Planta (1989) 178:157 163
9 Springer-Verlag 1989
Chromatographic and immunological evidence that chloroplastic and cytosolic pea (Pisum sativum L.) NADP-isocitrate dehydrogenases are distinct isoenzymes Ri-Dong Chen ~, Evelyne Bismuth 2, Marie-Louise Champigny 2, and Pierre Gadal 1 1 Physiologie V~g6tale Mol~culaire and z Photosynth6se et M6tabolisme, U R A CNRS D 1128, Universite Paris Sud, B~timent 430, F-91405 Orsay Cedex, France
Abstract. Two NADP-isocitrate dehydrogenase isoenzymes designated as NADP-IDH1 and NADP-IDH2 (EC 126.96.36.199) were identified in pea (Pisum sativum) leaf extracts by diethylaminoethylcellulose chromatography. The predominant form was found to be NADP-IDH, while NADP-IDH2 represented only about 4% of the total leaf enzyme activity. These enzymes share few common epitopes as NADP-IDH2 was poorly recognized by the specific polyclonal antibodies raised against NADP-IDH1, and as a consequence NADP-IDH2 does not result from a post-translational modification of NADP-IDH1. Subcellular fractionation and isolation of chloroplasts through a Percoll gradient, followed by the identification of the associated enzymes, showed that NADP-IDH1 is restricted to the cytosol and NADP-IDH2 to the chloroplasts. Compared with the cytosolic isoenzyme, NADP-IDH2 was more thermolabile and exhibited a lower optimum pH. The data reported in this paper constitute the first report that the chloroplastic NADP-IDH and the cytosolic NADP-IDH are two distinct isoenzymes. The possible functions of the two isoenzymes are discussed. Key words: Chloroplast (NADP-isocitrate dehydrogenase) Cytosol NADP-isocitrate dehydrogenase (isoforms) - 2-Oxoglutarate - Pisum (NADP-isocitrate dehydrogenase)
Introduction Although NADP-isocitrate dehydrogenase (NADP-IDH, EC 188.8.131.52) has been characterized in higher-plant tissues such as pea leaves, maize Abbreviations: BSA = bovine serum albumin; D E A E = diethylaminoethyl; N A D P - I D H : NADP-isocitrate dehydrogenase; NADP-IDH1 =cytosolic N A D P - I D H ; N A D P - I D H 2 = c h l o r o plastic N A D P - I D H
seeds and alfalfa root nodules (Omran and Dennis 1971; Curry and Ting 1976; Henson etal. 1980, 1986), its subcellular localization has not been unambiguously established nor has the presence of isoenzymes been reported. In all tissues studied so far, NADP-IDH has been found to be present in the cytosol (Elias and Givan 1977; Henson et al. 1986). In addition, enzyme activity was also reported to be present in chloroplast (Elias and Givan 1977), mitochondria (Curry and Ting 1976; Henson et al. 1980) or peroxisome fractions (Yamazaki and Tolbert 1970). In an attempt to resolve these contradictions, Randall and Givan (1981) used protoplasts from pea leaves as sources of subcellular organelles and found NADP-IDH activity to be present in both cytosol and chloroplasts, but not at a significant level in peroxisomes and mitochondria. In their determinations, up to 90% of the activity was located in the cytosol and about 10% associated with the chloroplasts. However, in these studies, no effort was made to further characterize the enzymes detected in the chloroplasts and the cytosol in order to determine whether or not the activities were caused by different isoenzymes. Previously, we have purified to homogeneity and characterized the cytosolic NADP-IDH from the cytosol of both pea roots and green leaves (Chen et al. 1988). Taking advantage of the antibodies raised against the cytosolic enzymes, we decided to reinvestigate the subcellular localization of the NADP-IDH in pea leaves using a novel immunological approach. In the course of this study, using intact and highly purified chloroplasts we have been able to confirm the presence of NADPIDH activity in chloroplasts. Furthermore, the comparison of the chromatographic behavior, immunological properties, optimum pHs and thermal stabilities of the NADP-IDHs from the cytosol and
R. Chen et al. : Isoenzymes of NADP-isocitrate dehydrogenase
chloroplasts unambiguously demonstrates that the NADP-IDH activities present in these compartments derive from two different proteins. Our data lead for the first time to the conclusion that higherplant leaves contain two isoenzymes of NADPIDH and that those isoenzymes are located in two different subcellular compartments. The possible metabolic role of the two isoenzymes is discussed in relation to their subcellular location. Material and methods Plant material. Pea plants (Pisum sativum L., cv. Finale) were grown in a greenhouse in plastic containers with vermiculite, as described previously (Chen et al. 1988). Natural light was supplemented to 16 h per day with artificial light (450 gmol. m - z . s- 1 at pot level). Seedlings were watered daily with tap water. The shoots were collected from two-week-old plants. Isolation and purification of chloroplasts. Shoots of two-weekold pea were used immediately after picking from the greenhouse. The washed shoots were homogenized in isotonic medium containing 330 mM sorbitol, 20 mM 2-(N-morpholino)ethanesulfonic acid (Mes; pH 6.5), 2 mM MgClz. The homogenate was filtered through a 20-gm nylon gauze and centrifuged for 30 s at 2200.g. The resulting pellet (crude chloroplasts) was suspended in a cation-free medium (330 mM sorbitol, 0.05 M 2-amino-2-(hydroxymethyl)-l,3-propanediol (Tris), pH 7.5) according to Nakatani and Barber (1977) and centrifuged for 20 s at 2200-g. The resulting pellet (intact chloroplasts) was suspended in the cation-free medium and layered on a linear Percoll (Pharmacia, Uppsala, Sweden) gradient for purification by isopycnic centrifugation according to Mourioux and Douce (1981). After centrifugation for 10 min at 5500.g, intact purified chloroplasts sedimented near the bottom of the tube while chloroplast fragments formed a band at the sample-gradient interphase (Fig. !)- The purified chloroplasts were diluted in 50 ml of the cation-free medium. The pellet of purified chloroplasts recovered after centrifugation (90 s, 3 500.g) was gently resuspended in the suspending medium (330mM sorbitol, 5 0 m M 4-(2-hydroxyethyl)-l-piperazineethane sulfonic acid (Hepes), pH 7.8, 0A 5% bovine serum albumin (BSA)). Chloroplast intactness was determined by ferricyanide-dependent Oz evolution in the presence of NH4C1 before and after an osmotic shock (Lilley et al. 1975). Chlorophyll content was determined according to Bruinsma (1963). Contamination of chloroplast preparations by cytosol, mitochondria and peroxisomes was assessed by determining the activity of the following marker enzymes: NADH-cytochrome c reductase for the cytosol (EC 184.108.40.206 ; Wray and Filner 1970); NADPH-ferricytochrome-oxidoreductase (EC 220.127.116.11) for mitochondria (Douce et al. 1973); hydroxypyruvate reductase (EC 18.104.22.168) for peroxysomes (Tolbert et al. 1970). Extraction and purification of NADP-IDH. Chloroplastic NADP-IDH: The purified chloroplasts were broken by osmotic shock in 10 mM Tris-HC1, pH 7.6. The chloroplast thylakoid membranes were sedimented by centrifugation for 30 min at 20000.g and discarded. Solid ammonium sulfate was added to the supernatant. The proteins precipitating between 30 and 70% of ammonium-sulfate saturation were redissolved in the following buffer: 10 mM potassium phosphate, pH 7.5, 2 mM sodium citrate, 1 mM MgC12, 10% glycerol and 14 mM 2-mercaptoethanol (hereafter named "buffer A"). The sample was
Fig. 1. Purification of isolated pea chloroplasts. The chloroplast preparation (2 mg Chlorophyll. m l - 1) was laid on top of the Pereoll gradient. Centrifugation was performed for 10 min at 5 500. g. A, purified chloroplasts sedimented near the bottom of the tube. B, chloroplast debris at the sample-gradient interphase
desalted by gel filtration on a Sephadex G-50 column (20 cm long, 2 cm i.d.) previously equilibrated with "buffer A " . The effluent containing the N A D P - I D H activity was chromatographed on a diethylaminoethyl (DEAE)-cellulose column (20 cm long, 1 cm i.d.). Elution was performed with a linear 0 to 0.4 M KC1 gradient prepared in 200 ml of "buffer A". The fractions containing N A D P - I D H activity were pooled and stored at - 2 0 ~ C. Cytosolic NADP-IDH: The cytosolic N A D P - I D H was purified to homogeneity from pea green leaves as previously described (Chen et al. 1988).
Enzymatic assay and protein measurements. The activity of N A D P - I D H was measured by following the reduction of N A D P spectrophotometrically at 340 nm. For routine assays the enzyme activity was determined at 30 ~ C in 100 mM potassium phosphate, pH 7.5 containing 5 mM MgClz, 2 mM isocitrate and 0.1 mM N A D P (Chen et al. 1988). Protein concentrations were measured according to Lowry et al. (1951) using BSA as a standard. Preparation of antibodies against pea cytosolic NADP-IDH and immunotitration assay. Antibodies against the pea cytosolic N A D P - I D H were raised and tested for their monospecificity as described by Chen et al. 1988. Samples of NADP-IDH1 and NADP-IDHz were obtained respectively from the fractions corresponding to the two peaks detected when intact chloroplastic preparations were submitted to DEAE-cellulose chromatography; N A D P - I D H from leaf homogenate was also partially purified through DEAE-cellulose chromatography. Immunotitration of N A D P - I D H was carried out by incubating the same N A D P - I D H activity with various amounts of rabbit antiserum raised against the cytosolic N A D P - I D H from pea leaves. The samples were incubated at 4 ~ C overnight and the immunocomplexes pelleted by centrifugation at 20000.g for 20 rain., the enzyme activity remaining in the supernatant was then determined. Effect of pH and temperature on the enzyme activity. The N A D P - I D H I and NADP-IDH2 prepared from DEAE-cellulose chromatography as described above were desalted with a G-50 column equilibrated with 50 mM potassium-phosphate
R. Chen et al. : Isoenzymes of NADP-isocitrate dehydrogenase
Table 1. Percentage intactness and distribution of marker enzymes and N A D P - I D H in the pea leaf homogenate, crude and intact chloroplast preparation and chloroplasts purified by Percoll gradients Preparation
Leaf homogenate Crude chloroplast Intact chloroplast Purified chloroplast
Enzyme activity (pmol- 1 (mg Chl- 1). h ~ of leaf homogenate)
10 48 99
NADP-Cytochrome c reductase
595.0 (100) 5.5 (0.9) 3.4 (0.4) 0.9 (0.1)
3.2 (100) 0.3 (11) 0.2 (6) 0.01 (0.3)
12.5 (100) 1.3 (9.3) 0.8 (6.4) 0.5 (4.0)
buffer, pH 7.5, containing 1% (w/w) BSA as described previously (Gonzalez-Villasefior and Powers 1985). No loss in activity was observed when the diluted enzyme was kept on ice for 5 h. The effect of pH was studied at 25 ~ C. Triethanolamine hydrochloride (100 mM) was used for kinetic studies at pH 6.5, 7.0 and 7.5 and 100 m M Tris-HC1 at pH 7.5, 8.0, 8.5, 9.0 and 9.5. The substrates and coenzymes were prepared in a buffer of corresponding pH. Heat-denaturation studies were performed in 50 mM potassium phosphate, pH 7.5 in the presence of BSA. Enzyme aliquots were placed in tubes (50 mm long, 6 mm i.d.) in triplicate and incubated for 20 min in a water bath at various temperatures from 20 to 65 ~ C. After heating, aliquots were immediately cooled on ice and assayed at 30~ for the remaining enzyme activity.
Intactness and purity of chloroplasts. On a chlorophyll basis the average yield of purified chloroplasts was about 4% of the total leaf chloroplasts (data not shown). The purification of pea chloroplasts is shown in Table 1. The percentage of chloroplast intactness as assessed by ferricyanide-dependent Oz evolution was from 90% to 99%. The peroxisomes, whose marker is hydroxypyruvate reductase, are essentially eliminated from the crude chloroplast fraction. In contrast, the intact chloroplasts were still heavily contaminated by mitochondria and cytosol as demonstrated by the activity of the marker enzyme NADP-cytochrome c reductase. It was only after sedimentation through a Percoll gradient, that chloroplasts were free of cytosolic, mitochondrial and peroxisomal contamination and could be considered as intact pure chloroplasts. The N A D P - I D H activity in the purified chloroplast preparation was found to be low. From the N A D P - I D H activity per mg chlorophyll of the purified chloroplast fraction, it can be calculated that in pea leaves, about 4% of the total NADPIDH activity is associated with chloroplasts. It must be pointed out that although low, the percentage of the N A D P - I D H associated with the chloroplasts is tenfold higher than that of the marker enzymes of the mitochondria, the peroxy-
g > p-
Fig. 2 a, b. Elution pattern of N A D P - I D H fi'om pea chloroplastic preparation and leave extracts after chromatography on DEAE-cellulose columns, a leaf extract; b chloroplast extract. l - - l , NADP-IDH1 ; rn--c~, NADP-IDHz
somes and the cytosol. This result constitutes a good indication but not a proof of the presence of N A D P - I D H inside the chloroplast, as a nonspecific binding of the cytosolic enzyme to the chloroplast envelope cannot be be ruled out at this stage.
Chromatographic behavior. Chromatography on DEAE-cellulose of the extracts from green pea leaves showed, in the experimental conditions reported in the legend of Fig. 2, a single peak of N A D P - I D H activity eluting at about 60 m M KC1 (Fig. 2a). However, when the extracts from the Percoll-purified chloroplasts were chromatographed on the same matrix, two clearly distinguishable peaks of activity were detected (Fig. 2b). Although the proportion between the two peaks varied with the purity of the chloroplast preparation, the first peak designated as IDH1 always eluted at the same position as that identified from crude extracts (Fig. 2a) while the second peak, termed IDH2 eluted at a higher KC1 concentration
R. Chen et al. : Isoenzymesof NADP-isocitrate dehydrogenase
50 TEMPERATURE ~
Heat-inactivation profiles of NADP-IDH from pea leaves. The enzymewas diluted with 20 mM triethanolamine/ chloride buffer, pH 7.4, containing 1% BSA. After heating the enzyme for 20 min at a given temperature, the samples were cooled rapidly on ice and assayed for NADP-IDH activity. m--m, thermal inactivationof NADP-IDH1 ; e - - e , thermal inactivation of NADP-IDH2
Fig. 3. Immunotitrationcurves of NADP-IDH from pea leaves. A constant amount of NADP-IDH activity (0.001 units) was incubated with increasing volumes of antiserum raised against the purified cytosolic NADP-IDH from pea leaves. Samples were incubated for 12 h at 4~ C and then centrifugedat 20000 .g for 20 min. The enzymeactivitywas assayedin the supernatant. n--m, NADP-IDH1 ; 9 o, NADP-IDH2
of about 120 raM. Considering the fact that IDH1 is much more active than IDH2, it is not surprising that in crude extracts only one peak is identified, the second being covered by IDH1. Taking into account these results and those published by other authors (Randall and Givan 1981), it is clear that NADP-IDH~, the predominant form, represents the cytosolic enzyme. Two hypotheses can be put forward to explain the presence of the minor form NADP-IDH2. One should first consider the possibility that NADP-IDHz could have arisen from an unidentified modification of NADP-tDH1 ; it might also correspond to another isoenzyme restricted to chloroplasts, as it is often the case when enzymes are localized in different cellular organelles (Hirel and Gadal 1981).
Immunological studies. To further assess these hy-
KC1, peak 2 in Fig. 2 b, was very poorly recognized by the antibodies, indicating that this isoenzyme shares few common epitopes with the cytosolic isoenzyme. On the basis of these chromatographic and immunological data, it can be concluded that the first peak referred to as NADP-IDH1 corresponds to the contamination of chloroplast preparations by the cytosolic enzyme. The second peak, defined as N A D P - I D H z , represents another isoenzyme which is indigenous to chloroplasts and is structurally different from the cytosolic NADPIDH. In that sense, the pattern of IDH-isoenzyme repartition agrees with the general scheme stating that each cellular compartment contains a specific isoenzyme, and differs from the pattern for fumarase which has been shown to be identical in the cytosol and the mitochondria of yeast (Kobayashi et al. 1983).
potheses, the immunological properties of the two N A D P - I D H s from a purified pea chloroplast preparation, as separated by chromatography on a DEAE-cellulose column, were compared with those of the cytosolic isoenzyme by using antibodies raised against the cytosolic N A D P - I D H from pea leaves. In Fig. 3, it is shown that the enzyme eluting at 60 m M KC1 (peak i in Fig. 2 b) exhibited an immunoprecipitation curve perfectly identical to that found for the N A D P - I D H purified on DEAE-cellulose (Fig. 2a), which corresponds to the cytosolic isoenzyme (Chert et al. 1988). In contrast the enzyme eluted at 120 m M
Thermal stability studies and pH optimum. The thermal stabilities of both N A D P - I D H isoenzymes are compared in Fig. 4. The chloroplastic NADPIDHz was less stable than the cytosolic NADPIDH1. The temperatures at which 50% of the enzyme activity was recovered after 20 min of incubation were 47~ for NADP-IDH2 and 51~ for NADP-IDH1. This result confirms that the two isoen~ymes are structurally different. The optimum pH was about 9.0 for the cytosolic isoenzyme and 8.5 for the chloroplastic form (Fig. 5). In addition, the shapes of the curves for
R. Chen et al. : Isoenzymes of NADP-isocitrate dehydrogenase
~" 100[ >.
65 7.0 7.5 8.0 8.5 9.0 9.5 pH
Fig. 5. Effect of pH on the activity of N A D P - I D H from pea leaves. Assays were performed in 100 mM triethanolamine/ chloride buffer for pH values from 6.5 to 7.5 and in 100 mM Tris-HC1 from 7.5 to 9.5. The profiles were obtained in the presence of Mg 2+, N - - m , N A D P - I D H 1 ; e - - e , NADPIDHz
the enzymes were also different, NADP-IDH1 exhibiting a very broad optimum whereas that for NADP-IDH2 was much sharper. Discussion
The pattern of isoenzymes and their intracellular localization are crucial clues for understanding their functions in the complexity of cell metabolism. Reliable techniques have been developed for subcellular fractionation in order to get highly purified preparations of one specific cellular organelle (Leech 1977). Usually, the procedures are satisfactory for enzyme localization if the enzyme of interest is either very active or restricted to a single cellular compartment. However, it remains difficult to demonstrate without ambiguity that a given isoenzyme is indigenous to a specific organelle, especially when its activity is low and when other compartments, susceptible to contaminating the preparation, exhibit very high activity. This is exactly the situation encountered for the NADPIDH isoenzymes. To overcome this difficulty, we developed a new strategy, combining subcellular fractionation, chromatographic separation of the isoenzymes and comparison of the immunological properties of the enzymes detected in the cytosol and the chloroplast preparation. A preliminary experiment, designed to immunoprecipitate the NADP-IDH from crude extract and crude chloroplast preparations using antiserum raised against the cytosolic NADP-IDH, strongly indicated that the NADP-IDH enzymes from the cytosol and
from the chloroplasts were different (data not shown). The next approach was to prepare intact and pure chloroplasts by the best method presently available. With such pure chloroplast preparations, devoid of substantial cytosolic contamination as assessed by the standard methods, it was firmly confirmed that part of the NADP-IDH activity was really associated with the chloroplasts, as reported previously by Randall and Givan (1981). In our studies, however, only about 4% of the total leaf activity was found in the chloroplast fraction as compared with 10% reported by. Randall and Givan (1981). The problem was then to determine whether the activity measured in the organelle preparation was due to the enzyme located in the organelle or reflected the activity associated with contaminating traces of the cytosolic compartment in which NADP-IDH activity is high (Chen et al. 1988). When purified on DEAE-cellulose, the NADP-IDH from purified chloroplasts was eluted from the column at a much higher ionic strength than the cytosolic enzyme (120 mM compared with 60 raM), bringing the first evidence that the cytosolic and the chloroplastic enzymes are different proteins: they were subsequently designated, respectively, as NADP-IDH1 and NADP-IDH2. The finding that NADP-IDH2 is not recognized by the antibodies raised against the cytosolic NADP-IDH1 confirms the above conclusion drawn from their chromatographic properties and is further evidence that NADH-IDH1 and NADPIDH2 are two different proteins as they share few common epitopes. This very poor recognition also shows that NADP-IDH2 is not the result of an unknown modification of NADP-IDH~, such as partial proteolytic degradation. It also excludes the possibility that NADP-IDH2 derives from the absorption or binding of the cytosolic NADP-IDH~ to the chloroplast envelope. The conclusion that two distinct isoenzymes are present is strengthened by the comparison of their comparative pH-activity profiles and thermostability curves. Recently, we have purified NADP-IDH2 to homogeneity and shown that its subunit molecular weight is much higher than that of NADP-IDH1 (data not shown). Our results confirm the subcellular localization reported by Randall and Givan (1981) but are not in agreement with the report that NADP-IDH was absent from chloroplasts and present only in peroxisomes (Yamazaki and Tolbert 1970) and mitochondria (Curry and Ting 1976; Henson etal. 1980). The failure to observe the chloroplastic form might be a consequence of its low activity. Three electrophoretically distinct forms of NADP-IDH
R. Chen et al. : Isoenzymes of NADP-isocitrate dehydrogenase
have been described in maize seeds but their subcellular localization has not yet been firmly established (Curry and Ting 1976). Multiple isoforms of NADP-IDH have also been observed in bacteria and animals (Colman et al. 1970; Illingworth and Tipton 1970; Ochiai etal. 1979; Gonzalez-Villasefior and Powers 1985). As is the case for many other isoenzyme systems the physiological significance of the existence of two forms of NADP-IDH in pea leaves remains unclear. The product of NADP-IDH activity is 2oxoglutarate, one of the substrates of glutamate synthase. In higher plants the chloroplast glutamine synthethase/glutamate synthase pathway is believed to be the main route for the assimilation of ammonia (Miflin and Lea 1976). Leaves of many higher plants contain a cytosolic and a chloroplastic isoform of glutamine synthethase. In pea leaves, however, the chloroplastic isoenzyme is the predominant form (Hirel and Gadal 1981). Furthermore, high rates of ammonia assimilation have been reported in these organelles (Dry and Wiskich 1983). Therefore, an immediate function of the chloroplastic NADP-IDH2 could be to supply 2oxoglutarate for glutamate synthesis. Unfortunately, the in vitro activity detected in chloroplasts appears to be insufficient to account for the production of the 2-oxoglutarate required for glutamate biosynthesis. An alternative origin for this ketoacid may be via the active cytosolic NADP-IDH1. It has been shown that 2-oxoglutarate can be easily transported into isolated chloroplasts (Woo 1983). Recently, Woo et al. (1987) have proposed a twotranslocator model for 2-oxoglutarate transport during NH3 assimilation in chloroplasts and have shown that the increase of 2-oxoglutarate in the chloroplast suspension medium leads to an increase of amino-acid synthesis and glutamate export from the organelles. The discrepancy between NADP-IDH1 and NADP-IDH2 activities, together with the existence of an efficient 2-oxoglutarate translocator on the chloroplast envelope, raise the question of the contribution of each isoenzyme to the supply of the substrate for glutamate synthase activity. In order to bring new insights on the relative importance of the two isoenzymes, as far as 2-oxoglutarate production is concerned, further investigations are in progress in our laboratory to study the kinetic and regulation properties of NADP-IDH2.
We are grateful to Dr. Vidal (our laboratory) for technical advice. We thank Dr. Cousin (INRA, Versailles, France) for supplying the pea seeds. The authors wish to thank Dr. MiginiacMaslow and Jacquot for critically reading the manuscript.
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