some minor modifications. Platymonas viridis cells (5 L suspension, late exponential growth period, 5 X 106 cells mL -1) were harvested by centrifugation (700 gn, 10 min), washed twice with 700 mL of medium A (1.5 mollL glycerol, 10 mmollL Tris/HCl, pH 7.2) and finally suspended in 700 mL of medium A supplemented with 0.06 mg mL -1 trypsin (SIGMA, type III), 0.5 mM ATP and 1 mM MgCh. The suspension was gently mixed for 25 min at room temperature. The treatment of the cells with trypsin caused a partial proteolytic cleavage of the glycoprotein cell wall, especially at the apical pole of cells close to the site of the fixation of flagella to the cell body. The following steps were carried out at 4 ·C using precooled solutions (4·C). The cells were harvested from the trypsin medium and washed twice with 700 mL of medium A supplemented with 12 J.Lg mL -1 soy-bean trypsin inhibitor. The final pellet was resuspended in 700 mL of a hypoosmotic medium B (0.5 mollL glycerol, 20 mmollL HEPES/BTP buffer (pH 7.2), 1 mmollL MgCh, 0.5 mmollL K1S 20 S, 1 mmollL DTT, 10 J.lg mL -1 PMSF, and 0.8 mmollL EGTA, supplemented with 0.5 mmollL ATP and 5 J.lg mL -1 TI. The cells were incubated in this hypoosmotic medium for 30 min. This treatment caused an exit of cytoplasm through the orifice in the cell wall created by proteolytic cleavage and finally a formation of «cytoplasmic droplets». Separation of cells from cytoplasmic droplets was carried out by centrifugation (3000 gn, 15 min). Plasma membrane vesicles formed in the supernatant and were harvested from the supernatant by centrifugation (15,000 gn, 15 min). The crude membrane pellet was suspended in 15 mL of medium B and layered onto a 15 % sucrose cushion prepared in medium B. Plasma membrane vesicles were collected from the top of the cushion after centrifugation (100,000 gn, 30 min). The suspension of PM vesicles obtained was diluted with medium C (0.5 mollL mannitol, 20 mmollL HEPES/BTP buffer (pH 8.0), 1 mmollL MgCh, 1 mmollL DTT, 10 J.lgmL -1 PMSF, and 0.4 mmollL EGTA} and the membranes collected by centrifugation (15,000 g, 15 min). The final

266

Yu. BALNOKIN, L. POPOVA, and H. GIMMLER

pellet was suspended in medium C, so that the protein concentration was between 1.2 and 1.5 mg mL -I. Freshly prepared vesicles (inside/out orientation, Popova and Balnokin, 1992; Balnokin et al., 1993) were used for all the experiments.

Determination ofsodium uptake Sodium ~take into the vesicles was measured by tracer techniques using 2 Na +; 0.2 mL samples were preincubated at room temperature in the reaction mixture (see below) for 15 min. Then ATP (TRIS salt) was added (2 mmollL). After 3 min of incubation of vesicles with ATp, sodium uptake was initiated by addition of 22NaCI. The final composition of the reaction media was: 0.5 mollL mannitol, 10 mmollL HEPES/BTP (pH 8.0), 100 mmollL N0 3-/BTP (pH 8.0), 10 mmollL MgS04' 0.5 mmol/L DTT, 0.2 mmollL EGTA, 1O~gmL-I PMSF, 5mmollL 22NaCI (0.25MBq), 2mmoi/L ATP (TRIS salt) and about 160 ~ vesicle protein (standard medium). In the control medium ATP was omitted and the volume replaced by water. Any alterations in the composition of the reaction medium are indicated in the legends of tables and figures. For sampling, 45 ~L of the suspensions were taken and vesicles separated from the medium by filtering through prewashed nitrocellulose filters (Synpor, pore size 0.6~m). Filters were washed three times with 1 mL of assay solution free of the label and ATP. The radioactivity retained on the filters was counted with a y-scintillation counter (Betthold, type 5002).

Determination ofATP-dependent formation of!J.pH ATP-dependent formation of the pH gradient across the vesicle membranes (inside acid) was assayed at room temperature by monitoring the changes in absorbance of the amino-acridine pH probe, acridine orange (AO), as described previously (Popova and Balnokin, 1992). The assay was performed in 2 mL of a reaction medium containing 0.5 mollL mannitol, 25 mmol/L N0 3-/BTP as buffer, 20 mmol/L MgC12' 8 ~g AO, and vesicles corresponding to about 30 ~g of protein.

Determination ofATP hydrolysis The ATP-hydrolysis by isolated plasma membrane vesicles was calculated from the amount of inorganic phosphate released from added ATP. Inorganic phosphate (Pi) was determined according to Carter and Karl (1982). ATPase activity was assayed at 22 ·C for 15 min in 0.5 rnL of a reaction medium containing 0.5 mol/L mannitol, 20 mmol/L MES/BTP buffer, 0.2 mmollL EGTA, 2 mmollL ATp, 10 mmollL MgS04' 6 ~mollL CICCp, and vesicles corresponding to about 5 ~ of protein. The protein content of the vesicles was determined according to Simpson and Sonne (1982) with bovine serum albumin (BSA) as a standard.

Table 1: Cytoplasmic Na+ and H+ concentrations in intact Platymonas cells at various pH values of the external medium. External NaCI concentration: 450 mM. Data reflect the means of 3 replicates samples with standard deviations. Data assigned with an asterisk were measured for P. subcordiformis by Kugel et al. (1987). Parameter

[Na+Ln (moI/L) [Na +] ou,![Na +] in pH.n* [H+] in (mollL) [H+] ou, (mollL) [H+] ou,![H+] in

External pH 5.5

7.2

9.0

0.023±0.008 19.6 7.4 4.0xl0- 8 3.2xl0- 6 0.8x102

0.019±0.007 23.7 7.5 3.2xlO- 8 6.3x1O- 8 1.97

0.026±0.O1O 17.3 7.6 2.5x1O- 8 10- 9 4.0xl0- 2

remained at levels between 17 and 24 mmollL as the pH of the medium was changed from 5.5 to 9.0 (Table 1). According to the data of Kugel et al. (1987), obtained with the related species P. subcordiformis, cytoplasmic pH remains practically constant at about pH 7.5 as external pH changes in the range from 5.5 to 9.0. Assuming the same capacity of the pH-stat mechanism for P. viridis cells and using the cytoplasmic H+ concentrations measured by Kugel et al. (1987) for P. subcordiformisone can estimate the ratio [H+]ou/[H+];n for P. viridis (Table 1). With [Na+]ou/[Na+];n and [H+]ou/[H+];n values in hand, we can estimate the possibility of Na + extrusion out of P. viridis cells mediated by both electroneutral and electrogenic Na + IH+ -antiporters . .:l~H+ -dependent Na + extrusion performed by a Na +IH+ antiporter is described by the following equation (Kotyk, 1983):

where R, T, F and .:lcp are the usual thermodynamic parameters, and n is the number of H+ transported in exchange for one Na+. In the case of an electroneutral exchange (n =1), the only driving force for Na + extrusion is a concentration component of the proton gradient and the equation (1) is simplified to: (2)

According to equation (2), the minimal external H+ concentration required to maintain the experimentally determined Na + gradient (Table 1) by means of an electroneutral Na +IH+ antiporter is in the range of (0.4-0.8) X 10- 6 mollL. So, an electroneutral antiporter is competent for the maintenance of the sodium gradients across the plasma membrane Results only in the case that the external pH is less than 6.1-6.4. At higher external pH values other transport systems are reSodium and proton gradients across the plasma membrane quired for the export of sodium. and the capacity ofthe antiporter If two or more protons exchange for one Na +, an electroThe cells of T. viridis are capable of keeping the cytosolic genic exchange occurs and the transmembrane difference of sodium concentration much lower than it is in the surround- the electrical potential, .:lcp, is an additional driving force for ing medium (Table 1) (for review see also Kirst, 1977b). This Na + export. From equation (1) it is possible to estimate the capacity of the cells is displayed at both acidic and alkaline minimal values of .:lcp required to maintain the experimenpH values of the medium. At the external sodium concentra- tally determined sodium gradients (Table 1) for an electrotion of 450 mmol/L, the cytoplasmic concentration of Na + genic antiport. The potentials are calculated for the stoichio-

ATP-driven sodium pump in Platymonas

metry of2 H+11 Na+ or 3 H+11 Na+ (n=2 and n=3, respectively) for various external pH values. The higher the external pH, the larger is the A

M of T. viridis, required to maintain sodium extrusion by electrogenic antiporters at pH 9 (Table 2), are much higher than A

ATP-driven 22Na+ uptake into PM vesicles In order to identify Na +-translocating systems in the PM of T. viridis and to elucidate their characteristics, the uptake of 22Na + into inside/out plasma membrane vesicles, isolated from this alga, has been investigated in the presence and absence of ATP at two different pH values (pH 7.0 and 8.0) (Fig. 1). The sodium uptake was very low at pH 7.0 and almost no stimulation by ATP was observed. At pH 8.0 the uptake in the absence of ATP was low, too, but ATP caused a Table 2: Minimal A

External pH

5.5

+ 151

n=2 n=3

+133

7.2

9.0

-47 -243 -14 -164

c

I

O;Q

E "0 E c

Time

(min)

Fig. I: 22Na + uptake into PM vesicles at pH 7.0 and 8.0. The uptake was assayed in the standard reaction medium, except that the pH was adjusted accordingly. (0) no ATP, pH 7.0; (e) + ATp, pH 7.0; (0) no ATp, pH 8.0; (_) + ATp, pH 8.0. Single, representative experiment.

267

significant increase of sodium uptake (Fig. 1). The rate of ATP-stimulated sodium uptake was in the order of 10 nmoles sodium taken up per mg of protein and minute. All subsequent experiments were carried out at pH 8.0. If ATP-dependent 22Na + uptake is mediated by a Na +/H+ anti porter, one would expect protonophores to inhibit the process. Therefore, the effect of 6 J.1mollL CICCP on the sodium uptake into PM vesicles was studied in the absence (Fig. 2 A) and presence (Fig. 2 B) of the permeant anion nitrate (100 mmol/L). When the reaction medium did not contain the permeant nitrate anions (Fig. 2 A), ATP-stimulation of sodium uptake (difference between filled and empty circels)·was small and the uncoupler CICCP increased the ATPdependent sodium uptake (difference between filled and empty triangles). The ATP-induced differences under the various conditions are replotted in Fig. 2 C. In the presence of 100 mmol/L nitrate (Fig. 2 B), ATP-dependent Na + uptake was larger than in the absence of nitrate and essentially unaffected by CICCP (compare also Fig. 2 C). Thus, protonophores do not inhibit ATP-dependent Na + uptake. Moreover, CICCP enhanced the rate of the uptake in the absence of permeant anions. The protonophore CICCP completely eliminates ApH generation across the PM of T. viridis (Popova and Balnokin, 1992) and, hence, the involvement of the AJ.1H+ -dependent Na +/H+ antiporter in the ATP-supported Na + accumulation. Thus, the results presented in Fig. 2 indicate directly that an ATP-driven, but pmf-independent Na +-translocating system operates in the PM of T. viridis. Most likely this system is a primary electrogenic Na +-pump (Na +-ATPase). Electrogenicity of the pump is indicated by the fact that CICCP or permeant anions such as nitrate enhance ATP-supported sodium uptake. Na + influx into the vesicles carried out by the supposed Na +-ATPase results in the generation of an electrical potential difference across the PM (positive inside), which inhibits further Na + uptake. Obviously, CICCP-mediated H+ efflux out of the vesicles discharges A
268

Yu. BALNOKlN, L. POPOvA, and H . GIMMLER 30

C

+ NO,·

c

i

+ Clcep, NO,·

20

Do

'"

ao E 40

+CICCP

40

"8

-~ I i

10

20

20

control

::I

o o Time

(min)

Time

(min)

4

12

8

Time

(min)

Fag. 2: The effect of the protonophore CICCP on 22Na + uptake into PM vesicles in the absence (A) and presence (B) of the permeant anion nitrate. Sodium uptake was assayed in the standard reaction medium with the following modifications: (A): without nitrate anions; buffer (HEPES/BTP) concentration was 30 mM. (B): mannitol was diminished to 0.3 M; buffer (HEPES/BTP) concentration was 30 mM; nitrate/BTP was 100 mM. (C): ATP-dependent sodium uptake was calculated from the differences between sodium uptake in the absence and presence of ATP (data from Figs. 2A and 2 B; data points are omitted since values reflect calculated rather than experimental data). (0) no ATP; (e) + ATP; (~) no ATP, 6/-lM CICCP; (.) + ATP, 6/-lM CICCP. Single representative experiment.

Table 3: The effect of ions, inhibitors and the protonophore

Jc

:=I

~

i f

A

100

Additions/Omissions

c

i

50

.2

0

!

!:c

5

6

7

8

9

Jc

:=I

If

• ie

.

~

B

22Na+ uptake (control = 100%)

none - nitrate + 61-1moUL CICCP - nitrate, + 61-1moUL CICCP + 10 mmoIlL KCI + 50l-1moIlL amiloride + 200l-1moIlL amiloride + 100 I-1moIlL orthovanadate

100 35 94

75

o

100

74

o

100

50

>-

~

~

CICCP on the ATP-dependent 22Na + uptake into PM vesicles isolated from P. viridis. The assay was carried out with the standard reaction medium.

6

6

7

8

9

pH

Fig. 3: pH-profIles of ATP-dependent ion translocatins (A) and of ATP-hydrolysis (B) in PM vesicles isolated from P. viridis. A: (e) ATP-dependent ApH formation; (.) ATP-dependent 22Na + uptake (difference in the uptake in the absence and presence of ATP). Sodium uptake was assayed in the standard reaction medium with the following modification: 100 mM HN0 3 adjusted with BTP to the indicated pH was used as a buffer. B: ATP-hydrolysis catalysed by the PM vesicles. All data are given in relative units. The rates of ATP-hydrol~is are usually in the order oflO0-200nmol ATP mg- I protein min -I, those of sodium uptake in the order of 10 nmol Na + mg-I protein min-I.

Amiloride, at concentrations known to inhibit Na + IH+ antiporters of various origins completely (Krulwich, 1983; Blumwald and Poole, 1985; Kleyman and Cragoe, 1988), did not affect sodium uptake into PM vesicles of P viridis at all (50 I1mol/L) or caused only slight inhibition (200 I1mollL) (Table 3). Orthovanadate, an inhibitor of transport ATPases that form phosphorylated intermediates in the course of the catalytic cycle (Serrano, 1990), causes considerable inhibitions of ATP-hydrolysis of PM-fractions isolated from P viridis (Balnokin et al., 1993). Table 3 demonstrates that orthovanadate also inhibits the ATP-dependent sodium uptake into the PM vesicles. K+ strongly suppresses ATP-dependent sodium uptake into the PM vesicles (Table 3).

Protein composition ofthe PM vesicles Unidimensional analysis of the proteins of the PM vesicles by PAGE demonstrated the existence of two abundant pro-

ATP-driven sodium pump in Platymonas teins with the molecular masses of 95 and 150 kDa (not shown, experiments carried out in cooperation with the group of Dietz, Wiirzburg).

269

pH values of the medium that represents conditions favourable for the functioning of H+ -ATPase and generation of ~pH across the vesicle membranes (Figs. 1 and 3 A). As shown earlier, at neutral pH Na + ions discharge ~pH formed by the H+ -ATPase across the vesicle membrane of P. viridis (Popova and Balnokin, 1992). This process was specific for Discussion Na and was inhibited by 50llmoi/L amiloride. The concluThe data of1:his investigation indicate that a primary Na +- sion was drawn from this observation that a ~IlH+­ pump operates in the PM of eukaryotic cells of T. viridis and dependent Na +IH+ antiporter operates in the PM of P. virithereby confirm recent results of Balnokin and Popova dis. Therefore, one should expect that an ATP-driven sodium (1994). The pump is suggested to be an orthovanadate-sensi- uptake into the vesicles, due to the operation of such an extive electrogenic Na +-ATPase. The operation of such a Na +- changer, also takes place at pH values between 6.0 and 7.0. ATPase in the PM is known already for a longer time to be a However, to our surprise this was not the case. There are two feature inherent in some prokaryotes living under high salin- feasible explanations for this phenomenon. (I) There may be ity. When external pH is more acidic than cytoplasm, these some unknown regulating factors that control the activity of organisms maintain a low level of Na + in the cytoplasm, due the Na +IH+ antiporter in intact cells, but which are absent in to a Na +IH+ antiporter operating in the PM. The presence of the experimental system of the isolated membranes. (2) Bethe Na +-ATPase in the PM is related to the adaptation of cause of the treatment of cells with trypsine and hypotonic these prokaryotes to alkalinization of their habitat (Skula- shocks, the vesicles were isolated with a functional H+chev, 1988; Avetisyan et al., 1991). Analogously, also in the ATPase and Na +-pump, but with a defect Na +IH+ antiporter eukaryotic cells of P. viridis, the Na +IH + antiporter probably that has lost the ability to transfer Na +. However, it must be expels Na + at acidic pH values of the aquatic environment. emphasised that such artefacts do not affect the main suggesThe Na +-ATPase, however, starts to operate at alkaline ex- tions of this paper, namely the existence of a primary Na +ternal pH when the ~pH across the PM is low or oppositely ATPase in the PM of P. viridis. The question arises whether this conclusion can be gendirected (more acidic inside the cells). Many salt lakes as well as the upper layers of the oceans inhabited by marine phyto- eralised for other eukaryotic halotolerant microalgae. A replankton have alkaline pH values: due to photosynthetic ac- view of the literature demonstrates that available data, even tivity of phytoplankton, the pH of the upper layers of the sea though mostly derived from in vivo experiments, are in agreement with our working hypothesis (Balnokin and Medvedev, water may rise to 9.0 (Vinogradov, 1989). The data of this paper are also in agreement with the pres- 1984; Wada et al., 1989; Galkina and Balnokin, 1992). For ence of a Na +-ATPase in the PM of halotolerant microalgae example, the Na + transport in intact cells of Dunaliella marias suggested by Wada et al. (1989) and Shono et al. (1995). tima (Balnokin and Medvedev, 1984) and P. viridis (Galkina These authors described a vanadate-sensitive ATPase activity and Balnokin, 1992) did not match the operation of a secin PM vesicles isolated from the marine unicellular alga He- ondary energized Na +IH+ -antiporter at alkaline pH. The terosigma akashiwo, which was synergistically stimulated by Na +-extrusion out of the cells demonstrated a low sensitivity Na + and K+. Wada et al. (1989) also demonstrated the pres- to ClCCP and occurred at an alkaline pH of the medium, ence of two phosphorylated polypeptides with molecular e.g. under conditions unfavourable for Na +-extrusion permasses of 95 and 150 kDa in the PM of this alga using acid formed by the Na +IH+ -antiporter. Studies on PM vesicles PAGE in combination with protein labelling with [y_ 32Pl_ isolated from Dunaliella (Gimmler et al., 1989 a) and PlatyATP and autoradiography. The 95 kDa polypeptide was monas (Balnokin et al., 1993) showed that the pH-profile of phosphorylated in the presence of Mg2+, whereas the phos- ATP hydrolysis by the PM preparations were extremely broad phorylation of the 150 kDa protein required both Mi and and did not correspond to the sharp pH-profile ascribed to Na +. Both proteins were dephosphorylated by the addition conventional plant H+ -ATPases of the p-type. This discreof K+ to the medium. The authors hypothesised that the pancy might be connected to the operation of two different 95 kDa polypeptide is an analogue of the higher plant H +- ATPases rather than one in the PM of halotolerant microalATPase and the 150 kDa protein a Na +-ATPase similar to the gae. The indications for the existence of a primary active Na + animal Na +IK+ -ATPase. Shono et al. (1996) were able to extrusion mechanism are also compelling for Dunaliella cells: transfer the sodium pump of Heterosigma into liposomes and These extremely halotolerant alga species thrive well at pH to measure Km-values of this enzyme. The apparent Km-value values higher than 8.0, maintain a cytoplasmic pH close to for ATP was 400llmollL and 7 mmol/L for sodium. As in our 7.0-7.2, independent of the external pH, are capable of keepsystem the Na +-transport was not affected by protonophores ing the internal sodium concentration much lower than the high external sodium concentration in the medium and exhibut inhibited by orthovanadate. In our experiments, ATP-dependent Na + accumulation bit membrane potential differences across the PM lower than was observed in the absence of K+ ions in the reaction me- - 100 mV at neutral to weakly alkaline pH values in the medium and when KCl was introduced into the medium, ATP- dium (Pick et al., 1986; Gimmler et al., 1988; 1989 b; Pick, dependent sodium uptake was completelJ inhibited (Table 1992). Thus, the very well characterised Na +IH+ antiporter 3). This may indicate that the primary Na -pump in the PM in the PM of Dunaliella (Katz et al., 1986, 1989, 1991) can similarly as it is the case in T. viridis, not contribute to soof P. viridis is not a Na +,K+ -ATPase. It remains to be elucidated why we failed to observe ATP- dium extrusion at alkaline pH. Furthermore it is of interest, dependent Na + uptake into the vesicles at neutral and acidic that most data on Na + elimination by D. salina and D. parva

270

Yu. BALNOKIN, L. POPOVA, and H. GIMMLER

seem to exclude the presence of a Na +IK+ pump (Pick, 1992). Acknowledgements

This investigation was supported by the Deutsche Forschungsgemeinschaft (SFB 251, Teilprojekt A2, H. Gimmler). We acknowledge that Y. Balnokin and L. Popova were financially supported by the SFB 251 and the Graduiertenkolleg (DFG) (Ka 456/5-7).

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