Plant Physiol. (1987) 84, 1402-14070032-0889/87/84/1402/06/$01 .00/0

Characterization of a Cytosolic Aconitase in Higher Plant CellsReceived for publication February 13, 1987

RENAUD BROUQUISSE, MIKio NISHIMURA', JACQUES GAILLARD2, AND ROLAND DOUCE*Laboratoire de Physiologie Cellulaire Vegetale, U.A. CNRS No. 576, DRF, CENG and USMG, 85 X,F-38041 Grenoble-Cedex, France

ABSTRACF

Protoplasts obtained from sycamore (Acer pseadplUatw) cell sus-pensions were found to be highly intact. If the protoplasts were taken upand expelled through a fine nylon mesh, all the protoplasts were rupturedleaving the fragile amyloplasts lagely intact. Aconitase hydratase (citratelisocitratel hydro-lyase, EC 4.2.13) activity of sycamore cells was asso-ciated with two protein , one present in the cytosol while thesecond is of mitochodril origi. Chromtography on DEAE-trisacryldid not separate the aconitae hydratase isoenzymes. EPR studies estab-lished that both isoenzymes exhibited an EPR sinal at g = 2.03 onceoxidized.

The tricarboxylic acid cycle enzyme aconitase (citrate [isocit-rate] hydro-lyase, EC 4.2.1.3) catalyzes the dehydration of bothisocitric and citric acids to form cis-aconitic acid, the reversereactions, and the interconversion of citric and isocitric acids(28). Hydration or dehydration occurs more readily thanisomerization. It is now well known that although aconitase doesnot catalyse a redox reaction it contains an iron-sulfur cluster(29). In the oxidized state, the enzyme is inactive and exhibitsan EPR3 signal at g = 2.03. In plant aconitase has been charac-terized in several tissues (for a review see Ref. 26) and moreprecisely in mitochondria (6).

Randall and Givan (27) concluded that the cytosolic fractionof plant cells exhibited a very active NADP-dependent isocitratedehydrogenase activity. Such a result raises the problem of theorigin of isocitrate and suggests, therefore, the presence of acytosolic aconitase in plant cells distinct from the mitochondrialaconitase. In this investigation we have used protoplasts fromsycamore cells as a source of subcellular fractions and haveconcluded that aconitase is localized in the cytosol and in themitochondria but not at significant levels in peroxisomes andplastids.

MATERIALS AND METHODS

Plant Material. The strain of sycamore (Acer pseudoplatanusL.) used in the present study was generously provided by J.

' Recipient of an award from the Japan Society for the Promotion ofScience and Centre National de la Recherche Scientifique under theJapan-France cooperative Science Programme, 1985. Present address:Nagoya University, Faculty of Agriculture, Chikusa, Nagoya 464, Japan.

2Present address: DRF/S.Ph-S.C.P.M., CENG, 85X, F-38041 Gren-oble-Cedex, France.

3 Abbreviations: EPR, electron paramagnetic resonance; Mops, (3[N-morpholino]propane sulfonic acid); PMSF, phenylmethylsulfonyl fluo-ride; TCA, tricarboxylic acid.

Guern (Plant Cellular Physiology, Gif sur Yvette, France). Thenutrient medium was prepared as described by Bligny (5) andcell suspensions maintained in exponential growth by frequentsubcultures.

Preparation of Protoplasts. Cells (20 g) were suspended in 200ml of their culture medium containing 0.5 M mannitol (finalosmolarity 0.55 osm), 1% (w/v) cellulase (Onozuka RS; YakultPharmaceutical Co., Nishinomiya, Japan), and 0.1% (w/v) pec-tolyase Y-23 (Seishin Pharmaceutical Co., Nishinomiya, Japan),adjusted to pH 5.7. The cells were incubated with constantshaking (20 cycles/min) at 25°C. Digestion ofyoung cells at 250Cfor 1 h resulted in a final yield of protoplasts near 100%. Afterdigestion, the suspension was filtered through one layer of Mir-acloth (Krantex, Alfortville, France) that retained any undigestedcell aggregates, and protoplasts were collected by centrifuging at150g for 10 min and then washed twice with 50 ml of culturemedium containing 0.5 M mannitol (suspension medium). Pro-toplasts were stored in suspension medium and were normallyused within 2 to 3 h.

Gentle Rupture of Protoplasts and Separation of the Organ-elles from the Cytosolic Fraction. Since sycamore cell protoplastshave an average diameter of 20 to 30 gm, a rapid and effectiveprocedure for the gentle rupture of intact protoplasts (i.e. forstripping the cell membrane) is to pass protoplasts through a finenylon mesh (20 ,um) affixed to the cut end of a 25-ml disposablesyringe (20). Thus, protoplasts (equivalent to 20.106 cells) arepelleted after dilution with suspending medium and resuspendedin about 5 ml of a basic medium: 0.5 M mannitol; 0.1% (w/v)BSA; 1% (w/v) insoluble polyvinylpyrrolidone (Kca 25, Serva);5 mM DTT; 10 Mm leupeptin; 5 mm phosphate buffer (pH 7.4);these last parameters were chosen because they correspond tothe phosphate concentration and the pH of the cytosolic com-partment previously determined by 31P nuclear magnetic reso-nance studies (22). If the protoplasts are taken up and expelledthrough the 20 Mm nylon mesh two times, they will be completelyruptured. In order to separate the cytosolic fraction from the cellorganelles, we subjected the broken protoplast fraction to cen-trifugation to yield a pellet largely free of cytosol and a superna-tant enriched in cytosolic enzymes. In order to prevent starchgrains from tearing through the plastid envelope, the centrifugalg force was gradually increased by using an automatic ratecontroller (Sorvall) and centrifugation was carried out in threesteps (100g for 5 min, 500g for 5 min, and 12,000g for 10 min;Sorvall rotor SS34). Each supernatant was centrifuged in a newtube, and the three successive pellets were combined together.This procedure ruptures all the protoplasts leaving the fragileamyloplasts and mitochondria largely intact (17).

Isolation of Amyloplastids. For the preparation of plastids,intact protoplasts resuspended in basic medium were passedthrough a fine nylon mesh as described above. The brokenprotoplasts were centrifuged for 10 min at 200g in a table topKubota KN-70 centrifuge (RS-4-rotor) and the pellet resus-pended in approximately 1 ml of wash medium containing: 0.5M mannitol; 0.1% (w/v) BSA; 1 mM EDTA; 10 mM Mops-NaOH

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CYTOSOLIC ACONITASE IN HIGHER PLANT CELLS

buffer (pH 7.5). The supematant was centrifuged for 5 min at1,OOOg (RS4-rotor, Kubota KN-70 centrifuge) and the pelletsuspended again in approximately 1 ml wash medium. The twosuccessive pellets were combined together and gently filteredthrough Miracloth. Amyloplasts thus obtained were purified byisopycnic centrifugation in a nontoxic silica sol (Percoll TM,Sigma) gradient which maintained isoosmotic conditionsthroughout the isolation procedure. Thirty-four ml Percoll me-dium (50% Percoll, 0.5 mm mannitol; 0.1% [w/v] BSA; 1 mMEDTA; 10 mM Mops-NaOH [pH 7.5]) were pipetted into eachof two centrifuge tubes. The tubes were placed in a precooledSorvall SS 90 vertical-rotor and centrifuged (at 3°C) at 10,500rpm (10,OOOg) for 100 min. At the conclusion of this step, acontinuous gradient of Percoll was obtained in each tube (24).Aliquots (2 ml sample) of the crude amyloplast suspension thenwere layered on the linear Percoll gradients. The tubes werecentrifuged for 10 min at 5,000g (at 3C) in the Sorvall SS 90rotor. The plastids containing starch grains were found in a broadyellowish band near the bottom of the tube while mitochondriaremained near the top of the tube. The heavy plastid fractionwas carefully removed by siphoning from the bottom of the tubeand pelleted after diluting at least 15-fold with wash medium bycentrifuging at 200g for 10 min. The supernatant was centrifugedfor 5 min at 1 ,OOOg and the two successive pellets were combinedtogether. The purified plastids were suspended in wash medium(final protein concentration 25-30 mg-ml-') and stored in anice-bath. The intactness of plastid preparation was evaluated bygluconate 6-P oxidation using a spectrophotometer according toJoumet and Douce (16).

Isolation of Mitochondria. The broken protoplasts were cen-

trifuged for 10 min at 800g in a table top Kubota KN-70centrifuge (RS-4-rotor) in order to remove the bulk of amylo-plastids. The supernatant was centrifuged for 20 min at I0,000g(Sorvall; SS-34 rotor) and the mitochondrial pellet resuspendedin approximately 0.5 ml of wash medium. Mitochondria thusobtained still heavily contaminated with plastids were purifiedby isopycnic centrifugation in density gradients of Percoll as

previously described by Neuburger et al. (25) except that theautomatic rate controller (Sorvall) must be used. The preparationof mitochondrial extract (matrix enzymes) was carried out ac-cording to Brouquisse et al. (6).

Isolation of Peroxisomes. All the attempts we have made toisolate pure peroxisomes from sycamore cells were unsuccessful.This was attributable to the fact that sycamore cell peroxisomesexhibited almost the same density than mitochondria. Further-more, a close examination of sycamore cells by electron micros-copy indicated that the number of peroxisomes per cell is ex-

tremely low compared to that of mitochondria or plastids. Wehave therefore prepared large amounts of peroxisomes frompotato tubers according to Neuburger et al. (25) using continuousPercoll gradients.Measurement of Enzyme Activities. All assays were first per-

formed on blanks to detect any unspecific drift or endogenoussubstrate or enzyme contamination. They were then optimizedwith respect to the concentration of each component and to thepH of the reaction mixture. We verified that under these condi-tions activities were linear with respect to time for at least 2 minand were proportional to the amount of protein in the range of20 to 300 ttg ml-'. For the reactions that were followed with a

spectrophotometer (Kontron Uvikon 810), the coupling enzymesystems-were checked not to be rate limiting. Unless otherwisestated, all enzymes were assayed at 25°C, in 0.5 ml final volumeand the buffer used was either 50 mm Tricine-NaOH (for pHvalue above 7.4) or 50 mm Mops-NaOH (for pH value under7.4). References to the procedures are given together with anyfeatures of the reaction mixtures that differed from those in thereferences. Aconitase activities were assayed either by the coupled

assay of Rose and O'Connel (28) in which NADP reduction ismeasured or by following the disappearence of cis-aconitate at240 nm as a function of time (12). ADP-glucose pyrophosphor-ylase (EC 2.7.7.27): pH 8.0; 1 mM PPi, 5 mM NaF, 1 mM ADP-glucose, 5 mM glycerate 3-P, 5 mM MgC12, 2 mM DTT, 10 AMglucose 1,6-bisP, 500 AM NADP, 0.8 unit P-glucomutase, and0.7 unit glucose 6-P-dehydrogenase (1 1). Catalase (EC 1.1 1.1.6)was assayed with and 02 electrode (31). Fumarase (EC 4.2.1.2)(15). Malate synthase (EC 4.1.3.2) and isocitrate lyase (EC4.1.3.1) (7). Alcohol dehydrogenase (EC 1.1.1.2) (3). Citratesynthase (EC 4.1.3.7) (30). Acyl-CoA dehydrogenase (EC1.3.99.?), enoyl-CoA hydratase (EC 4.2.1.17), 3-OH acyl-CoAdehydrogenase (EC 1.1.1.35), and thiolase (EC 2.3.1.9) (13).Acyl-CoA synthetase (EC 6.2.1.3) (18).

Latency for These Measurements. Enzyme activities of sam-ples of intact (in the presence of 0.5 M sucrose) and ruptured (byadding 0.025% Triton X-100) cell fraction were measured. Theactivity in the ruptured preparation (T) minus that in the intactpreparation (I) is called latent and is expressed as a percentageof the activity of the ruptured preparation (100- [T-I]/T) to givelatency (2 1).EPR Measurements. These were recorded on a Varian E109

spectrometer coupled to a Hewlett Packard 9826 calculator. Thesamples were cooled with a liquid-helium transfer system (OxfordInstrument, ESR900) for variable temperatures down to 4.2 K.Temperature was monitored with a gold-iron/chromel thermo-couple about 2 cm below the bottom of the EPR sample in theflowing helium gas stream. The magnetic field was calibratedusing a varian gaussmeter. Samples of mitochondrial extract orcytosolic fraction concentrated by ultrafiltration through a low-absorption hydrophilic YM membrane (30,000 mol wt cutoff

Table I. Subcellular Localization ofFumarase (mitochondrial marker),ADP-glucose Pyrophosphorylase (Plastidial Marker), Catalase

(peroxisomal marker), Alcohol Dehydrogenase (cytosolic marker), andAconitase in Mechanically Ruptured Sycamore Cell Protoplastsfollowed

by Centrifugation (12,000gfor 10 min)Preparation of intact and broken protoplasts (total extract) and cen-

trifugation of intact organelles, were carried out as described under"Materials and Methods." These data are from a representative experi-ment and have been reproduced four times.

Enzyme Total extract Distribution

Supematantpellet

nmol/min- 106 cells % total activityFumarase 26 1 96ADP-glucose pyrophos-

phorylase -1 5 106Catalase 520 8 80Alcohol dehydrogenase 34 90 7Aconitase 3 46 61

Table II. Latency ofAconitase in Total Extract (Lysates) and CellSubfractions ofSycamore Cell Protoplasts Preparedfrom Suspension

CulturesSpecific activities and latency were measured, in five different experi-

ments as described under "Materials and Methods." These data are froma representative experiment.

Cell Compartment Total Latency ProteinActivty Laec Prti

nmol/min % mgTotal extract 400 60 27Supernatant 180 2 18Pellet 240 95 8

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Plant Physiol. Vol. 84, 1987

Table III. Measurements of Various Markers and Aconitase in Washed and Purified Cell Organelles(Mitochondria and Plastids) Isolatedfrom Sycamore Cells

The separate preparations of cell organelles are described in the text.Associated Percoll-purified Percoll-purified

Enzyme Cell Compartment Mitochondria PlastidsAlcohol dehydrogenasea Cytosol 1 bCatalase (nmol 02/min *mg Microbodies 5000 200

protein)Fumarasea Mitochondria 1100 10ADP-glucose pyrophosphory- Plastids 1 75

lase'Aconitasea Mitochondria 1 lOC I

a Specific activity expressed as nmol substrate consumed/min mg protein. b Not detected.c Considerable latency (98%) was found for aconitase in mitochondrial preparation.

Table IV. Enzyme Activities in Potato Tuber PeroxisomesSpecific activities were measured in four experiments, as described

under "Materials and Methods." The variability of all activities did notexceed 20%. Note that in contrast with glyoxysomes (1) potato tuberperoxisomes do not possess glyoxylate cycle enzyme activities includingaconitase.

Enzyme Specific Activitynmol/min *mg protein

Catalase 2,260,0009-OxidationAcyl-CoA synthetase 104Acyl-CoA dehydrogenase 52Enoyl-CoA hydratase 1913-hydroxyacyl-CoA dehydrogenase 253Thiolase 86

Glyoxylate cycleMalate synthase aCitrate synthase 0.2Aconitase aIsocitrate lyase a

a Not detected.

Centricon, Amicon) (220 M,l 0.5-2 mg protein) were placed inEPR quartz tubes, frozen rapidly in liquid N2 and stored at 77K until assayed.

RESULTS

Intracellular Location of Aconitase. This was investigated inprotoplasts from suspension cultures of sycamore cells. Thefollowing markers enzymes were used: mitochondria, fumarase;plastids, ADP-glucose pyrophosphorylase; peroxisomes, catalase;cytosol, alcohol dehydrogenase. The gentle rupture of intactprotoplasts passed through a fine nylon mesh followed by cen-trifugation carried out in three steps (see "Materials and Meth-ods") produced a supernatant that contained an appreciableproportion of the aconitase activity (Table I). This was not dueto mitochondrial and plastidial contaminations of the superna-tant, or to the location of the aconitase in peroxisomes as nomore than 1% of the fumarase, 5% of the ADP-glucose pyro-phosphorylase, and 8% of the catalase were in the supernatant(Table I). The observation that almost all the alcohol dehydro-genase activity was in the supernatant is consistent with thelocation of at least some ofthe aconitase in the cytosol. Further-more, very little latency was found for alcohol dehydrogenase incarefully prepared lysates of protoplasts (not shown) indicatingthat almost all the protoplasts have been ruptured.

Further evidence that a very substantial fraction of aconitasein sycamore cells is not within a membrane bounded cell orga-

nelle is provided by the low latency of the enzyme in carefullyprepared lysates (total extract) of protoplasts (Table II). Aftercentrifugation, very little latency was found for the aconitasepresent in the supernatant whereas a considerable latency of theenzyme was observed in the pellet containing intact cell orga-nelles. These results demonstrated that at least two aconitasehydratase isoenzymes are present in intact plant cells. One isreadily released after stripping the cell membrane and is presentin the cytosol, the other is confined within a membrane boundcell organelle.

Aconitase from sycamore cell cytosol showed Michaelis-Men-ten kinetics with both citrate and cis-aconitate at pH 7.4 (opti-mum pH; apparent Km values for citrate and cis-aconitate wereabout 130 and 36 uM, respectively). The apparent Ki value ofcytosolic aconitase for fluorocitrate was 5 gM. The aconitaseextracted from the mammalian cell cytosol (14) was much lessinhibited by fluorocitrate than was the aconitase found in plantcell cytosol. Furthermore, plant cytosolic aconitase is fully activeand does not require activation by anaerobic incubation for 30min at room temperature with an excess of Fe2" and a thiol suchas DTE or cysteine (19).With the aim to localize further aconitase in the pellet con-

taining cell organelles, intact mitochondria, and amyloplastidswere isolated from sycamore cells. The evidence for intactnessof purified plastid preparation was confirmed by the measure-ment of the latency of gluconate 6-P dehydrogenase activities(16). In 10 experiments, the latency thus measured on freshlyprepared sycamore cell plastids ranged from 90 to 95% with amean value of 92% (not shown). Assays of various markers(alcohol dehydrogenase, catalase and fumarase [Table III])showed that sedimentation of amyloplastids through a continu-ous Percoll gradient almost completely eliminates cytosolic andmitochondrial contamination. Table III clearly indicates thataconitase was not associated with sycamore cell amyloplastids.A feature of purified mitochondria from sycamore cells was

their high rate of 02 consumption with tricarboxylic acid cyclesubstrates and external NADH (17). Percoll gradient almostcompletely eliminates cytosolic and plastid contaminations (Ta-ble III). The only marker which did show large activity wascatalase indicating a contamination with intact peroxisomes. Inaddition, the rate of KCN-sensitive cytochrome c-dependent 02uptake in intact mitochondria was only 1 to 2% ofthat recordedfor osmotically shocked mitochondria, giving an apparent per-centage of intactness of 95 to 98% (not shown). As expected,aconitase activity present in the mitochondrial preparationshowed considerable latency (Table III). Aconitase from syca-more cell mitochondria showed Michaelis-Menten kinetics withboth citrate and cis-aconitate at pH = 7.1 (optimum pH; appar-ent Km values for citrate and cis-aconitate were about 120 and30 Mm, respectively). This enzyme was very sensitive to inhibition

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CYTOSOLIC ACONITASE IN HIGH

by fluorocitrate. The apparent Ki value of mitochondrial aconi-tase for fluorocitrate was 3 gM when citrate was the substrate.The kinetic parameters of aconitase from sycamore cell (theseresults) and potato tubers mitochondria are similar to those ofits counterpart in cytosol (see above). Quantitative considerationof our data shows that the absolute maximum activity for mito-chondrial aconitase under optimum conditions (Table III) ex-ceeds the maximal rate at which intact mitochondria oxidizecitrate (state 3 02 consumption: 40 nmol/min -mg protein). Ingood agreement with previous studies (6) aconitase freshly re-leased from sycamore cell mitochondria is fully active and doesnot require activation.

It is most unlikely that peroxisomal contamination contributedsignificantly to the total aconitase activity present in the mito-chondrial pellet. In support ofthis suggestion, Table IV indicatesthat aconitase was not associated with potato tuber peroxisomesalthough they contain all the enzymes (Acyl-CoA synthetase,Acyl-CoA dehydrogenase, enoyl-CoA hydratase, 3-OH-acyl-CoAdehydrogenase and thiolase) involved in fatty acid fl-oxidation(1). These results are in perfect agreement with those ofGerhardt(13).

Consequently, these results together strongly support the pres-ence of at least two aconitase isoenzymes in plant cells, onepresent in the mitochondria while the second is of cytosolicorigin.

Partial Purification of Aconitase Isoenzymes from Plant Cells.

IER PLANT CELLS 1405

FIG. 1. Elution pattern of sycamore cell mitochon-drial (A) and cytosol (B) aconitate hydratase fromDEAE-trisacryl. Aconitase was eluted at 4°C with acontinuously increasing Tris-citrate gradient. For de-tails see text. Note that cytosolic and mitochondrialaconitase eluted almost together.

The mitochondrial (matrix enzyme) and cytosolic extracts (29mg protein) supplemented with OgM leupeptin and 1 mM PMSFwere applied to a 0.8 x 20 cm column of DEAE-trisacrylequilibrated in 10 mM Tris-HCl (pH 7.2). Aconitase was elutedat 4C with a continuously increasing Tris-citrate gradient (10-75 mM) (pH 7.2). Typical runs using extracts prepared fromprotoplasts are shown in Figure 1. It was of interest that similarelution patterns were noted for cytosolic and mitochondrialaconitase isoenzymes. In both cases recoveries ofenzyme activityplaced on the column were almost quantitative and no significantimprovement in the recoveries could be obtained by activationof the eluted fractions by Fe2+-cysteine (not shown). This was insharp contrast with the results of Eanes and Kun (10) concerningthe separation and characterization ofaconitase isoenzymes frompig tissues. In this case, chromatography on DEAE-cellulosereadily separates the two isoenzymes.

Electron Paramagnetic Resonance Characterization of PlantAconitases. EPR studies have established that mammalian mi-tochondrial aconitase exhibited an EPR signal at g = 2.03 onceoxidized by a potent oxidant such as peroxodisulfate (2). TheEPR spectra of peroxodisulfate-treated aconitase isoenzymes iso-lated from plant cells (Fig. 2) revealed the presence of a nearlyisotropic signal with a low field maximum at g = 2.03 whichidentifies the [3 Fe] cluster. Both spectra have a shoulder in thecenter of the resonance absorbance. Interestingly, the EPR spec-trum of mitochondrial aconitase resembles that of the cyto-

FRACTIONS0 5 10 15 20 25

0.2 A 1.5

00.1 0

A 1~~~~.5

0L5

0~~~~~~~~~~~~

° ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~cnE 02

mUo.2 B ~ 1.5

co w~~~~~~~~~~~~~~0.1 ~i

0 5 10 15 20 25

FRACTIONS

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Plant Physiol. Vol. 84, 1987

MAGN

2.06 2.04 2.02 2.00 1.98

G - VALUE

a

b+c

IETIC FIELD

GAUSS )

3200 3250 3300 3350

FIG. 2. First derivative EPR spectra of sycamore cell aconitase in theoxidized state preincubated with peroxodisulfate. a, Total extract; b,cytosolic eluate (see Fig. IB; 1,9 mg protein); c, mitochondrial eluate(see Fig. IA; 0,6 mg protein); b + c, cytosolic + mitochondrial eluates.Total extract and eluates concentrated by ultrafiltration (see "Materialsand Methods") was suspended to an appropriate protein concentrationin suspending medium containing 1 mM peroxodisulfate. Two min afterperoxodisulfate addition, samples were transferred to a quartz EPR tubeand rapidly frozen at 77 K. Conditions ofmeasurement were: modulationamplitude, 5 Gauss; modulation frequency, 100 KHz; microwave fre-quency, 9250 MHz; microwave power, 0.5 mW; temperature, 16 K.Note that the EPR spectrum of mitochondrial aconitase resembles thatof the cytoplasmic aconitase.

plasmic aconitase. The EPR signal of plant aconitases and mam-malian cytoplasmic aconitase (2), however, differs in some detailfrom that of the mammalian mitochondrial one (6).

DISCUSSION

The separation of the intact organelles from the lysed proto-plasts clearly indicates that aconitase activity is essentially limitedto the mitochondria (about 50%) and cytosol fractions (50%).Interestingly, Dickman and Speyer (8) first described two acon-itases in rat liver tissues: 'cytoplasmic aconitase' occurring in thesupernatant fraction after homogenization and 'soluble' aconi-tase partially released from mitochondria by the freezing andthawing of the particles. The plant cytosolic aconitase does notappear to be absorbed to the outer surface of the mitochondrialouter membrane since aconitase activity in the mitochondrialpreparation showed considerable latency. The present investiga-tion provides evidence for the existence of an iron-sulfur centerin both aconitase isoenzymes yielding an EPR signal typical ofoxidized three Fe clusters with a peak at a g value of approxi-

mately 2.03 in the oxidized state. Furthermore, aconitase releasedaerobically from sycamore cell mitochondria is fully active anddoes not require activation. The same thing holds true withcytosolic aconitase. In contrast, aconitase from mammalian mi-tochondria after purification requires addition of Fe2" for fullactivity (19). This activation is complex involving both reductionand iron insertion (19). Preliminary results carried out in ourlaboratory indicated that the plant enzymes, when purified, alsoloses activity but it was not always completely clear from ourresults, whether the enzyme was only reversibly deactivated orirreversibly inactivated.The physiological significance of a very active cytosolic acon-

itase in plant cells is unclear. This enzyme may play a key rolein the organic acid metabolism. When stored reserves of citratewithin the vacuole are mobilized, aconitase and cytosolicNADP+-isocitrate dehydrogenase (28) could allow their rapidpartial oxidation via conversion of the citrate to a-ketoglutarate.a-Ketoglutarate, thus formed, can be completely oxidized in thenormal reactions of the TCA cycle. However this pathway is ofdoubtful significance because according to Birnberg et al. (4) themajority (or all) of the extramitochondrial citrate is synthesizedin the mitochondria originally. It is also possible that the cytosolicaconitase and NADP+-isocitrate dehydrogenase could play asignificant role in the metabolism of fast growing cells by pro-ducing carbon skeletons for biosynthetic purposes (e.g. aminoacid synthesis) (23). Under these circumstances, the presence ofglutamin synthetase, glutamate synthase, aconitase, and NADP+-linked isocitrate dehydrogenase in plant cell cytosol affords anexcellent explanation for net ammonia assimilation at the ex-pense ofa-ketoglutarate deriving from cytosol. Since all the plantmitochondria isolated so far possess specific NAD+-linked malicenzyme (9), malate synthetized in the cytosol by a sequenceinvolving PEP carboxylase and malate dehydrogenase can beconverted into citrate in the mitochondria under the control ofNAD+-linked malic enzyme and citrate synthase. There mightbe, therefore, endogenous controls polarizing the malate-citrateexchange facilitating citrate efflux (4). If this exchange is 100%electrogenic the driving force is provided by the electrochemicalpotential difference of the proton (A,uH). This last point is underinvestigation in our laboratory.

It is classically considered that the primary function of theoxidative portion of the pentose phosphate pathway is to supplyNADPH for use in reductive biosynthetic reactions in the cyto-plasm (for review see Ref. 9). The results presented in this paperdemonstrate that additionalNADPH in the plant cytoplasm maybe produced by citrate oxidation. Under these circumstances,the NADP-linked dehydrogenase enzymes involved 'compete'for the same pool of cytoplasmic NADP. Unfortunately, therelative importance of the two modes of NADP productioncannot be assessed at present.

Acknowledgments-We thank Dr. Etienne-Pascal Journet and Jacques Joyardfor helpful suggestions.

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* * * * * * *

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