Aspects of Dimethylsulfoniopropionate Effects on Enzymes Isolated from the Marine Phytoplankter Tetraselmis subcordiformis (Stein)

J. Plant Physiol. Vol.

138. pp. 85-91 {1991}

Aspects of Dimethylsulfoniopropionate Effects on Enzymes Isolated from the Marine Phytoplankter Tetraselmis subcordiformis (Stein) T.

GRONE

and G. O.

KIRST

Department of Marine Botany FB 2, University of Bremen, P.O. Box 330440, D-2800 Bremen 33, Federal Republic of Germany Received September 25, 1990 . Accepted January 22, 1991

Summary The compatibility of glucose-6-phosphatedehydrogenase, glutamatedehydrogenase, malatedehydrogenase (MDH) and a proteolytic enzyme isolated from Tetraselmis (platymonas) subcordiformis with organic and inorganic solutes was tested in vitro. Only MDH showed considerable NaCl-sensitivity. All four enzymes exhibited no or only little inhibition of activity in the presence of up to 100 mM DMSP-Cl. MDH was further characterized. MDH hydrating activity under substrate saturation was equally inhibited by 100 mM NaCI or DMSP-Cl. Activity rates at low oxalacetate concentrations in the presence of DMSP-CI or equimolar NaCI were also reduced compared with controls without additional solutes. However, DMSP-CI was less inhibitory than NaCl. The inhibition by NaCI was also mitigated by increasing substrate concentration. The ratio of reducing to oxidizing MDH-activity was altered by NaCI from 7 with no NaCI present to 28 at 500 mM NaCl. DMSP-CI (200 mM) altered this ratio to a lesser degree than equimolar NaCl. A possible regulation mechanism of MDH is altered by NaCI but not by DMSP-Cl. It is proposed that NaCI inhibits MDH-activity and alters regulation of the enzyme by malate. Presence of DMSP-CI or increased substrate levels can minimize these effects.

Key words: Compatible solutes, dimethylsulJoniopropionate, NaCl stress, Tetraselmis (Platymonas) subcordi· /ormis enzymes. Abbreviations: DMSP = dimethylsulfonipropionate; P = proteolytic enzyme; GDH = glutamatedehydrogenase; G-6P-DH = glucose-6-phosphatedehydrogenase; MDH = malatedehydrogenase; DTE = dithioerythritol; Tris = Tris(hydroxymethyl)aminomethane.

Introduction The tertiary sulfonium compound dimethylsulfoniopropionate (DMSP) has been detected in numerous marine micro- and macroalgae (Ackman et al., 1965; Reed, 1983; Karsten et al. 1990) from different habitats. Although it occurs in all classes of marine algae investigated it is present in very different concentrations up to 15 mg S g-l dry weight (Holligan and Kirst, 1989). While many studies have been conducted on dimethylsulfide, a breakdown product of DMSP found in the atmosphere (Andrae et al., 1983), the © 1991 by Gustav Fischer Verlag, Stuttgart

biological role of DMSP in algae is still obscure. Challenger (1948) and Dubnoff and Borsook (1948) considered DMSP as a methyl-group donator. Sieburth (1959) described antibiotic effects of the DMSP breakdown product, acrylic acid. For algae containing high intracellular levels of DMSP, interest has focused on its possible role as an osmolyte (Reed, 1983; Dickson and Kirst, 1986; Kirst, 1990). In the Prasinophyte, Tetraselmis subcordiformis, a phytoplankton algae abundant in coastal waters and well investigated with respect to salinity tolerance, the average concentration of DMSP at seawater salinity was in the range of

86

T. GRONE and G. O. KIRST

140 mM (Dickson and Kirst, 1986). These levels are high enough to be of osmotic significance. The same authors demonstrated that DMSP together with glycine betaine, homarine and mannitol accounted for 56 % of the osmotic potential of T. subcordiformis. Changes of external salt concentrations resulted in subsequent shifts in intracellular DMSP levels (Dickson and Kirst, 1986). The intracellular location of DMSP has not been demonstrated as yet. For T. subcordiformis, it may be deduced from the small volume of the vesicles and the absence of a vacuole that DMSP is located in the cytoplasm/chloroplast (Kirst and Kramer, 1981). Enzymes of marine algae (Gimmler et aI., 1984) and halophytes (Flowers et aI., 1977), in contrast to those of halophilic bacteria (Hochachka and Somero, 1973), are NaCI sensitive. Therefore, it was suggested that algae substitute the detrimental NaCI by more compatible solutes such as potassium ions (Wiencke, 1984), glycerol, mannitol, proline, betaine and/or possibly DMSP (Kirst, 1990). These compounds were termed «compatible» solutes by Brown (1976). This paper describes the effect of DMSP on in vitro enzyme activity to address the question whether DMSP is a compatible solute or not.

Material and Methods Growth conditions Tetraselmis (platymonas) subcordiformis (Stein) strain 161-la (Gottingen culture collection, FRG) was cultivated at 23°C under a 14 h light, 10 h dark regime. Light was supplied by two fluorescent tubes with a photon flux density of 70 ~mol m - 2 sec - I. A modified ASPmedium was used as described by Richter and Kirst (1987). NaCl concentration was 400 mM. Initial cell concentration was made to approximately 0.5 x 106 cells per mL. Preparation of crude extract Algae were harvested after 4 - 5 days of growth. Approximately 700 mL algae were centrifuged gently (600 x g, 5 min) and then washed with fresh medium. Algae were centrifuged again and resuspended in ice cold Tris-HCl buffer (pH 7.5, 100 mM) containing 1 mM DTE. The suspension was passed twice through a French Press at 16,000 PSI followed by centrifugation at 20,000 x g for 20 min. The resultant supernatant was brought to 30 % saturation with ammonium sulfate and centrifuged again for 20 minutes at 20,000 x g. The supernatant was brought to 75 % saturation, centrifuged and the resultant pellet was stored at -30°C until use (at maximum 7 days). All preparations were performed at 4°C. The pH was checked for constancy during the ammonium sulfate precipitation.

Enzyme assays Enzyme preparations were dissolved in a small volume of the test buffer shortly before the assay. All stock solutions with osmolytes were prepared freshly. pH was measured and corrected to enzyme test pH levels with KOH where necessary. pH, substrate and temperature optima were ascertained for GDH and the proteolytic activity in preliminary experiments. MDH and G-6P-DH were measured according to the procedure of Richter (1987). Salts and osmolytes were added to the assay 5 min before starting the reaction.

Standard composition of the tests is given below, but substrate and osmolytes were varied as given in the legends to the figures. Buffer concentrations for assays with added osmolytes were lower than given for the standard assays but did not fall below 10 mM. Activity determinations were performed with a spectrophotometer (Shimadzu UV 2 A). Proteolytic enzyme assay: Proteolytic activity was measured with azocasein (Sigma) as a substrate. The assay contained 10 ~M ZnCh and MgCh, 0.5mM DTE, 66mM Na-acetate buffer (pH 5.0) and 3 mg/ mL azocasein. The reaction was stopped after 90 min of incubation at 40°C by adding an equal volume of HCl0 4 (14 %). Activity was determined as acid soluble protein at 340 nm after centrifugation of samples at 3000 x g for 10 min. GDH (E.C. 1.4.1.4): The test assay contained 240~ NADPH, 75 mM NH4-acetate, 61 mM Tris-HCl (pH 7.5), 50 ~M EDTA and 4.3 mM ketoglutaric acid. Assays were started with ketoglutaric acid. Enzyme activity was measured as NADPH oxidation at 366 nm. No NADH dependent activity was detected. G6P-DH (E.C. 1.1.1.49): The assay contained 1.5mM NADP, 1 mM glucose-6-phosphate and 79 mM Tris-HCl buffer (PH 7.5). Enzyme activity was measured at 340 nm. MDH (E.C. 1.1.1.37): The hydrating reaction was measured with 150 ~M NADH, 1 mM oxalacetate and 79 mM Tris-HCl buffer (pH 7.5) in the assay. Dehydrating activity was measured with an assay containing 3 mM MgCh, 200 ~M NAD, 10 mM malate and 66 mM Tricine buffer (PH 9.0).

Protein measurement Protein was measured following the procedure of Lowry et al. (1951).

Chemicals DMSP-Br was prepared according to Challenger and Simpson (1948). Thin layer chromatography showed a single spot, and identity and purity of the sample were also confirmed by NMR-spectroscopy. DMSP-Cl was kindly supplied by D. M. J. Dickson.

Concentrations and osmotic potentials Solute concentration is refered to as mM throughout this paper. However, as most osmolytes are salts and some had to be pH-adjusted, osmolarities were different from concentrations. For comparison, salt concentrations and corresponding osmotic potentials are shown in Table 1. The greatest difference in osmotic potential between equimolar concentrations of DMSP-Cl and NaCl was 9 %.

Results

Tetraselmis subcordiformis is an euryhaline algae with internal NaCl concentrations not exceeding 100 mM in equilibTable 1: Solute concentrations and osmotic potentials in media used for MDH assays. Assay

solute concentration [mM]

osmotic potential [mosmol· kg-I]

NaCl

100 200 500

323 484 952

50

255

DMSP-C!

100 200

349 532

Dimethylsulfoniopropionate effects on enzymes

[%)

;€ 0

G-6P-DH

P

MDH

(IlH

25

25

15

15

~ 5

5

15 c-

S2

E"

o.

01 ::l..

"'C

8

.~

'E .~

0

£

7C

x

100

87

x

I:

o

« z

60

~

u

'" E ,., QJ

C

N

c::

control mannitol. DMSP-Cl KBr NaCl betaine DMSP-Br

QJ

20

50 100 200

SO 100200

SO 100 '200 50 100:;00

DMSP -[I [mM)

t0

[%)

G-6P--OH

Ma-t

P

GDH

100

Fig. 2: Influence of various organic and inorganic solutes on malatedehydrogenase activity (hydrating activity) from Tetraselmis sub· cordiformis. The test was performed under substrate saturating conditions with a preincubation period of 5 min. The mean of 4 assays is shown. SD values were in all cases less than 17 % of the mean. Concentrations of organic and inorganic solutes were: ~ 25 mM, [[[]] 50 mM, 0 100 mm, • 200 mM, [QJ 500 mM.

~

"

"0

.t

6

u

1::~

60

~

u

'" Qj

E >-

N

c:: Qj

20

100 :;00 SOO 100 '200 SOO 100 2OOS00 100 200 SOO NaG [mM)

Fig. 1: Effect of NaCI and DMSP-CI on the activity of four enzymes of Tetraselmis subcordiformis. All enzymes were measured (after (NH4) S04 precipitation) under substrate saturation conditions. Control rates without NaCI or DMSP-Cl were set to 100 %. Absolute activity rates for assays without added solute were: 32.9 nmoles NADH min - I Ilg protein - I for MDH, 578 nmoles NADH min - l/Lg protein - 1 for G-6P-DH, 14.6nmoles NADPH min - I /Lg protein - I for GDH and 2.45 U mg protein - I for the proteinase. 1Unit = 0.01 E 340 min - I . G-6P-DH = glucose-6-phosphatedehydrogenase, MDH = malatedehydrogenase, P = proteolytic activity, GDH = glutamate-dehydrogenase. The mean of 3 -4 assays is shown. SD values were in all cases less than 15 % of the mean.

rium in salinities up to 1000 mM N aCl (Dickson and Kirst, 1986). This concentration range, and even higher concentrations of NaCI, did not affect G-6P-DH, GDH and the proteolytic enzyme if tested at substrate saturation. Maximal inhibition achieved in the presence of 500 mM NaCI or 200mM DMSP for these enzymes was about 30% (Fig. 1). Only MDH showed considerable loss of activity: 100 mM NaCI reduced MDH activity by 3S %, seawater NaCI concentrations of 500 mM by 70 %. MDH activity was not much reduced with 100 mM DMSP-CI; however, 200 mM

DMSP-CI caused a similar inhibition as 200 mM NaCl. Thus DMSP in the concentration range of up to 100 mM did not have adverse effects on the four tested enzyme activities. Furthermore, only MDH seems in need of a reduction of high NaCllevels as the enzyme reacts sensitive to NaCl. Data obtained with glycine betaine up to 200 mM and mannitol up to 500 mM also showed high activities of all four enzymes comparable to those with DMSP-CI of equimolar concentrations. However, slight preferences of the enzymes for particular solutes were noted (Fig. 2 and unpublished data). DMSP is stable as a bromide or chloride salt. Therefore, DMSP could not be supplied to the assay without CI - or Br-. Furthermore, when the pH of the stock solution was adjusted with KOH to that of the assay buffer, K + was added. As a result, an assay with 200 mM DMSP contained besides 200mM Cl- or Br - additionally 240mM K+. The effect of ionic additions in the presence of DMSP on enzyme activities was therefore investigated. Fig. 2 shows that the enzyme activity was greatly affected by the accompanying anion of DMSP: DMSP-Cl was about 30 % less inhibitory than DMSP-Br. This is in agreement with the Hofmeister sesries of chaotropic salts (Br - >Cl- >N0 3 - >1- for anions). Addition of KBr resulted in a similar pattern to that observed with DMSP-Br. This confirms that Be had severe adverse effects on the enzyme activity. Experiments with K2HP0 4 showed that 200 mM K + must not result in MDHinhibiton if the counterion is compatible. Thus the anions CI- and Br- exert a greater inhibitory effect on MDH than the cations K +,DMSP+ and betaine + . The osmotic stress caused by DMSP on MDH is actually a combined DMSP/KCI stress with a total osmotic potential of up to 532 mosmol kg - 1 for the 200 mM DMSP assay. Nevertheless, MDH in the presence of up to 100 mM DMSP had more activity than in the presence of equal amounts of glycine betaine and mannitol (Fig. 2), two widely accepted

88

T. GRONE and G. O. KIRST

Table 3: Effect of malate, NaCI and DMSP-Cl on MDH (hydrating activity) from Tetraselmis subcordiformis. Units for V max are nmoles NADH . min -I . ttg- I protein and mM oxalacetate for Km.

a. control

Assay control control+4mM malate 100mM NaCI 100mM NaCI+4mM malate 100 mM DMSP-CI 100mM DMSP-CI+4mM malate

b. NaCl

c. DMSP-[l

•

9

3

3

9

15

oxalacetate [mM"1]

Fig. 3: Double reciprocal plot of rates of MDH (hydrating activity) of Tetraselmis subcordiformis versus concentration of oxalacetate in the presence of DMSP-CI, NaCl and malate. 0 = assay without malate; • = assay with 4 mM malate as an inhibitor. a) assay without additional solutes, b) MDH-activity in the presence of 100mM NaCl, c) MDH-activity in the presence of 100 mM DMSP-Cl. The mean of 2 - 4 assays is shown. Table 2: MDH activity (hydrating activity) as a function of oxalacetate concentration, effects of NaCI and DMSP-Cl. enzyme activity [nmol NADH· min-I ·ttg- I [mM] protein] + 100 mM DMSP-CI +100mM NaCI control oxalacetate 5.0 2.7 0.020 14.2 8.6 0.043 20.3 5.1 13.5 0.100 904 25.8 21.5 21.0 0.700 31.3

compatible solutes (Reed et aI., 1985; Pollard and Wyn Jones, 1979). Maximum activity in the presence of DMSP-CI was slightly higher than in the presence of NaCI at 100 mM. Inhibition was similar with 200 mM NaCI or DMSP-CI in the assay buffer. The above data were obtained from experiments under substrate saturation conditions. The inhibitory effects of the various osmolytes are better compared and more adequately described by Michaelis Menten kinetics than solely by measurements at substrate saturation. Therefore, the data of Fig. 1 must be interpreted with care. Of more physiological significance are substrate limitation conditions that

32.6 30.3 26.3 18.5 24.0 22.5

Km 0.043 0.064 0.185 0.103 0.077 0.105

Table 4: Effect of NaCI and DMSP-CI on MDH hydrating and dehydrating activity. The ratio of hydrating to dehydrating activity is shown for assays using (NH4)zS04 precipitated extracts from Tetra· selmis subcordiformis. Assays were carried out under optimal incubation conditions. Assay

15

Vmax

control 100mM NaCI 200mM NaCI 500mM NaCI 50 mM DMSP-CI 100 mM DMSP-CI 200 mM DMSP-CI

MDH activity: ratio hydration/dehydration NADH consumption] [ NADH formation 7.2 10.6 16.9 28.3 8.6 11.9 lOA

occur for many enzymes in vivo. Experiments with varying substrate concentration are summarized in Fig. 3 and Table 2. Under substrate limitation conditions around and below the Km-value (0.043 mM oxalacetate) MDH is less inhibited in the presence of DMSP compared with NaCl. The enzyme activity in the presence of 100 mM NaCI at Km was only 10% of the control. Substitution of NaCI by DMSP-CI instead resulted in a doubling of MDH activity, reaching 35 % of the control value. In contrast, DMSP-CI was as inhibitory as NaCl under substrate saturation (0.77 mM oxalacetate). However, at this oxalacetate concentration (0.77 mM) 100 mM NaCI or 100 mM DMSP-CI caused only a 33 % inhibition of MDH activity. Both NaCI and DMSP-CI affected MDH by lowering the Vmax and raising the Km (Fig. 3 and Table3), which is characteristic of mixed inhibition. NaCI (100 mM) resulted in a 19% lower Vmax whereas DMSP-CI decreased Vmax by 26%. In contrast, large differences were found in the affinity of MDH to oxalacetate: NaCI increased the Km more than fourfold, DMSP-CI only two-fold. In addition to enzyme activity and affinity, maintenance of enzyme regulatory principles and turnover balances are of great significance to cells under stress conditions. For a first approximation the ratio of malate formation to oxalacetate formation was used as an indication of turnover balance. There was a seven-fold faster malate synthesis than oxalacetate synthesis under conditions without osmolytes and with optimum substrate concentrations in the assay (Table4). NaCI changed this ratio remarkably. With 200 mM NaCI there was a 17-fold higher rate of malate syn-

Dimethylsulfoniopropionate effects on enzymes

I

+

~

....a c

a '.;:::-

89

20 Q

400

pM oxalacetate

O~--~~----~--~----~--~----~~------~

roo

Fig. 4: Effect of 4 mM malate on activity of MDH (hydrating activity) as a function of substrate concentration. The malate effect is shown in relative units in relation to the same assay without malate. ( x ) control, (0) 100mM NaCl, (.A) 100mM DMSP-Cl. Absolute activity of controls without malate at 500 JLM oxalacetate was 31.3 nmoles NADH min -1 JLg protein - 1.

. ~ II t;~-

~ ~ §

E

20

~ 2-

..[)

-E

40

o~

thesis. With 200 mM DMSP-CI the ratio remained at a level more similar to the control. Hydration and dehydration were targeted similarly. To check for solute effects on a possible regulation pattern of MDH, malate inhibition of oxalacetate hydrating MDH was investigated. Malate (4 mM) resulted in mixed inhibition of MDH (Fig. 3, Table3), reducing Vmax and raising Km values. Apart from the lower turnover rate, the presence of 100 mM DMSP-CI did not alter this effect. Malate in the presence of DMSP-CI acted in a similar fashion: Vmax was reduced to 93 % of the value without malate and Km was increased by 37 % (93 % and 48 % from the control, respectively). In the presence of NaCl the enzyme responded very differently to malate: Vmax and Km were decreased, which is an uncompetitive inhibition according to Michaelis Menten kinetics (Fig. 3). While in the presence of DMSP-CI, MDH remained sensitive to malate inhibition within the substrate concentration range, enzyme activity was even stimulated by the «inhibitor», malate, if oxalacetate concentrations were low and 100 mM NaCI was present (Fig. 4). Discussion The enzymes of Tetraselmis subcordiformis tested were not NaCI sensitive to the same extent. A proteolytic enzyme, G-6P-DH and GDH were little inhibited in vitro, even with high NaCI concentrations in the assay. Priebe and Jager (1978) found no significant inhibition of a GDH enzyme activity from Atriplex species up to 800 mM NaCl. Wieneke (1984) demonstrated high KCI insensitivity of pyruvate kinase extracted from Porphyra umbilicalis and Gimmler et al. (1984) showed that Dunaliella parva contained highly tolerant as well as salt sensitive enzymes. These studies as well as the data presented here indicate that salt tolerance is a quality of individual proteins and not of the whole system. MDH has often been found to be salt sensitive during in vitro studies (Pollard and Wyn Jones, 1979; von Willert, 1974). Our results are consistent with these findings. Especially at low substrate concentrations MDH is inhibited

severely by NaCl, and inhibition could partly be overcome by raising the substrate levels. NaCl affected enzyme activity by reducing enzyme affinity and maximal velocity. This mechanism is classified as mixed inhibition. With other organisms NaCI effects may be different. Wieneke (1984) observed an uncompetitive influence on pyruvate kinase. von Willert (1974) showed competitive action of low salt concentrations on a decarboxylating MDH while he did not define a different inhibition at higher NaCI concentrations. Treichel et al. (1974) demonstrated a competitive mode of inhibition for a phosphoenolpyruvate carboxylase. In comparison, up to 100 mM DMSP in the assay was compatible with all four enzymes tested. Even 200 mM DMSP-CI was compatible with high enzyme activity except in the case of MDH. However, a considerable part of this MDH inhibition is due to the accompanying anion as indicated by the assays run with DMSP-Br and DMSP-Cl. Enzyme activity following NaCI inhibition may be restored by increasing substrate levels (Greenway and Setter, 1977) and/or enhancement of enzyme synthesis (Gimmler et aI., 1984). The effect of NaCl on enzyme regulation, in contrast, should be more severe than its effects on enzyme activity, since biochemical pathways may become unregulated. Provided that malate regulation of MDH-activity is of relevance in vivo, substitution of NaCl by a compatible solute could be of vital importance. In the presence of NaCI, malate turns from being an inhibitor to an activator when substrate levels are low (ca. 80 /-tM oxalacetate or lower). In the presence of DMSP-CI, however, such a change was not observed. DMSP-CI acted as a genuine compatible solute (Brown, 1976) and thus supports the idea of such a role in Tetraselmis

subcordiformis.

MDH is involved in malate synthesis in the cytoplasm, synthesizing the NADH-shuttle between cytoplasm and mitochondria, and also participates in oxalacetate production in the Krebs cycle. A balance between both processes is of significance. NaCl altered the ratio between production and consumption of malate by MDH whereas 200 mM DMSP-CI resulted in a smaller change in this ratio. Together with data taken from the literature our results are sum-

90

T. GRONE and G. O. KIRST

~rate

R

aj

~

oxalacetate MDH') malate

oxalacetate M[J-I NaG

oxa~cetate

filH~

malate

(fl1t!

~ oxalacetateo~~ malate

oxalacetate MOH/OMSP malate

Fig. 5: A proposed model of regulatory patterns for MDH of Tetra· selmis subcordiformis. Only the cytoplasm and mitochondria are considered as compartments of the cell. a) Situation before salt stress: malate pool is regulated by product inhibition of MDH and «sink» processes. b) In the presence of NaCI, malate accumulates because of different effects on malate production and consumption by MDHs and loss of product control. c) In the presence of DMSP-CI, product inhibition is restored. Both MDH reactions are affected equally. ¢ -+ - --+ symbolize different fluxes of substrates; -&- a translocato'r f~r malate; R = regulation; For further details, see text.

marized in the hypothetic model given in Fig. 5. MDH acts in two ways: as part of the Krebs cycle in the mitochondria and in supplying malate in the cytoplasm as a transport metabolite for NADH across the mitochondria membrane (Fig. 5 a). The enzyme is regulated via product feedback inhibition. If high levels of NaCI are present in the cytoplasm and in the mitochondria (Fig. 5 b) activities of both processes decline, oxalacetate production being more effected. If no other processes interfere, malate will accumulate. In an early stage of salt adaptation this accumulation may even be advantageous because it raises substrate levels and helps to overcome NaCI inhibition (Greenway and Setter, 1977). In the long run, however, this will uncouple malate production from consumption processes such as the Krebs cycle. In the presence of NaCI, in conjunction with oxalacetate levels below 80/tM, feedback regulation by product inhibition will not be possible. With increasing cellular exclusion of NaCI by ion pumps and the increase of DMSP and other compatible solutes during the process of salt adaptation (Dickson and Kirst, 1986) feedback inhibition is restored again (Fig. 5 c). However, a reduced activity may remain. A few metabolically important but salt sensitive enzymes may already require the cytoplasmic exclusion of NaCl. MDH represents such an enzyme because of its key role

mentioned above. The intracellular NaCl concentration of Tetraselmis subcordiformis in osmotic equilibrium never exceeded 50mM (Dickson and Kirst, 1986). Thus the 100mM NaCI used in these in vitro assays may be intolerable in osmotic equilibrium under natural in vivo conditions. Although DMSP-CI is thought to be compatible with enzyme regulation, there was some inhibition of MDH activity. The effect of compatible solutes on enzymes activities is controversaly discussed in the literature. Pollard and Wyn Jones (1979) found no inhibition of MDH from different sources by glycine betaine up to 500 mM. Ginzburg (1987) reports inhibition, stabilisation or counter-action by glycerol in Dunaliella enzyme assays. Richter (1987) did not observe any influence of up to 800 mM mannitol on several enzymes of Tetraselmis subcordiformis. However MDH, G-6PDH and phosphoglukomutase showed slight to medium inhibition in his experiments. Enzyme inhibition by NaCI may even be compatible for the whole organism as demonstrated for the inhibition of mannitol-1-phosphatedehydrogenase (M-1P-DH), the mannitol degrading enzyme (Richter and Kirst, 1987). The inhibition of this enzyme by NaCI in T. subcordiformis increases the pool of this compatible solute in the algae (Richter and Kirst, 1987). However, an advantage by NaCl-inhibition of enzymes seems to be exceptional and unlikely for enzymes of the primary metabolism. The observed inhibition of MDH activity by DMSP-CI may be overcome in vivo by constituting specialized osmolyte-enzyme systems by compartimentation. As mentioned previously Tetraselmis subcordiformis has a range of organic solutes available. Change in regulation patterns and inhibition of MDH activity could be buffered (partly) by two mechanisms: substrate levels could build up or NaCI may be substituted by DMSP-Cl. For low substrate concentrations below and around Km , DMSP-CI was more compatible than NaCl. At high substrate levels there seemed to be little difference. Thus DMSP exhibits best compatibility where it is needed most and is able to contribute to salt tolerance mechanisms in this species. Acknowledgements We would like manuscript.

to

thank D. N. Thomas for critical reading of the

References ACKMAN, R. G., C. S. TOCHER, and J. McLACHLAN: Occurrence of dimethyl-fJ-propiothetin in marine phytoplankton. J. Fish. Res. Bd.23, 357 -364 (1965). ANDRAE, M. 0., W. R. BARNARD, and J. M. AMMONS: The biological production of dimethylsulfide in the ocean and its role in the global atmospheric sulfur budget. Environmental Biogeochem. 35, 167 -177 (1983). BROWN, A. D.: Microbial water stress. Bacteriol. Rev. 40, 803 - 846 (1976). CHALLENGER, F. and M. 1. SIMPSON: Studies on biological methylation 12. J. Chern. Soc. 43, 1591-1597 (1948). DICKSON, D. M. J. and G. O. KIRST: The role of dimethylsulfoniopropionate, glycine betaine and homarine in the osmoacclimation of Platymonas subcordiformis. Planta 167, 536-543 (1986).

Dimethylsulfoniopropionate effects on enzymes DUBNOFF, l W. and H. BORSOOK: Dimethylthetin and dimethyl-~­ propiothetin in methionine synthesis. l Bio!. Chern. 176, 789 -798 (1948). FLOWERS, T. J., P. F. TROKE, and A. R. YEO: The mechanism of salt tolerance in halophytes. Ann. Rev. Plant Physiol. 28, 89-121 (1977). GIMMLER, H., R. MADEN, U. KIRCHNER, and A. WEGAND: The chloride sensitivity of Dunaliella parva enzymes. Z. f. Pflanzenphys. 114(2),131-150 (1984). GINZBURG, M.: Dunaliella: a green algae adapted to salt. In: CALLOW, l A. (ed.), Adv. Bot. Res. 14, 95-193, Academic Press (1987). GREENWAY, H. and T. SETTER: Effects of chloride salts at high concentrations on glycolysis in vitro. l Exp. Bot. 28,545-558 (1977). HOCHACHKA, D. W. and G. N. SOMERO: Strategies of biochemical adaptations. W. B. Saunders Company, Philadelphia (1973). HOLLIGAN, P. M. and G. O. KIRST: Marine algae as a source of dimethylsulfide emissions to the atmosphere. In: UNESCO technical papers in marine sciences 56, 64-68 (1989). KARSTEN, U., C. WIENCKE, and G. O. KIRST: The ~-dimethylsulfo­ niopropionate content of macroalgae from Antarctica and southern Chile. Bot. Mar. 33, 143 -146 (1990). KELLER, M. D., W. K. BELLOWS, and R. R. L. GUILLARD: Dimethylsulfide production in marine phytoplankton. In: SALTZMANN, E. S. and W. l COOPER (ed.), Biogenic sulfur in the environment, 167 -182, American Chern. Soc. (1989). KIRST, G. 0.: Salinity tolerance of eukaryotic marine algae. Ann. Rev. Plant Physiol. Plant Mol. BioI. 40, 21-53 (1990). KIRST, G. O. and D. KRAMER: Cytological evidence for cytoplasmic volume control in Platymonas subcordiformis after osmotic stress. Plant, Cell and Environ. 4, 455-462 (1981). LOWRY, O. M., N. S. ROSENBROUGH, A. L. FARR, and R. l RANDALL: Protein measurement with the folin phenol reagent. l BioI. Chern. 193, 265-275 (1951).

91

POLLARD, A. and R. G. WYN JONES: Enzyme activities in concentrated solutions of glycine betaine and other solutes. Planta 144, 291-298 (1979). PRIEBE, A. and H. l JAGER: Responses from amino acid metabolising enzymes from plants differing in salt tolerance to NaCl. Oecologia 30,307 -315 (1978). REED, R. H.: Measurement and osmotic significance of ~-dimethyl­ sulfoniopropionate in marine macroalgae. Mar. BioI. Lett. 4, 173-181 (1983). REED, R. H., L. R. DAVISON, l R. CHUDEK, and R. FOSTER: The osmotic role of mannitol in the phaeophyta: an appraisal. Phycologia 24, 35-47 (1985). RICHTER, D. F. E.: Untersuchungen zum D-Mannitolmetabolismus in Platymonas subcordiformis Hazen. PD-thesis University of Bremen (1987). RICHTER, D. F. E. and G. O. KIRST: D-mannitoldehydrogenase and D-mannitol-1-phosphate dehydrogenase in Platymonas subcordi/ormis: some characteristics and their role in osmotic adaptations. Plant a 170, 528 - 534 (1987).

SIEBURTH, l M.: Antibacterial activity of antarctic marine phytoplankton. Limno!. Ocean. 4, 419-424 (1959). TREICHEL, S. P., G. O. KIRST, and D. l VON WILLERT: Veranderung der Aktivitat der Phosphoenolpyruvat-Carboxylase durch NaCI bei Halophyten verschiedener Biotope. Z. f. Pflanzenphysiol. 71(5),437 -449 (1974). VON WILLERT, D. l: Der Einflull von NaCI auf die Atmung und Aktivitat der Malatdehydrogenase bei einigen Halophyten und Glykophyten. Oecologia 14,127-137 (1974).

WIENCKE, c.: The response of pyruvate-kinase from the intertidal red algae Porphyra umbilicalis to sodium and potassium ions. J. Plant Physiol. 116,447-453 (1984).