Compared properties of phosphoenolpyruvate carboxylase from dark- and light-adapted Sorghum leaves: use of a rapid purification technique by immunochromatography

Plant Science, 81 (1992) 37-46 Elsevier Scientific Publishers Ireland Ltd.

37

Compared properties of phosphoenolpyruvate carboxylase from dark- and light-adapted Sorghumleaves: use of a rapid purification technique by immunochromatography Martine Arrio-Dupont, NaYma Bakrim, Cristina Echevarria*, Pierre Gadal, Pierre Le Mar6chal and Jean Vidal Laboratoire de Physiologic VOgktale Mol~culaire, URA CNRS 1128, Universitk de Paris-Sud. Centre d'Orsay, batiment 430, 91405 Orsay cedex (France)

(Received July 3rd, 1991; revision received August 29th, 1991; accepted September 18th, 1991)

Two C4-type phosphoenolpyruvate carboxylase (EC 4.1.1.31, PEPc) forms of Sorghum leaves, differing by their phosphorylation state (the phosphorylated form being predominant during the day), have been purified from dark- and light-adapted leaves by using an immunoaffinitychromatography column. The phosphorylation state of these purified PEPc forms was assessed by incubation with either the endogenous PEPc protein-serine kinase, or the catalytic subunit of the cAMP-dependent protein kinase and alkaline phosphatase on the dark and light enzyme, respectively.HPLC gel filtration studies showed that both forms are tetrameric at concentrations used throughout the work. Comparison of the kinetic properties at pH 7.0, 7.3 and 8.0, indicated a cooperativity for phosphoenolpyruvate (PEP) at all pHs considered, with a Hill number of 2 for both forms and higher Vm (2- to 4-fold) and K m for the light form. Malate inhibition mainly affected the Vm of the dark form, whereas it was an allosteric inhibitor for the light enzyme. The results established that phosphorylation is the molecular basis which mediates the functional properties of the enzyme. In a medium mimicking an illuminated mesophyll cytosol (2.5 mM PEP, 5 mM glucose-6-P, 20 mM malate and 10 mM MgCI2 (pH 7.3), the activity of the enzyme from light-adapted leaves was twice that of the dark one. This increased activity due to phosphorylation may be of importance for the regulation of C4 photosynthesis. Key words: phosphoenolpyruvate carboxylase (Sorghum leaves): immunoadsorbent; regulatory protein phosphorylation: photosynthesis: day/night photoregulation

Introduction The photosynthetic CO2 fixation process in C 4 plants is initiated in the cytoplasm of leaf mesophyll cells by the c a r b o x y l a t i o n of p h o s p h o e n o l pyruvate [l].The soluble enzyme PEPc catalyses the conversion o f P E P a n d H C O 3- to oxaioacetate a n d inorganic phosphate. This enzyme which is usually purified as a tetramer, is MgZ+-depen Correspondence to: M. Arrio-Dupont, Laboratoire de Physiologic V6g&ale Mo16culaire, URA CNRS 1128, Universit~ de Paris-Sud, Centre d'Orsay, batiment 430, 91405 Orsay cedex, France. *Present address: Dpto. Biologia Vegetal y Ecologia, Facultad de Biologia, Universidad de Sevilla, Av. Reina Mercedes, 41012 Sevilla, Spain.

C4

dent and is regulated by several cell metabolites: malate is an inhibitor, while glucose 6-phosphate and glycine are activators [2,3]. More recent works have shown that PEPc is photoregulated through a process which confers an increase in Vm and a decrease in sensitivity to malate r e t r o i n h i b i t i o n in the light form [4-10]. The molecular basis for p h o t o a c t i v a t i o n has been f o u n d to be a day/night p h o s p h o r y l a t i o n cycle in both maize [4,5] a n d S o r g h u m leaves [8]. Phosphate is covalently linked to a seryl residue close to the N - t e r m i n u s of the protein both in vivo [4,5,8,1 1] and in vitro when incubated in the presence of M g A T P a n d either the e n d o g e n o u s soluble P E P c - p r o t e i n serine kinase [12,13] or the catalytic s u b u n i t of the c A M P - d e p e n d e n t protein kinase from bovine

0168-9452/92/$05.00 © 1992 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland

38 heart [14]. The consistent changes in the enzymatic properties observed upon in vitro phosphorylation of the dark enzyme were a good indication that this post-translational modification was the mechanism to which light-induced changes in catalytic properties of C4 leaf PEPc may be attributed [4,12,13]. Nevertheless, some authors observed a correlation between the oligomerization state of the maize enzyme and its catalytic activity [15-19] and have proposed that in vivo photoregulation occurs through changes in its quaternary structure. As a consequence of PEPc phosphorylation, two different enzyme forms exist in the leaf cytosol; furthermore, the extent of this process is dependent upon the light irradiance [4]. Most of the previous works devoted to the purification and the determination of PEPc functional characteristics have ignored this situation and the corresponding reported data are thereby questionable. More recent works have taken into account the existence of enzyme polymorphism; surprisingly, a quite limited number of them have been devoted to the determination of the specific properties of the purified enzyme forms [10]. Another point which is a source of conflict when the enzyme properties are to be considered is the fact that the enzyme has a very strong tendency to aggregate and deaggregate, depending on the protein concentration and environmental conditions, pH, metabolites, etc. [15,20,21]. Finally, the sensitivity of PEPc to proteolytic degradation has been emphasized and great care must be taken to protect it from this detrimental effect in the course of purification [21]. The main objective of the present work has been to reexamine some relevant functional properties of PEPc enzyme forms, well-defined in terms of protein purity and integrity, phosphorylation state and quaternary structure. Data are discussed in relation to the physiological context of Ca photosynthesis. Materials and Methods

Plant material Sorghum vulgare var. Tamaran, was grown from seed in an illuminated growth room (14-10 h photoperiod, 28°C day, 18°C night, 300 mE m -2

s -I, PAR). Leaves from 2-3-week-old plants were used for enzyme preparation. For purification of dark and light PEPc, the leaves were harvested by the end of the dark period and after a 2 h exposure to light irradiance of 700 mE m -2 s- i, respectively.

Purification of PEPc by classical methods All steps were performed at 4°C except hydrophobic chromatography which was done at room temperature. Sorghum leaves (50 g fresh wt) were chopped and homogenized in 8 vol. extraction buffer (25 mM Tris-HCI, pH 6.8, 5% glycerol, 14 mM mercaptoethanol, 0.2 mM PMFS, 50 mM KF) containing 2% insoluble PVP. The extract was filtered through an 80/~m nylon net and submitted to ammonium sulfate fractionation; the fraction obtained between 30 and 60°/° saturation was purified by hydroxylapatite as described by McNaughton et al. [21]. Active fractions were pooled and brought to 60% saturation with ammonium sulfate. The protein pellet was collected by centrifugation, dissolved in 100 mM potassium phosphate buffer (pH 7.0), containing 1.8 M ammonium sulfate and purified by HPLC hydrophobic chromatography on a TSK phenyl 5 PW column; the column (7.5 × 75 mm) was equilibrated with the above buffer and eluted with a linear 40 ml gradient of decreasing ionic strength, 1.8-0 M ammonium sulfate at a flow rate of 0.5 ml/min. PEPc activity was eluted between 0.1 and 0 M ammonium sulfate. Active fractions were brought to 60% ammonium sulfate and stored at 4°C in the presence of 5'¼, glycerol and 0.1 mM PMFS.

Preparation of the immunoadsorbent column Rabbits were immunized as previously described [22]. Prepurified immune serum (1.5 ml, 30 mg protein) was loaded onto a 4 ml protein ASepharose column equilibrated with 50 mM phosphate buffer (pH 7.4) containing 0.15 M NaCI, then the unfixed protein fraction was washed out with 30 ml of the same buffer. The total immunoglobulin G was then eluted by 0.2 M citrate buffer (pH 2.4), and rapidly precipitated by ammonium sulfate (50"/,, saturation). The pellet of centrifugation was dissolved in a minimum volume

39 of 0.1 M Hepes buffer (pH 8.0), and desalted on a P10 column equilibrated with the Hepes buffer. The anti-PEPc lgG (7 mg total lgG in 3.5 ml) was bound to Affigel 15, according to the method described by the manufacturer (bulletin no. 1099 from BioRad). After coupling, the gel was poured in a small Poly-Prep column, washed with 25 mM Tris/200 mM glycine buffer (pH 8.3) for 1 h, 2 vols. of H20, and then equilibrated in 50 mM phosphate buffer (pH 7.4), containing 0.15 M NaCI. The immunoadsorbent column was stored at 4°C in the presence of diluted NaN 3.

otherwise, the standard reaction mixture (1.0 ml) contained 100 mM H e p e s - N a O H buffer (pH 8.0), 2.5 mM PEP, 5 mM MgCI2, 4.5 mM NaHCO3, 0.2 mM NADH and 10 units malate dehydrogenase. The reaction was started by adding PEPc. For the malate test, activity was recorded in the same medium as above, except that the pH was 7.3, in the absence or presence of 1 mM L-malate. At this pH value, the PEP concentration is suboptimal. Soluble protein content was determined according to Bradford [23] using bovine serum albumin as the standard.

Preparation of PEPc by immunoadsorption chromatography All steps were carried out at 4°C except immunoadsorption which was done at room temperature. Five grams of fresh leaves from either darkened or illuminated plants were cut with scissors and homogenized in 25 mi of 100 mM M O P S - K O H buffer (pH 6.8), 5 mM L-malate, I mM EDTA, 10 mM MgCIz, 14 mM mercaptoethanol, 10% glycerol and 2% (w/v) insoluble PVP (plus 20 mM KF for illuminated samples) and filtered through a nylon net. The suspension was brought to 40% saturation with ammonium sulfate and centrifuged for 35 min at 100 000 × g. The clear supernatant was then brought to 60% ammonium sulfate saturation and centrifuged at 30 000 x g for 15 min. The pellet was dissolved in a minimum volume of 0.15 M NaCI, 50 mM phosphate buffer (pH 7.4), clarified by centrifugation at 30 000 × g and the supernatant (2-3 ml) was loaded onto the immunoadsorbent column. The column was thoroughly washed with 35-40 ml 0.15 M NaCI/50 mM phosphate buffer (pH 7.4). PEPc was then eluted by a mild two-step washing procedure with 20% (v/v) glycerol/H20. The first peak of activity was obtained by elution with three column volumes (10-12 ml), then the elution was stopped for 30 min and restarted. A high PEPc activity was obtained in the subsequent 1.5 ml.

In vitro incubation of purified PEPc with either endogenous protein kinase or alkaline phosphatase A soluble PEPc-protein serine kinase fraction was prepared from illuminated Sorghum leaves according to Jiao and Chollet [12]. Purified PEPc (6 #g) was incubated with this fraction (5 mg kinase extract per mg carboxylase) at 30°C, in 50 #l of medium containing 50 mM T r i s - H C l buffer (pH 8.0), 1 mM dithiothreitol (DTT), 1 mM ATP, 5 mM MgCI2, for 2 h. In some cases, the catalytic subunit of cAMP-dependent protein kinase (4 units/6#g of PEPc) from bovine heart was used at pH 7.3 in Hepes buffer. Incubation with alkaline phosphatase was performed as in [10].

Assays of PEPc activity and soluble protein PEPc activity was assayed spectrophotometrically at 340 nm by coupling to exogenous NADHmalate dehydrogenase at 30°C. Unless noted

Oligomerization state of the purified PEPc Gel filtration was carried out at room temperature on a TSK 3 000 column (7.5 x 300 mm) equilibrated with 0.1 M phosphate buffer (pH 7), containing 0.1 M NaCI, linked to an HPLC system. The eluted proteins were detected by their absorbance at 280 nm. The column was calibrated with marker proteins ferritine, Mr 440 000 and catalase, M r 230 000. Materials NADH, protein kinase A, calmodulin free alkaline phosphatase, L-malate and glucose-6-P were from Sigma. Na PEP and malate dehydrogenase were from Boehringer. TSK Phenyl 5 PW columns, protein A-Sepharose and PLO columns were from LKB-Pharmacia. Affigel 15 and PolyPrep 2 ml columns were obtained from BioRad and TSK 3 000 column from Beckman.

40 Results and D i s c u s s i o n

40

Purification of the dark and light jorms of PEPc

35

The purification of PEPc by h y d r o p h o b i c H P L C (see Fig. 1 for a typical elution diagram) led to a degree of purity similar to those recently described [10,21], using ion exchange chromatography (mono Q). However, the whole procedure was relatively long and the addition o f protease inhibitors was found essential for maintenance o f the regulatory properties o f the enzyme [21]. When subjected to non-denaturing polyacrylamide gel electrophoresis, this preparation showed a highly predominant protein band ( > 9 5 % ) with PEPc activity [22] and a few minor bands o f higher mobility which have been attributed to PEPc proteolysis. The dark-form prepared by this method had a high specific activity in standard conditions 23 ± 3/~mol min-~ mg protein-l; nevertheless its sensitivity to malate inhibition was variable. C o m p a r e d to classical purification procedures, immunoadsorption c h r o m a t o g r a p h y is probably

tO

1.8

@ ¢ 0 U

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£ Z f;

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40

min.

Elution t i m e

Fig. !. Purification of PEPc on a TSK Phenyl 5PW column. The elution was performed at a flow-rate of 1.2 ml/min, with a 40 rain linear ammonium sulfate gradient from 1.8 to 0 M, in 0. I M phosphate buffer (pH 6.5). Proteins were detected by absorbance at 280 nm ( -- ) and PEPc by its enzymatic activity (--). The fraction between the arrows was collected as purified PEPc.

r~ ,m

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Fig. 2. lmmunoadsorbtion chromatography of PEPc. Crude extracts from either darkened or illuminated Sorghumleaves were fixed onto a 3.5 ml immunoadsorbent column. Active PEPc was recovered from the column by a two-step elution procedure with 20% glycerol in water. The first elution step was performed with ll ml of 20% glycerol, then the elution was stopped for 30 min (-II-) and restarted, --, dark enzyme; - -, light enzyme.

the fastest way o f obtaining the enzyme and detrimental effects are minimized. Nevertheless, the protein phosphatase activity is high in Sorghum leaves so that K F had to be added in light extracts to maintain PEPc phosphorylation. The binding capacity o f the i m m u n o a d s o r b e n t column was approx. 8 0 - 9 0 units PEPc, i.e. most o f the activity present in extracts. A typical elution diagram, for both PEPc forms, is shown in Fig. 2. The lightform is easier to detach from the column and this trend was observed on several i m m u n o a d s o r b e n t columns prepared with the same immune serum. In the retarded peak, the activity was 1.5-2 times higher than the activity eluted in the first peak. In both peaks, the recovery o f active PEPc was usually 20-40%. Removal of the remaining enzyme requires elution by low pH citrate buffer, however it is irreversibly inactivated [24]. Using the standard conditions, specific activity was found to be 22 :e 2 (10 determinations) and 25 -4- 3 (3 determina-

41 tions) #mol min-~ mg protein -l for dark- and light-forms, respectively. As judged by electrophoresis o f denaturing and non-denaturing polyacrylamide gels and immunoprecipitation with either anti-PEPc or anti-pyruvate,orthophosphate dikinase (EC 2.7.9.1) [25], the protein was o f a high degree o f purity. Furthermore, the sensitivity of the dark-enzyme to malate inhibition was maintained. It has been previously shown by electrofocusing that our PEPc preparation was devoid o f p y r u v a t e , o r t h o p h o s p h a t e dikinase contamination [8]. Although the yield o f the immunoaffinitybased preparation is one half that o f classical preparations, this disadvantage is largely compensated for by the quality o f the preparation and the short time required to obtain the purified enzyme.

1

Oligomerization state of both PEPc forms prepared by the immunoadsorbent method Gel filtration performed at high protein loading (200/~1 o f 0.3 mg/ml purified enzyme) in buffer without glycerol gave a single peak with an apparent M r corresponding to a tetrameric aggregation state (Fig. 3). In the case o f the light form, the presence of 0.5 M NaCI does not induce the formation o f the dimeric species; it has been previously observed, in the dark form from maize leaves, that 0.2 M NaCI induces the dissociation o f the tetramer into dimers [21]. Freshly prepared Sorghum PEPc forms, stored at 4°C in 20% glycerol, are essentially tetrameric; dimers and m o n o m e r s appeared after several days o f aging and were correlated with a loss o f activity. Furthermore, aging in the absence o f glycerol was faster [22].

Interconversion of light- and dark-PEPc forms The,differential malate sensitivity o f the PEPc forms [4,5,9] was used as a test to study their reciprocal interconversion. Incubation o f the purified light enzyme with alkaline phosphatase increased the sensitivity to malate retroinhibition at pH 7.3, to a level similar to that observed for the enzyme prepared from darkened leaves (Fig. 4, curve A) and decreased its activity (l.3-fold). Conversely, the activity o f the dark enzyme at pH 7.3 was increased ( × 1.3) by incubation with ATP, MgCI 2 and the PEPc protein-serine kinase

~~

0

10

20

0

10

m,n

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Elution time

Fig. 3. Aggregation state of purified PEPc. Samples were submitted to gel filtration through a TSK 3 000 SW column. The elution was performed with a 0.1 M phosphate buffer (pH 7.0), containing 0.1 M NaCI, at a flow-rate of 0.5 ml/min. Marker proteins were: TI, ferritine M r 440 000; T2, catalase M r 230 000. (1) After elution by 20% glycerol/H20 of the immunoadsorbent column. (2) Same as 1, after a 30 min incubation at 4°C in 20% glycerol/H20 (v/v) containing 0.5 M NaCI. (3) Same as 2, after an overnight incubation.

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Fig. 4. Alkalinephosphatase and protein kinase effect on the dark and light PEPc forms. The malate sensitivity of PEPc at pH 7.3, 2.5 mM PEP and 5 mM MgCI2 were tested as a function of the time of incubation with either alkaline phosphatase for the enzyme from illuminated leaves (curve A, r-I) or ATPMg and an endogenous protein kinase for dark enzyme (curve B, I).

42

Table I. Effect of pH on the activity of PEPc purified from dark and light S o r g h t o , leaves. PEPc activity was assayed spectrophotometrically at 340 nm by coupling to exogenous NADH-malate dehydrogenase at 30°C. The reaction mixture ( 1.0 ml) contained 100 mM H e p e s - N a O H buffer at the pH studied, 5 mM MgCI 2, 4.5 mM NaHCO 3, 0.2 mM NADH, 10 units malate dehydrogenase and various PEP concentrations. The reaction was started by adding PEPc (2-5 t~g determined by the Bradford method). pH

7.0 7.3 8.0

Vm /~mol m i n - I m g - I protein Dark

Light

4.5 ± 0.5 19.0 4- 2 22.0 ± 3

13 + 2 33 + 3 43 + 4

Vm light/ Vm dark

4.4 1.8 1.8

fraction (extracted from Sorghum leaves according to the method described for maize) and its malate sensitivity was decreased (Fig. 4, curve B). Similar results were obtained by using the protein kinase A from bovine heart, as previously observed on maize PEPc [14]. In this case the reaction was quite slow but nonetheless induced both PEPc phosphorylation and the change in properties.

Enzymatic properties of purified light and dark PEPc Kinetic properties as a function of pH and PEP concentration. The saturation kinetics with respect to total PEP were studied at three pHs (7.0, 7.3 and 8.0), in the presence of 5 m M total MgCI 2. At all the pHs studied, the kinetics are sigmoidal for both forms, and the Hill number close to 2. The Vmax of the light enzyme is twice that of the dark enzyme at p H 8.0 and four times that of the dark enzyme at pH 7.0 (Table I). The variation of Vm/Km as a function of the p H suggests that two H ÷ are involved in the activity of the dark enzyme, and only one for the light one; this may be correlated with the presence of a phosphate group per subunit on the light-enzyme. As observed in crude extracts from maize [6,9] or Sorghum leaves (unpublished results), both forms are activated 2 - 4 times by 5 m M glucose-6-P. Glucose-6-P increases the Vm value and is an allosteric activator [2,3].

Sensitivity of purified PEPc to malate inhibition. Malate is an inhibitor of the Ca PEPc activity and is believed to play an important role in the regulation of PEPc during Ca photosynthesis [I,26,27].

K,n PEP (mM) dark

light

Km light/ K m dark (mM)

1.60 1.25 0.96

1.60 1.45 1.25

0 0.2 0.3

The inhibition by malate of the dark- and light forms from Sorghum leaves has been studied as a function of L-malate and PEP concentration at pH 7.3, a plausible value for the cytoplasmic pH [28,29], in the presence of 5 mM MgCI_~. Figure 5A and B compare the activities of the dark- and light-forms as a function of L-malate concentration, for 0.5 and 2.5 mM PEP. The effects of malate as a function of PEP concentration are summarized in Table II. For the dark enzyme, Vm is decreased markedly, with no change in the cooperativity (Hill number and Km), whereas malate behaves as an allosteric inhibitor on the light form, the Hill number and the Km for PEP are increased, the estimated Vm is constant. This is confirmed by the effect of malate concentration observed for two PEP concentrations (Fig. 5B), K0.5 for malate does not depend upon PEP concentration for the dark enzyme and there is no cooperativity, whereas K0.5 for malate increases with PEP concentration and a cooperativity is observed for the malate effect on the light enzyme (the Hill number relative to malate is 2 in the presence of 2.5 mM PEP and 1.6 for 0.5 mM PEP). In the case of PEPc isolated from a CAM-plant, analogous differences have been observed between the sensitive (day) and insensitive (night) forms to malate inhibition and this had been correlated with the oligomerization state of the enzyme [16]. However, it has been clearly demonstrated that for PEPc from t h e C4-plant maize, the oligomerization state of the dark-enzyme is not directly involved in its sensitivity to malate inhibition [21]. Darkand light-forms of Sorghum PEPc are apparently

43

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Fig. 5. Effect of malate concentration on dark and light PEPc forms: (A) Variations of dark- (@) and light- (O) PEPc activity, in the presence of 2.5 m M PEP, 5 m M MgC12 and 4.5 mM NaHCO3, as a function of L-malate concentration. (B) Variations of the ratio between the activities with/without malate as a function of L-malate concentration for two PEP concentrations; O and A light enzyme; @ and A, dark enzyme; A and &, 0.5 mM PEP; O and @, 2.5 m M PEP.

less sensitive to dissociation into dimers than the corresponding maize forms and the changes in the regulatory properties are more likely due to differences in the phosphorylation state. Effect of magnesium. MgCI 2 is a very hygroscopic salt, therefore the Mg 2÷ concentration was not always accurately controlled which may explain some variations from preparation to preparation ([10] and our first results). MgCI~ was then thoroughly dessicated. There is some controversy about the true substrate of PEPc. According to O'Leary, Mg 2÷ is bound to the maize enzyme with a high affinity which in turn increases the affinity of the enzyme for its substrate, the trianion form of PEP [2]. On the other hand, it has been suggested that the true substrate is the PEP-Mg 2÷ complex [30]. It was not the purpose of the present work to prove one or other hypothesis, but we have observed that very low activity is always seen in the absence of Mg2÷; furthermore, in the absence of malate, the activity is the same in the presence of either 5 or 10 mM MgC12. Another effect of Mg 2÷, as already observed on crude extracts from the C 4 plant Digitaria sanguinalis [27], is to counteract the inhibition of the enzyme by malate. We have observed the same apparent behaviour in the presence of 10 mM MgC12, this can be explained if malate-Mg 2÷ does not bind to the enzyme. A quantitative study of the magnesium ion effect, would require a determination of the affinity constants of Mg 2÷ for all the components of the assay [6].

Table II. Effect of malate on the activity of PEPc purified from dark and light Sorghum leaves at pH 7.3. PEPc activity was assayed as in Table I. The reaction mixture (I ml) contained 100 m M H e p e s - N a O H buffer at pH 7.3, 5 m M MgCI 2, 4.5 mM N a H C O 3, 0.2 mM NADH, 10 units malate dehydrogenase and various PEP concentrations.

L-Malate (mM)

0 I 2

Dark

Light

K m PEP (mM)

n

I"m (~tmol rain - t mg -I)

Km PEP (mM)

n

I"m (#mol min -t mg - t )

1.25 1.25 1.25

2 2 2

19 4.8 3.1

1.45 2.7 4.0

2 2.2 2.5

33 33 ° 33"

aEstimated values.

44

Effect of phosphorylation on the activity of PEPc in reconstituted ¢Ttosol The enzyme was assayed in mixtures of metabolites at concentrations believed to be present in the mesophyll cytosol in the light [6,31], i.e. 6 mM glucose-6-P, 10 mM MgC12, 2.5 mM PEP and 20 mM malate (pH 7.3). In this simulated cytosol, the activity of the phosphorylated Sorghum PEPc was double that of the dark-form of PEPc (three determinations).

Concluding Remarks The C4 plant PEPc undergoes a day/night phosphorylation cycle in vivo [4,5,8]. Therefore, two forms of the enzyme, which differ by their phosphorylation state, occur in the cytosol of leaf mesophyll cells. For a better understanding of the day/night regulation of PEPc, a purification protocol which maintains the enzyme in an undegraded form. and with unaltered regulatory properties, has been devised. To this end we used a fast immunoaffinity-based purification procedure which minimizes detrimental effects exerted by phosphatases and proteases. Applying this technique to crude extracts of dark- and light-adapted leaves (2-h exposure to high light irradiance), led to highly pure PEPc forms, as judged by denaturing polyacrylamide gel electrophoresis, in less than 3 h of effective working time. The phosphorylation state of both forms was assessed by reciprocal interconversion after incubation with either the endogenous PEPc-kinase or an alkaline phosphatase and measuring the change in malate sensitivity of PEPc. As was previously observed [21], a partially proteolysed PEPc did not display a typical resp= onse to phosphorylation, therefore the interconversion of the Sorghum forms indicated the conservation of enzyme integrity. Finally, both enzyme preparations were submitted to gel filtration chromatography on TSK columns and shown to be fully tetrameric. These preparations constitute the enzyme source for the determination of regulatory and functional properties. For the three pHs studied, a cooperativity for the substrate PEP, at saturating level [2] of NaHCO3, and for a constant total Mg 2÷ concen-

tration of 5 mM, was observed. The Hill number was close to 2, in agreement with the values of 1.9 and 1.8 already described for both purified forms from maize leaves [10], but contrary to the absence of cooperativity observed for the partially purified enzyme at pH 8 [32]. Consistent with previous data [4,9,10], the well-known differential retroinhibition by the metabolite L-malate was confirmed, being significantly higher for the dark-form of PEPc, at a pH presumed to be physiological, i.e. 7.3 [28,29]. Using an enzyme prepared from maize of poorly defined physiological state and culture conditions [2,33], the malate effect has been found to be a mixture of competitive (V type) and uncompetitive (K type) inhibitions. Here we show that the inhibition of PEPc from darkened Sorghum leaves is predominantly of the V type whereas that of the phosphorylated form isolated from illuminated leaves is of the K type. Therefore it appears that phosphorylation of Sorghum leaf PEPc, which has been demonstrated elsewhere to occur on Ser 8 [11], at the protein N-terminus, has a profound influence on its catalytic behavior. It is still not known to what extent the phosphorylation state influences the strength of the interactions between the PEPc subunits. Undestructive techniques [15,20] are favorable for this type of study. The activity of PEPc in leaf extracts is several times higher than that required by the observed rate of photosynthesis. However, it is now admitted that the in vivo environmental conditions of the enzyme do not allow its full capacity to be expressed. In particular, cytosolic pH as well as photosynthesis-related metabolites are thought to adjust their activity to the demand of the BensonCalvin cycle whose rate is primarily determined by light and the availability of chemical energy. In addiiion to this metabolic control, phosphorylation of PEPc which increases both the K i malate and the Vm of the enzyme could be a critical factor influencing the C4 photosynthesis efficiency. We have shown that, in a medium simulating the composition of an illuminated cytosol, phosphorylation induces a twofold increase in the PEPc activity. Current work is being carried out to clarify the actual role of PEPc phosphorylation in C 4 photosynthesis.

45

References I 2

3

4

5

6

7

8

9

10

11

12

13

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