Arch Microbiol (1988) 150:109 - 112

Archives of

Micraldelegy �9 Springer-Verlag 1988

Glycerol-3-phosphatase and the control of glycerol metabolism in Dunaliella tertiolecta cells Dirk Belmans and Andr6 Van Laere

Department of Plant Physiology and Biochemistry, Laboratory for Developmental Biology, Kardinaal Mercierlaan 92, B-3030 Heverlee (Leuven), Belgium

Abstract. Glycerol-3-phosphatase (EC 3.1.3.2.1) was studied by following the release of radioactive glycerol from L-(U- 14C)glycerol-3-phosphate in DunaIiella tertiolecta enzyme extracts. The reaction showed a neutral pH optimum and had an absolute requirement for Mg 2+. The substrate saturation curve was hyperbolic with an apparent Km value for glycerol-3-phosphate of 0.7 mM in the absence of phosphate. Inorganic orthophosphate was a competitive in- hibitor of the enzyme with an estimated Ki of 0.1 raM. The glycerol-3-phosphatase reaction was blocked nearly com- pletely by millimolar C a 2 + concentrations. C a 2 + inhibition did not depend on the presence of calmodulin in the reaction medium. The characteristics of glycerol-3-phosphatase are discussed in relation to the regulation of the cyclic glycerol metabolism in Dunaliella cells during periods of osmotic stress.

Key words: Calcium - Dunaliella tertioIecta - Enzyme kinetics - Glycerol - Glycerol-3-phosphatase - Meta- bolic regulation - Osmoacclimation

Evidence exists that the glycerol-3-phosphate dehydroge- nase is a control point in the glycerol cycle. Indeed, a tran- sient increase of the glycerol-3-phosphate content was found during the period of elevated glycerol synthesis following an hyperosmotic shock, whereas the triosephosphate content slightly decreased during the same period (Belmans and Van Laere 1987). The rising glycerol-3-phosphate concentration could seriously affect the flux rate via glycerol-3-phos- phatase depending on the characteristics of the enzyme.

In this paper we report substrate saturation kinetics and some other characteristics of the glycerol-3-phosphatase from Dunaliella tertiolecta. Using phosphate detection, which is poorly sensitive at low substrate concentration, Sussman and Avron (1981) found a rather high apparent Km of 2.7 or 8.5 mM, depending on the Mg 2+ concentration, for the glycerol-3-phosphatase of Dunaliella salina. We assayed the activity of the enzyme for Dunaliella tertiolecta by measuring the liberation of glycerol from L-(U14C) - glycerol-3-phosphate, which is very sensitive and reproduc- ible at the submillimolar concentrations which prevail in vivo.

DunalielIa tertiolecta is a unicellular, wall-less, green alga that, when confronted with a decrease or an increase of the waterpotential of the surrounding medium, accumulates or dissimilates, respectively, the compatible solute glycerol to restore osmotic equilibrium (Ben-Amotz and Avron 1973). To understand the osmoacclimating mechanism in Duna- liella cells a clear insight into the enzymology of glycerol metabolism is essential.

A glycerol cycle with dihydroxyacetonephosphate as the central metabolite most probably is operational in Dunaliella cells (Wegmann 1979). The glycerol synthetic pathway con- sists of two enzymes: a glycerol-3-phosphate dehydrogenase (Haus and Wegmann 1984a, b; Marengo et al. 1985) catalyzing the reduction of dihydroxyacetonephosphate to glycerol-3-phosphate and a glycerol-3-phosphatase (Suss- man and Avron 1981), converting glycerol-3-phosphate to glycerol. Glyceroldehydrogenase (Ben-Amotz and Avron 1974) and dihydroxyacetonekinase (Lerner et al. 1980) are believed to be responsible for the turnover of glycerol to dihydroxyacetonephosphate during adaptation of the cells to lower ambient solute concentrations.

Offprint requests to: D. Belmans

Materials and methods

Algal culture

Dunaliella tertiolecta (strain 19/6, kindly supplied by Prof. K. Wegmann, University of Tiibingen, FRG) was grown at 0.1 molar NaCI as described previously (Belmans and Van Laere 1987).

Preparation of enzyme extract

One liter algal culture in the late exponential growth phase (3 " 10 6 cells/ml) was harvested by centrifugation (2 rain at 2,000 x g). Before homogenization the cells were washed twice with 100 ml of a cold 100 mM Tris-HC1 buffer pH 7.5. Crude enzyme extracts were prepared by disruption of the cells in ice-cold extraction buffer (50 mM Tris pH 7.5 con- taining 1 mM DTT, 2 mM EDTA and 500 mM glycerol to stabilize the glycerol-3-phosphatase). The cells were broken by vigorous shaking for 2 min with 1.5 g glass beads (0.5 mm diameter) in 2 ml of the extraction buffer. Cell debris were removed by centrifuging the homogenate for 15 min at 40,000 x g in a pre-cooled Spinco centrifuge. The super- natant was quickly desalted by centrifuging through a

110

2O

o E = k

2

._= 10

I

-1 1 21.5 1/S (mM -1)

Fig. 1. Lineweaver-Burk plots of initial reaction velocities of glycerol-3-phosphatase at different substrate and Pi concentrations�9 The concentrations of Pi were: (0) none; (A) 0.1 raM; (rq) 0.3 raM; (L) 1 mM. The lines shown were drawn with the parameters obtained by fitting the non-transformed data to a hyperbola

Table 1. The effect P i o n kinetic constants of glycerol-3-phosphatase from Dunaliella tertiolecta. The reaction assay mixture contained 30raM Hepes pH7.1, l mM EDTA, 50raM KC1, 5mM magnesiumacetate, 10% PEG, 300,000 cpm L-(U-14C)glycerol-3 - phosphate and unlabeled substrate concentration varying from 0 to 10 raM. Apparent Km and Vm,~ values were calculated after plotting 1/V against 1/S. Lineweaver-Burk plots were calculated from the computer fitted hyperbolic stubstrate saturation curves

Concentration Apparent Km for Vmax of Pi (raM) Glyc.-3-P ( m M ) (gmol/min - mg prot)

0 0.7 0.63 0.1 1.4 0.63 0.3 2.3 0.72 1 4.5 0.77

Sephadex G25 column equilibrated with extraction buffer. All operations were performed at 0 - 4 ~ C.

Determination of enzyme activity

Glycerol-3-phosphatase (EC 3.1.3.2.1) was measured by the release of radioactive glycerol from g-(U-14C)glycerol-3 - phosphate at 25~ as described by Van Schaftingen and Van Laere (1985). Unlike the assay based on phosphate determination this method can easily be used at sub- millimolar substrate concentrations. The assay mixture contained 30 mM Hepes pH 7.1, 1 mM EDTA, 50 mM KC1, 5 mM magnesium acetate, 10% P(oly)E(thylene)G(lycol), 300,000 cpm g-(U-14C)glycerol-3-phosphate (from Amers- ham, Bucks, UK) and unlabeled L-glycerol-3-phosphate at the indicated concentrations. The reaction rates were calculated after subtraction of the blank which were low in all cases. Enzyme activities are expressed in gmol �9 min-1 �9 (mg protein)-1. Protein content was determined by the method of Lowry et al. (1951). Protein concentrations in the crude extracts were in the range of 6 - 8 mg/ml.

The kinetic data obtained were directly fitted to an hyperbola (Enzfitter: Elsevier Biosoft, Cambridge, UK) to

0.4

Z_--

i 0.3

~ 0 . 2

& o.~

0 10 50 100 Magnesium concentration (mM)

Fig. 2. The effect of different Mg z + concentrations on the activity of glyceol-3-phosphatase from Dunaliella tertiolecta. Mg z+ was added to the reaction mixture in the form of (O) MgC12 or (A) magnesiumacetate. Glycerol-3-phosphate concentration was 0.5 mM

estimate Km and Vmax- All values reported represent the average of at least two determinations.

Results

Like glycerol-3-phosphatase from Phycomyces (Van Schaf- tingen and Van Laere 1985), the glycerol-3-phosphatase from Dunaliella cells is difficult to purify since the enzyme quickly loses activity (Sussman and Avron 1981). This is especially the case when during purification protein concen- tration becomes low. Therefore we chose to evaluate the properties of the enzyme using crude extracts�9

pH optimum

The liberation rate of glycerol from glycerol-3-phosphate was maximal at pH 7 of the assay reaction medium. At this neutral pH optimum 0.3 ~tmol glycerol-3-phosphate was dephosphorylated per minute and per mg protein at a glycerol-3-phosphate concentration of 0.5 raM. Glycerol-3- phosphatase was almost completely inactive when the pH of the reaction mixture was lower than 5 or higher than 9 (not shown).

Substrate saturation kinetics

Glycerol-3-phosphatase showed hyperbolically shaped saturation curves with respect to the concentration of glycerol-3-phosphate. By fitting the data directly to a hyperbola an apparent Km of 0.7 mM was found for gly- cerol-3-phosphate in the absence of inorganic orthophos- phate (Fig. 1). Maximal reaction velocity (Vm,x) under these

t 0.3

~ 0 . 2 > ,

"7

0,1

$

I I I I

1 3 5 10 Calcium concenfrafion (raM)

Fig. 3. The effect of different concentrations of Ca 2 + on the activity of glycerol-3-phosphatase from Dunaliella tertiolecta. Ca 2 + was added in the form of (0 ) CaC12 or (A) calciumacetate. Glycerol- 3-phosphate concentration was 0.5 mM

Table 2. The effect of calcium and calmodulin on glycerol-3- phosphatase activity in Dunaliella tertioleeta extracts. The reaction mixture contained 30 mM Hepes pH 7.1, 1 mM EDTA, 50 mM KC1, 5 mM magnesiumacetate, 10% PEG, 300,000 cpm L-(U14-C) - glycerol-3-phosphate and 0.5mM unlabeled glycerol-3-phos- phate

Addition of Specific enzyme activity (pmol/min. mg protein)

Control 0.45 3 mM Ca 2 + 0.080 1 gM R24571 0.46 t p.M R24571 + 3 mM Ca 2+ 0.071 30 U calmodulin 0.43 30 U calmodulin + 3 mM Ca 2+ 0.071

conditions was 0.63 ~tmol/min - (mg protein)-1. Addition of increasing concentrations of inorganic phosphate to the reaction medium resulted in a marked inhibition of the enzyme at the lower substrate concentrations (Fig. 1). Vmax was hardly affected suggesting competitive inhibition by phosphate. An overview of the kinetic constants (apparent Km and Vmax values) under these conditions is presented in Table I. Using these and other similar data a Ki of 0.1 m M was calculated.

Effect of divalent cations

Glycerol-3-phosphatase had an absolute requirement for Mg 2+ (Fig. 2). Less activity was found when Mg 2+ was added with C1- as its counterion than after addition of Mg 2+ in the form of magnesium acetate (Fig. 2).

o L ca.

"T .E E

o E : I_

[ l o sL I

0.25

111

100 200 300 400 500

Salt concanfrafion (raM)

Fig. 4. The effect of different concentrations of various monovalent cations on the activity of glycerol-3-phosphatase from Dunaliella tertiolecta. Cations were added as (O) NaC1; (O) KC1; (ll) sodiumacetate or ([]) potassiumacetate

Glycerol-3-phosphatase was extremely sensitive to the presence of Ca 2 + in the reaction mixture. Release of glycerol from glycerol-3-phosphate was significantly inhibited by Ca z + concentrations of 1 m M (Fig. 3) even in the presence of 5 m M Mg z +. Lower Ca 2 + concentrations (pmolar range) had no effect on the enzyme activity. Neither the addition of the calmodulin inhibitor R24571 ( 1 0 - 6 - - 1 0 - 9 molar), nor the addition of spinach calmodulin ( 1 0 - 1 0 0 Units) to the glycerol-3-phosphatase reaction mixture changed the inhibition characteristics of Ca 2 + (Table 2).

Effect o f rnonovalent ions

Enzyme activities measured in the presence of different con- centrations of Na +, K + and C1- are presented in Fig. 4. Inhibition was strongest when C1- was present in the assay mixture, suggesting, as was the case with Mg z+, that C1- is the main inhibitory species.

Discussion

Glycerol-3-phosphatase proved to be very labile in cell-free extracts. Its activity rapidly decreased after a few hours and was strongly influenced by the protein concentration of the enzyme extract (not shown). Even in the presence of enzyme stabilizing factors as glycerol (500 raM) and DTT (1 raM), it was very difficult to attain an appreciable purification factor using current purification methods. Therefore, so as to mimic more closely the situation in vivo (Marengo et al. 1985), we determined glycerol-3-phosphatase activities with concentrated crude enzyme extracts.

Interference of acidic or alkaline non-specific phos- phatases with the glycerol-3-phosphatase reaction could be

112

excluded since the dephosphorylation rate of glycerol-3- phosphate showed a clear optimum at neutral pH and was near zero at a pH lower than 5 or higher than 9.

Using a more sensitive assay we found a lower apparent Km of 0.7 mM for glycerol-3-phosphate than Sussman and Avron (1981). Our Value is in the range of physiological glycerol-3-phosphate concentration under steady-state con- ditions, whereas inorganic ortbophosphate in millimolar concentrations greatly increases the apparent K,, (Table 1).

The glycerol-3-phosphatase activity was strictly depend- ent on the presence of Mg 2 + in the reaction mixture (Fig. 2). Sussman and Avron (1981) suggested that the real substrate of the glycerol-3-phosphatase is a glycerol-3-phosphate- Mg 2+ complex.The inhibition of the phosphatase reaction at millimolar Ca 2 § concentrations (Fig. 3) might be due to a competition with Mg 2§ for a specific binding site on the glycerol-3-phosphatase (Sussman and Avron 1981). The Ca z +-calmodulin complex did not affect the Ca 2 + inhibition characteristics of the enzyme (Table 2). Calcium inhibition might have physiological significance, since the calcium content in Dunaliella cells has been reported to vary between I and more than 30 mM, depending on the Ca z+ and Na + concentration in the growth medium (Pick et al. 1986). Although most of the Ca z+ might not be in a free form, a sequestration from, or release of, Ca 2 + in the cytosol might clearly affect in vivo activity of the glycerol-3-phosphatase.

The fact that the affinity of glycerol-3-phosphatase for its substrate was strongly diminished by increasing concen- trations of inorganic phosphate in the assay mixture (Table 1), might also have important consequences for the in vivo activity of the glycerol-3-phosphatase and its role in the control of the rate of glycerol synthesis. At a Pi concentration of I mM [ = the overall phosphate concentra- tion in Dunaliella tertiolecta cells (Belmans and Van Laere 1987)], half maximal reaction velocity of the glycerol-3- phosphatase was reached only at a substrate concentration of 4.5 mM (Table 1). Since cellular glycerol-3-phosphate concentration was much lower (approx. 0.6 mM) during steady-state conditions, the glycerol-3-phosphatase activity will be rather low and hardly any glycerol will be synthesized. However, Belmans and Van Laere (1987) found that the cellular glycerol-3-phosphate concentration transiently in- creased severalfold, to reach values of 5 raM, immediately after an hyperosmotic shock. This event might greatly in- crease the flux rate via glycerol-3-phosphatase and in doing so be responsible for the increased synthesis of glycerol during the stress period.

Acknowledgement. We are grateful to Luc Slegers for skilful help with the experiments.

R e f e r e n c e s

Belmans D, Van Laere A (1987) Glycerol cycle enzymes and in- termediates in Dunaliella tertiolecta cells during hyperosmotic stress. Plant Cell Environ 10:185-190

Ben-Amotz A, Avron M (1973) The role of glycerol in osmotic regulation of the halophilic alga Dunaliella parva. Plant Physiol 51 : 875- 878

Ben-Amotz A, Avron M (1974) Isolation, characterization and partial purification of a reduced nicotinamide adenine di- nucleotide phosphate-dependent dihydroxyacetone reductase from the halophilic alga Dunaliella parva. Plant Physiol 53 : 628- 631

Haus M, Wegmann K (1984 a) Glycerol-3-phosphate dehydrogenase (EC 1.1.1.8) from Dunaliella tertiolecta. I. Purification and kinetic properties. Physiol Plant 60: 283 - 288

Haus M, Wegmann K (1984b) Glycerol-3-phosphate dehydroge- nase (EC 1.1.1.8) from Dunaliella tertiolecta. II. Influence of phosphate esters and different salts on the enzymatic activity with respect to osmoregulation. Physiol Plant 60:289-293

Lerner HR, Sussman I, Avron M (1980) Characterization and partial purification of dihydroxyacetone kinase in Dunaliella salina. Biochim Biophys Acta 615:1 - 9

Lowry OH, Rosebrough NJ, Farr AL, Randall RJ (1951) Protein measurement with the Folin phenol reagent. J Biol Chem 193 : 265- 275

Marengo T, Lilley RMcC, Brown AD (1985) Osmoregulation in Dunaliella. Catalysis of the glycerol-3-phosphate dehydroge- nase reaction in a chloroplast enriched fraction of Dunaliella tertiolecta. Arch Microbiol 142: 262- 268

Pick U, Karni L, Avron M (1986) Determination of ion content and ion fluxes in the halotolerant alga DunalielIa salina. Plant Physiol 81:92-96

Sussman I, Avron M (1981) Characterization and partial purifica- tion of DL-glycerol-l-phosphatase from Dunaliella saIina. Bio- chim Biophys Acta 661:199-204

Van Schaftingen E, Van Laere A (1985) Glycerol formation after the breaking of dormancy of Phycomyees blakesleeanus spores. Role of an interconvertible glycerol-3-phosphatase. Eur J Bio- chem 148: 399- 404

Wegmann K (1979) Biochemical adaptation of Dunaliella tertiolecta to salinities and temperature changes. Ber Dt Bot Ges 92: 43 -54

Received September 14, 1987/Accepted January 25, 1988