Partial purification and characterization of stomatal

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analysis revealed that the stomatal enzyme had two bands (M, of 11()(KK) and. 112000), crossreacting with PEPCase antibodies raised against PEPCase from ...

PHYSIOLOGIA PLANTARUM 87: 96-102, Copenhagen 1993

Partial purification and characterization of stomatal phosphoenoipyruvate carboxylase from Viciafaba Martin Denecke, Margot Schulz, Christoph Fischer and Heide SchnabI

Denecke, M.. Schulz. M,. Fischer, C. and SchnabI, H. 1993. Partial purification and characterization of stomata! phosphoenolpvruvate carbosylase from Vicia faba Physiol. Plant. 87; 96-102. Stomatal phosphoenoipyruvate carboxylase (PEPCase EC, extracted from abaxial epidermal peels of Vieia faba L. cv. Fruhe WeiBkeimige. was partially purified by ammoniumsulfate precipitation, and molecular sieve (Sepharose S-4(K)) and ion exchange (DEAE-Sepharose) chromatography. The partially purified enzyme, essentially free of a PEPCase isoform existing in mesophyll and epidermal cells, had a specific activity of 3(K) nkat m g ' protein at 2.S°C. Western immunobtot analysis revealed that the stomatal enzyme had two bands (M, of 11()(KK) and 112000), crossreacting with PEPCase antibodies raised against PEPCase from Kalanchoe daigremontiana. The native molecular mass of the enzyme (467000) points to a tetrameric subunit structure. The temperature optimum was found to be 35°C; cold treatments of PEPCase before assaying were accompanied by inactfvation. The energy of aetivation was calculated to 51 kJ moL'. 'The kinetic behaviour of the enzyme at fixed MgCl; concentrations is characterized by a pH optimum between pH 8.0-8.2 with or without 1 mM maiate or 5 mM glucose-6-phosphate (Glc-6-P). but a combination of both effectors resulted in a shift of the optimum to pH 7,6. The enzyme showed a pH sensitive inhibition by 1 miM malate and an activation by Glc-6-P, At low pH (6-7). Glc-6-P was able to compensate for the malate induced inhibition of the enzyme. Malate and G!c-6-P both affected K^ip^p, drastically and influenced V^^ at pH 7, but not at pH 8.3. The inhibition constant of malate was determined to be 1.2 mM at pH 7. From the Dixon plot, a competitive inhibition of malate was assumed under defined assay conditions. Key words - Glucose-6-phosphate, kinetics, malate. phosphoenoipyruvate carboxylase. purification, stomata, Vicia faba. M. Denecke. M, Schulz and H. Sehnabl fcorresponding author), tnstilulfiir Landwirtsehaftliche Botanik. Univ. Bonn, Meckenheimer Allee 176. W-53l)0 Bonn I. Germany.

, introduction Phosphoenoipyruvate carboxylase (EC, occurring universally in higher plants, catalyses the irreversible carboxylation of phosphoenoipyruvate (PEP). The cytoplasmic enzyme plays an important regulatory role in the photosynthetic pathways of C4 and CAM plants. In these plants, specific PEPCase isoforms are found, which differ in their malate-dependent kinetic properties (Kluge et al. 1988, Echevarria et al. 1990, Jiao and ChoUet 1991). The enzyme also has various functions in C3 plants, including anapieurotic COj fixation and involvement in N, assimilation (O'Leary 1982,

Schuller et al. 1990). During the past two decades. attention has been focused on the role of PEPCase during physiological processes of stomatal movements, It is generally accepted that PEPCase catalyses the first step in malate production, a metabolite accumulating during stomatal opening that compensates K* uptake during the swelling of guard cells (SchnabI and Kottmeier 1984a,b, Kottmeier and SchnabI 1986, Outlaw 1990, SchnabI 1992). Whereas a large number of C4 and CAM isozymes, and C3 PEPCases from whole leaf tissue or from phototrophically cultured green tobacco ceils have been purified and intensively characterized, no stomatal

Received 14 July, 1992; revised 25 September, 1992 96

Physiol. Planl. 87. 1»3

PEPCase has been similary studied after purification (Miziorko et al. 1974, OXeary 1982, Sato et al, 1988). Biochemical properties of guard cell PEPCase were investigated with crude extracts either from guard cell protoplasts or from guard cell pairs dissected from freeze dried leaves (Schnabl and Kottmeier 1984a, Kottmeier and Schnab! 1986, Tarczynski and Outlaw 1990). However, determination of the enzyme activity in crude extracts can he influenced by endogenous effectors such as malate, Mg-', PEP, and K*, and modulations of PEPCase activity are to be expected in their presence. The swelling process of guard cell protoplasts is correlated with a decrease of the Kn^pj^p, as shown by Kottmeier and Schnabl (1986). Moreover, Michaike and Schnabl (1990) measured oscillations of PEPCase activity during the K*-induced volume increase of guard cell protoplasts. To avoid contaminations of low molecular weight effectors. Schnahl and Kottmeier (1984a) analyzed properties of stomatal PEPCase with desalted crude extracts from guard cell protoplasts. Outlaw et al. (1979) reported on guard cell PEPCase properties studied by histochemical techniques. However, the importance of PEPCase as a regulatory enzyme during stomatal opening and the various possibilities of activity modulations (e.g., light, abscisic acid, maSate, K*, pH) requires further investigation of PEPCase properties after purification. Starting with epidermal peels, we will focus here on the development of a purification procedure that yields an enzyme preparation essentially free from the isozymic forms associated with the mesophyll or epidermis (Schuiz et al. 1992). Furthermore, we will present some properties of the partially purified stomatal PEPCase,

guard cells were disrupted. Leupeptin was found to conserve 80% of the enzyme activity during a period of 8 h at room temperature. The homogenate was filtered through a 20 \im nylon net and centrifuged at 12000 g for 10 min. The supernatant was saturated to 33% with (NH4)2SO4 and allowed to equilibrate for 45 min. The resulting suspension was centrifuged at 47(K)0 g (45 min) and the pellet discarded. Solid (NHJ^SOj was added to bring the supernatant to 70% saturation. Equilibration and centrifugation were performed as described above. Further purification steps were performed at room temperature. The ammoniumsulfate precipitate containing the PEPCase activity was dissolved in extraction buffer and chromatographed on a Sephacryl S-4(K1 column (105x1.4 cm), previously equilibrated with extraction buffer. Protein was eluted with the same buffer at a flow rate of 0.2 ml min '. Active fractions were combined and concentrated by uitrafiltration (Amicon cell. PM-30 membrane) to a final protein concentration of 1 g I ^ The concentrate was applied to a DEAE-Sepharose column (11 x 1 cm), preequilibrated with 50 mM Tris-HEPES buffer, pH 7. containing 5 mM DTE, 1 mM MgCU and KXI mM KCI. The fraction containing PEPCase activity was eluted with a linear gradient from KK) to 500 mM KCI in Tris-HEPES buffer at a flow rate of 0.6 ml min '. Active fractions were combined and concentrated as described above to a final protein concentration of 0.1 g I '. Subsequently, chromatography on DEAE-Sepharose was repeated.

PEPCase assay

The activity of the enzyme was measured spectrophotoAbbreviation.^- - Glc-6-P, glueose-6-phosphate; MDH, malate metrically in an assay medium as described by Schuiz et dehydrogenase; PEP, phosphoenolpyruvate; PEPCase, phosa!. (1992). The assay mixture consisted of KM) mM Trisphoenolpyruvate carhoxylase. HEPES. pH 8.3. 10 mM KHCO,. 5 mM MgCl,. 0.2 mM NADH, 4 mM PEP, 33.3 nkat MDH, and 10-.50 ^1 enzyme solution to a final volume of 1 ml. The producMaterials and methods tion of 1 nmol of oxaloacetate per min at 25°C was Plant material defined as one unit of enzyme activity. Vicia faba L, cv. Fruhe WeiBkeimige (Zwaan-Pannevis. Kleve. Germany) was grown for three weeks from seed as previously described (Schuiz et al, 1992). Fully expanded leaves were removed during the light period. Abaxial epidermal tissue was peeied and collected in ice cold 10 mM sodium-ascorbate within 1 h and frozen in liquid nitrogen. Epidermal peels were stored at -2O''C for maximally 3 weeks. Puriflcation of stomatal PEPCase

Unless otherwise noted, initial purification steps were performed at 4°C. Epidermal tissue (20 g) was ground in a mortar with 80 ml 100 mM Tris-HC! buffer, pH 7.5. containing 10% (v/v) glycerol, 1 mM MgCl2, 10 mM KCI, 2 mM EDTA, 20 mM sodium-ascorbate, 5 mM dithioerythreitol (DTE), and 20 ^.M leupeptin until all Phvskil. Plant. 87. 1W.1

Characterization of PEPCase Initial characterization of PEPCase (temperature optimum, pH optimum, K„,(p^p, values in presence of several effectors. K,,^,.,,.,,^,, at pH 7) was performed with the partially purified enzyme. The kinetic data were fitted with the aid of a computer program (Biosoft Enzfitter. Milltown. USA). The native molecular weight of PEPCase was determined by molecular sieve chromatography using a Sephacryl S-.300 high resolution column, calibrated with the following standards; dextran blue (M, 2000000), bovine thyroglobine (M, 669(WK)). horse ferritin (M, 450000). bovine catalase (M, 240(KKI), and rabbit muscle aldolase (M, !60(K)0). Molecular mass of PEPCase subunits was determined by SDS-po!yacrylamide gel electrophoresis and western blot analysis. 97

Tab. 1. Purification of phosphoenolpyruvate carboxylase. Crude = crude extract. 33-70% = (NH4),SOj precipitation, S-40fl = Sephacryl S-4(X), DEAE = DEAE Sepharose, PM 30 — ultrafiltration. Step

Total Total Specific Yield Yield Increase protein activity activity protein activity (-fold) (mg) (nkat) (nkat' (%) (%)' (%)


Crude 33-70% S-40() DEAE PN 30 DEAE

51.8 19,6 3.9 0.7 0.7 0.4

188.3 161.7 115.0 50.0 51.6 12.0

3.1 8.2 28.9 66.6 70.0 311.4

KM) 37 7.6 1.4 1,4 0.1

100 86 6! 26 27 6

1 2.3 8.2 18.2 19.8 88,1

SDS-electrophoresis and western immunoblot

SDS-polyacrylamide gel electrophoresis was performed according to Laemmli (1970). Protein samples were soiubilized in sample buffer, boiled for 4 min and cleared by centrifugation in a microcentrifuge at 5000 g. Proteins were separated in a Mini-Protean II vertical slab unit (Bio Rad, Miinchen, Germany) on 10% polyacrylamide gels. The Bio Rad low molecular weight marker kit complete with E. coli /J-galactosidase {M^ 116000) (Sigma, Miinehen, Germany) was used for determination of molecular mass. Gels were either stained with Coomassie brillant blue or electrotransferred to nitrocellulose membranes (pore size 0.45 \im}. Electrotransfer was carried out following the method of Towbin et al. (1979). Membrane sheets were incubated for 1 h with PEPCase antibodies raised against a PEPCase preparation purified from Kalanchoe daigremoiitiana. For visualization of the bound primary antibody, a 30 min incubation was performed with goat anti-rabbit antibodies conjugated to alkaline phosphatase.

zyme extraction was carried out in the presence of Mg"*, glycerol and sodium-ascorbate, which had stabilizing effects on PEPCase activity. Without Mg^*, using the standard assay mixture, only 177o of the activity could be obtained. Dtiring storage of PEPCase solutions, the activity could be conserved for a further 5 days by glycerol and sodium-ascorbate. Mg-* was therefore added to all buffers used for the chromatographic purification steps. Crude extracts usually showed PEPCase activity in the range of 3.3 nkat mg"' protein. The enzyme was concentrated from crude extracts by (NH4)2SO4 precipitation. The inhibitory effect of NH; ions at high concentrations was completely abolished during molecular sieve chromatography. The latter, and further chromatographic steps, were performed at room temperature sitice in preliminary studies we observed a loss of PEPCase activity after cold treatments (data not shovi'n). Because of proteolytic activity released from disrupted tissue, it was not possible to extract the enzyme at room temperature, although PEPCase degrading proteases were inhibited by leupeptin. Chromatography on Sephacryl S-4CK1 resulted in an 8 fold purification and 60% of the original activity could be recovered (Tab. 1). A separation of the major activity peak from most of the protein was obtained (Fig. 1). However, the enzyme showed another peak of low activity coinciding with the main protein peak, as indicated by the elution profile. From molecular mass determinations, the second peak of activity should present a monomeric form of the enzyme. Only fractions belonging to the major activity peak were collected for further purification. DEAE-Sepharose chromatography

Standard methods and chemicals Proteiti was determined by the method of Bradford (1976) using BSA as a standard. Chemicals were obtained from Boehringer, Mannheim, Germany (MDH, PEP) and from Sigma. Reagents for electrophoresis were purchased from Bio Rad. Materials for molecular sieve chromatography were from Pharmacia (Miinchen, Germany).

Results Partial purification of stomatal PEPCase

The distribution of PEPCase in the different cell types revealed the possibility of using epidermal peels (88% of PEPCase activity in guard cells) for enzyme extraction (Schulz et ai. 1992). For an effective purification, a minimum of 20 g of epidermal tissue had to be tised. During storage over 3 weeks at —20°C, the loss of PEPCase activity in frozen peels was negligible. En98


Elution voluiDB (ml) Fig. 1. Molecular sieve filtration after (NH4)2SO4 precipitation (;33%-70% saturation). The elution profiles of PEPCase activity ( • ) and protein ( • ) are shown. Physiol, Plant, 87, 1993

bodies when gels were immunobiotted (Fig. 3). These bands were demonstrated to be those characteristic of guard cell protoplasts (Schuiz et al. 1992). It is therefore assumed that the activity of the partially purified enzyme belongs to PEPCase isoforms or subunits, which are involved in stomatal movements. Properties of PEPCase .. 300

_ 100


v o l u m e (ml)

Fig. 2. Ion exchange chromatography (DEAE-Sepharose) of PEPCase after molecular sieving. PEPCase activity eluted at 300 mM KCI. A smaller peak of activity pre-eluted at 130 mM KCI. PEPCase activity (•), protein (•) and KCI gradient t)-130 and i3(KiOO mM KCI (dashed line) are given.

was performed to remove further contaminating protein. Using the linear gradient described in Materials and methods, PEPCase activity eluted at about 250 mM KCI in a major peak (Fig. 2). At 100 mM KCI another, smaller peak of activity pre-eluted, which was not combined with the other active fractions. Repeated chromatography on DEAE-Sepharose resulted in an 88-fold purification of PEPCase with a final yield of 6% activity (Tab. 1), Although the enzyme was not purified to homogeneity as indicated by SDS-electrophoresis (data not shown), only two bands with molecular masses of 110000 and 112000 cross-reacted with PEPCase aotiB

-110 kDa

I'ig. 3. PEPCine-immunoblot of (.A) crude extract, (B) protein concentrate, after chromatography on DEAE-Sepharose. Lane B shows an enrichment of the three PEPCase bands compared to the crude extract (A). Lane C illustrates that oniy the two stomatal PEPCase bands (M, of 110000 and 112000) are obtained after purification. Physiol Pliinl. 87. t

Molecular weight determination

The elution volume of PEPCase from Sephacryl S-300 coincided closely with that of horse ferritirj exhibiting a molecular mass of 450000. The molecular mass of the native enzyme was estimated to M, 465000, the monomeric form to about 110000. The electrophoretic mobility of the denatured PEPCase on SDS-gels indicated a subunit molecular mass of 110000 for the band with the strongest immunoresponse. A tetrameric composition would be consistent with ail experimental observations made on C4 and CAM PEPCases, Temperature optimum Tbe temperature optimum was found to be 35°C. Assaying the enzyme at higher temperatures was accompanied by a drastic decrease in activity. From Arrhenius plots (log V^j,, vs K^') an energy of activation of 51.5 kJ mol"' was calculated. No discontinuity was obser\'ed in the plot, as was found for some C4 PEPCases. Kinetic properties and pH optima

Apparent K^ values for PEP were determined without and in the presence of 1 mM malate and 5 mM Glc-6-P at pH 8.3 and 7 (Tab. 2). When PEPCase was assayed at pH 8.3, a K^ of 0.059 + 0.003 mM was calculated' from Lineweaver-Burk plots. However, a much larger K^ was observed at pH 7 (0.35 ± 0.15 mM). Also. V,^, was significantly reduced at pH 7 compared to at pH 8.3 (Tab. 2). In the presence of 1 mM malate, a further drastic increase of K^ was found (pH 7), whereas ¥„„ was not affected significantly (Tab. 2). At pH 8.3, malate had no significant influence on K^. but V^, was reduced. Glc-6-P had a stimulating effect on PEPCase activity at pH 7. Additions of 5 mM Glc-6-P caused a decrease of K,, and an increase of ¥„„ (Tab. 2). At pH 8.3, K^ and ¥„,„ were nearly unaffected by Glc-6-P, Obviously, the effector Glc-6-P compensates for the increase of K^ due to the lower pH. Furthermore. Glc-6-P was able to suppress the maiate-induced increase of K» at pH 7, partly to 100%. When PEPCase was assayed in presence of 1 mM malate and 5 mM Glc6-P, a K^ of 0.06 ± 0.01 mM was obtained and ¥ „ , increased slightly. The addition of 1 mM malate and 5 mM Glc-6-P at pH 8.3 reduced K,,, whereas V^,,, was nearly unaffected. PEPCase activity was profiled as a function of pH (Fig. 4). An optimum of activity was found at pH 8.1K8.2. when PEPCase was measured under standard conditions, but with varying pH of the assay mixture.

Tab. 2. Effect of glucose-6-phosphate and raalate on nkat mg '. Means ± so.

pH 7 pH8.3

p^ and V^y^ of phosphoenolpyruvate carboxylase. K^ in mM; V^,.,^ in

Without effector

+ 5mM

0.35±0.15 7.2±0.5 O.(I6±O.(X) 15.7±().7



I3.2±0.5 0.05±0.01 16.2±0.7

+ 1 mM malate

+ I mM malate -^ 5 mM Glc-6-P

0.85 ±0.16 8.3±0.8 0.04±0.(K) 10.6±!).4

0.06±0.01 9.9±0.6 O.(I4±O.OO I5.2±O.3

Similar pFi optima were obtained by supplementing the assays with 1 mM malate or 5 mM Glc-6-P. However, a combination of 1 mM malate and Glc-6-P resulted in a shift of the pH optimum to pH 7.6. pH dependent PEPCase activity is affected considerably by malate and especially by Glc-6-P at low pH. In the presence of 1 mM malate. low activity was found at pH 6. but 5.8 nkat mg ' protein was measured when also 5 mM Glc-6-P was added. When the latter effector was supplemented alone, the activity was found to be 10.8 nkat mg"' protein. Generally PEPCase activity was up to 100% higher at low pH when determined in presence of G-6-P. which also reduced malate inhibition in the lower pH range (pH 6-7). The Kj for malate was determined by Dixon plot. A value of 1.2 mM w as obtained, when malate concentrations were varied between 0 and 5 mM at fixed PEP concentrations (0.5, 1, 2, and 4 mM PEP) and assays were performed at pH 7. Under these conditions, kinetic analysis indicated a competitive inhibition by malate (Fig. 5).

Stomatal PEPCase was partially purified by molecular sieve and ion exchange chromatography. yielding a specific activity of at least 300 nkat mg ' protein. A similar specific activity was achieved with spinach leaf PEPCase after a 7-step purification procedure (Miziorko et al. 1974). Despite precautions (room temperature. MgCU). a small part of PEPCase obviously dissociated to the monomeric form during chromatography on Sephacryl-400, causing some loss in final activity. However, the major part of the activity was lost after chromatography on DEAE-Sepharose, due in part to the splitting into two peaks of the total applied activity. As expected, molecular sieve chromatography was not suitable for separating PEPCase isoforms. This is in agreement with the results of Outlaw et al. (1979), who described a coincidental elution of PEPCase activity from Sephadex G-200 performed with enzyme preparations obtained from epidermal peels and leaflets of

Fig. 4. The pH optimum of PEPCase in the presence of 1 mM malate ( • ) , 5 mM Glc-6-P ( • ) , 1 mM malate -I- 5 vaM Glc-6-P ( • ) and without effectors (O).

Fig. 5. Dixon-plot for the determination of K; (malate). Inhibitor concentrations (malate) were varied between 0 and 5 mM at fixed PEP concentrations: 0.5 (D). 1 ( • ) , 2 ( • ) and 4 mM PEP (O). Assays were performed at pH 7. The K^ (maiate) was 1,2 mM,



Physin! Plant 87.

Vicia faba. As illustrated in Fig. 2, chromatography on DEAE-Sepharose resulted in a separation of a PEPCase isoform appearing in epidermal and mesophyll cells and eventually also in guard cells (compare Schulz et al. 1992). Our results confirm the observations of Ting and Osmond (1973a,b), who described existence of different forms of PEPCase in leaves of C4 and C3 plants. These isozymes could be partly distinguished by certain kinetic properties but more particularly by their elution behaviour on DEAEcellulose, The separation of stomatal PEPcase from an epidermal and a mesophyllic form was an important prerequisite for studying its kinetic and other properties. The remaining two PEPCase bands, detectable after western immunoblot, should represent either very similar forms or subunits with slightly different molecular masses that cannot be distinguished by SDS-PAGE, Nimmo et al. (1986) reported that highly purified PEPCase day and night forms from Bryophyilum fedtschenkoi were indistinguishable by chromatography on Mono Q and Superose 6. The two forms also showed identical behaviour on SDS and native polyacrylamide gels, but were different in malate sensitivity. The latter was accompanied by phosphorylation and dephosphorylation, which was supposed to be the only difference between the two forms. However, phosphorylation of a protein often produces changes in electrophoretic mobility (Laird et al. 1991). Both day and night forms exhibited the same subunit-composition of M, 123 000 and 112(X)0. It is therefore possible, that stomatal PEPCase activity eluted from DEAE-Sepharose at 250 mM KCl contained two co-purified forms representing a mixture of phospho/dephospho-PEPCase. This possibility will be clarified in further experimentation. It cannot be excluded that the tetrameric enzyme is composed of two different subunits, a problem which still has to be resolved. The native molecular mass of the enzyme (465 (UK)) was comparable to PEPCase from other plants (O"Leary 1982, Fijikuna et al, 1991). The temperature optimum was found to be 35°C. Higher temperatures caused a rapid loss in activity. An instability of the enzyme above 35°C was also observed by Outlaw et al. (1979). Also low temperature decreased the activity, when the enzyme was assayed at 25°C after cold treatment. As no discontinuity was observed in Arrhenius plots, a cold inactivation is assumed. Giucose-6-phosphate and malate are well known effectors not only of C3, C4 and CAM PEPCase, but also of guard cell PEPCase (Meyer et al. 1989, Wedding et al. 1989). In contrast to C3, C4 and CAM alloenzymes, kinetics of stomatal PEPCase were never obtained with a (partially) purified enzyme. Kinetic studies using crude extracts can only be preliminary, as interactions with endogenous effectors that may be present in different concentrations in the cells depending on physiological state, are not assessable. This might be one reason for contradictory results, especially concerning K^ valPhysiol. Planl. 87. 1993

ues, presented by different authors (Kottmeier and Schnabl 1986, Outlaw 1990), In the present investigation, we therefore focused on a re-investigation of K^jpEpi and V^,, both in the presence and absence of malate and Glc-6-P, as well as pH dependency at fixed Mg-* concentrations. Glc-6-P activates PEPCase in C4 and CAM plants by lowering the K^jpip, and by a slight increase in V^,, (O'Leary 1982, Wedding et al. 1989), Furthermore, Glc-6-P balances malate-induced inhibition of the enzyme. Under the assay conditions as presented, stomatal PEPCase showed a comparable behaviour: Glc-6-P induced changes of K^|p[;p, to smaller values, slightly affected the velocity of the reaction, and reactivated the enzyme in presence of malate. which is in agreement with the results of Donkin (1982). This behavioural pattern, however, could be demonstrated only at pH 7. but not at pH 8.3. where effects of Glc-6-P were insignificant. Also at pH 8.3, malate had no striking effect on K^. The pH-dependent activity profiles of the enzyme in the presence and absence of the effectors fitted well to the kinetic data. The K^, values obtained with the partially purified PEPCase are similar to the values published by Kottmeier and Schnabl (1986). Thus alkaline pH generates not only a decrease of K^iptPi but also an increase in V^,,^. Our results support the assumption that PEPCase can act as a pH-stat during stomatal opening (Smith and Raven 1979). For Vicia faba guard cell PEPCase, Outlaw (1990) calculated a K^(Ms.pEP) of 0.07 + 0.03 mM at pH 8.5. The K^ obtained by Tarczynsky and Outlaw (1990), however, was about 100% higher (0.16 + 0.02 mM), One reason for this discrepancy might have been due to the different types of plant material used for the studies (extracts from protoplasts and from microdissected guard cells). The inhibitory effect of malate on stomatal PEPCase is well documented (Outlaw 1990, Schnabl and Kottmeier 1984a,b, Kottmeier and Schnabl 1986). Malate concentrations causing 50% inhibition have been published by Outlaw (1990) and Kottmeier and Schnabl (1986). Values presented vary from 50 to 700 uAf malate, depending on the assay conditions. In the present paper, the inhibition constant for malate was determined to be 1.2 mM with the above-mentioned concentrations of PEP and malate. It is possible that K^ may depend on assay conditions such as pH, Mg-*, or K* concentrations, which will have to be part of future studies. The kinetic analysis points to a competitive inhibition when the substrate PEP was varied between 0.5 and 4 mM. Malate was found to be a competitive inhibitor for crude stomatal PEPCase by Raschke et al. (1988) and Outlaw (1990). Sato et al. (1988) reported on a mixed-type inhibition by malate for PEPCase purified from photomixotrophically cultured tobacco cells, as we!l as for the purified enzyme of Hevea brasiliensis (Jacob et al. 1979). We have obtained some indication that at very low PEP concentrations the type of inhibition can change (data not shown). Further investigations are needed to explain this preliminary observa101

leaf phosphoenolpyruvate carboxylase: purification, properties and kinetic studies. - Arch. Biochem. Biophys. 163: 378-389. NimmD, G. A., Nimmo, H. G., Hamilton, I. D., Fewson. C. A. & Wilkins, M. B. 1986. Purification of the phosphorylated night form and dephosphorylated day form of phosphoenolpyruvate carboxylase from Bryophyllum fedtschenkoi. - Biochem. J. 239: 2LV220. O'Leary. M. 1982. Phosphoenolpyruvate carboxylase: an enzymologist's view. - Annu. Rev. Plant Physiol. 33: 297315. Acknowledgements - We thank Prof. M. Kluge, !nstitut fiir Outlaw, W. H. 1990. Kinetic properties of guard cell phosphoenolpyruvate carboxylase. - Biochem. Physiol Pflanz. Botanik der Technischen Hochschuie Darmstadt. Germany. 186: 317-325. for the gift of polyclonal PEPCase antibodies and T. Klock- , Mancheter. J., DiCamelli, C. A. 1979. Histochemical enbring for assistence. 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tion, e.g., whether interactions of PEPCase isoforms with different malate sensitivity but otherwise identical properties might be involved, or whether an allosteric behaviour of PEPCase in the presence of suboptimal PEP concentrations is responsible for this discontinuity. In the first case, the two PEPCase bands found after western immunoblotting should be considered as possible isoforms.

Edited by C. H. Bornman 102

Physkit. Plant 87. 1993

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