Catharanthus roseus Cells - ZfN - Max-Planck-Gesellschaft

Phosphate Starvation and a Glycolytic Bypass Catalyzed by Phosphoenolpyruvate Carboxylase in Suspension-Cultured Catharanthus roseus Cells M ayum i N agano*, A k ik o H achiya** and H iroshi Ashihara Department of Biology, Faculty of Science, Ochanomizu University, 2-1-1, Otsuka, Bunkyo-ku, Tokyo, 112, Japan Z. Naturforsch. 49c, 742-750 (1994); received June 20/August 25, 1994 Madagascar Periwinkle, Phosphoenolpyruvate Carboxylase, Pyruvate Kinase. Glycolysis, Phosphate Starvation Pathways involved in the conversion of phosphoenolpyruvate (PEP) to pyruvate, the final step in glycolysis, were investigated after transfer of stationary-phase cells from suspension cultures of Catharanthus roseus to fresh complete or phosphate (Pi)-deficient Linsmaier and Skoog medium. In addition to pyruvate kinase (PK). enzymes that can function in an alterna tive pathway, namely, PEP carboxylase (PEPC), NAD-malate dehydrogenase and NADmalic enzyme, were present in significant amounts in C. roseus cells. The activity of PEPC in Pi-starved cells was more than twice that in cells in the complete medium (Pi-fed cells), while that of PK in Pi-starved cells was lower than that in Pi-fed cells. No significant differences were observed in the levels of NAD-malate dehydrogenase and NAD-malic enzyme between these two types of cell. At cytosolic pH, the K m value of PEP (45 |i m ) for PEPC was lower than that for PK (100 (.i m ) . The activity of PEPC was inhibited by malate, citrate, aspartate and ATP. The activity of PK was also inhibited by ATP, but to a lesser extent. The cellular levels of PEP, adenylates and malate, which are substrates and effectors of PK and PEPC, in Pi-fed and Pi-starved cells suggest that the contribution of PEPC to the metabolism of PEP increased in Pi-starved cells in vivo. Evidence for operation of a bypass from malate to pyruvate in vivo was supported by the rapid release of 1 4 C 0 2 from organic compounds derived from fixed N aH 1 4 C 0 3 and from [4-1 4 C]malate.

Introduction The m etabolic adaptation o f respiratory path w ays to p hosphate (Pi) deficiency have recently b een investigated in cultured cells o f Brassica nigra and C atharanthus roseus (D u ff et al., 1989; Li and A shihara, 1990; T h eod orou et al., 1992; T heod orou and Plaxton, 1993; N agano and A sh i hara, 1993). D u ff et al. (1989) indicated that ad en ylate-ind ep en dent, alternative en zym es o f glycolysis, inducible by Pi starvation, such as PPi: fructose-6-phosphate phosphotransferase (PFP, EC 2.7.1.90) and p hosp hoenolpyruvate p hosp ha tase (P E P ase, E C 3.1.3.60), bypass n ucleotid e

Part 45 in the series, “Metabolic Regulation in Plant Cell Culture”. * Present address: Research Institute, Morinaga Milk

Industry, Higashihara, Zama. 228, Japan. ** Present address: Research Institute for Bioresources,

Okayama University, Kurashiki, 710, Japan. Reprint requests to Dr. H. Ashihara. Telefax: +81-3-3942-2815. 0939-5075/94/1100-0742 $ 06.00

p hosphate-dependent glycolytic reactions in Pistarved cells. A lthough little change in the m axi m um activity o f PFP is observed during Pi star vation o f C. roseus cells (Li and A shihara, 1990; N agano and A shihara, 1993), PFP bypass the reac tion catalyzed by p hosphofructokinase (PFK , E C 2.7.1.11) as a result o f the fine control o f the activi ties o f PFK and PFP (N agan o and A shihara, 1993). Thus, m echanism s of m etabolic adaptation to Pi deficiency seem to differ am ong plant sp ecies (D u ff et al., 1989; Li and A shihara, 1990; T h eo d o rou and Plaxton, 1993; N agano and A shihara, 1993). C om pared with inform ation available about B. nigra cells (T heodorou and P laxton, 1993), little is know n about the conversion o f P E P to pyruvate in C. roseus cells, although it has b een suggested that this step is also im portant for regulation o f glycolysis (C opeland and Turner, 1987; K ubota and A shihara, 1990). We found previously that the activity o f p hosphoenolpyruvate carboxylase (PE PC , EC 4.1.1.31) is higher in an extract o f Pistarved Catharanthus roseus cells than in an ex-

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M. Nagano et al. ■Glycolytic Bypass Catalyzed by PEPC

tract o f cells supplied with 1.25 m M Pi (N agano and A shihara, 1993). A n increase in the level of PE P C during Pi d eficien cy has also b een found in su spension-cultured B. nigra cells (D u ff et al., 1989), rape roots (H uffland et al., 1992) and a green alga (Schuller et al., 1990 a). In the present study, w e exam in ed w heth er PEPC can function as an alternative en zym e in glycolysis and can by pass the reaction catalyzed by pyruvate kinase (PK , E C 2.7.1.40) in Pi-starved C. roseus cells. From the results ob tain ed , w e discuss the physio logical significance o f the PEPC bypass.

Materials and Methods C ell cultures Suspension cultures o f cells o f Catharanthus roseus (L .) G. D o n w ere m aintained heterotrophically and subcultured at 7 day intervals in com p lete LS m edium (L insm aier and Skoog, 1968) that con tained 2.2 jam 2,4-dichlorophenoxyacetic acid and 3% sucrose (A shihara et al., 1988a). For preparation o f P i-deficient cultures, portion (7 ml) o f the su spension o f 7-day-old cultures w ere trans ferred to 43 ml aliquots o f the fresh LS culture m edium excluding Pi in 300 ml E rlenm eyer flasks. The flasks w ere incubated on a horizontal rotary shaker (90 strokes m in -1 , 80 mm am plitude) at 27 °C in the dark. P reparation o f en zy m e s f o r determ inations o f m axim u m activity C. roseus cells ( 1 .5 - 2 .0 g fresh w eight) w ere col lected and w ashed w ith distilled water by vacuum filtration through a layer o f filter-paper on a B uchner funnel. W ashed cells w ere h om ogenized with 5 - 1 0 vol. o f extraction m edium as follows. The m edium for assays o f PE P C contained 50 m M im id azole-H C l (pH 7.2), 0.1% (v/v) 2-m ercaptoethanol, 2 m M M gC l2 and E D T A . The m edia for assays o f N A D -m a la te d ehydrogenase (N A D M D H , E C 1.1.1.37) and N A D -m alic enzym e (N A D -M E , E C 1.1.1.39) w ere the sam e as that for assays o f P E P C but they contained 2 m M M nCl2 as an additional inorganic salt and 5 m M dithiothreitol (D T T ) instead o f 0.1% 2-m ercaptoethanol. F urtherm ore, 0.5% (w /v) Triton X -100 was added to the extraction m edium for assays of N A D -M E . The m edium for extraction o f PK

con sisted o f 50 mM potassium p h osp hate buffer (pH 7.6), 2.5 m M M gC l2, 2 m M E D T A , 2 m M DTT, 50 m M NaF, 0.1 m M PEP, 1 m M phenylm ethylsulfonylfluoride (PM SF ), 20% glycerol and 2.5% (w /v) p olyvin ylpolypyrolydon e (P V P ). Each h om ogen ate was centrifuged at 3 0 ,0 0 0 x g for 20 m in at 2 °C. A portion o f the supernatant (2.5 m l) was d esalted on a colum n o f S ephadex G -25 (P D -1 0 colum n; bed volu m e, 9.0 ml; Pharm a cia, U ppsala, S w ed en ) that had b een equilibrated with the appropriate extraction buffer, excep t in the case o f PK, w hen the colum n was equilibrated with 50 m M potassium p hosp hate buffer (p H 7.1). The fraction containing eluted protein (3.5 m l) was used im m ediately for assays o f enzym atic activities. Purification o f P E P C PE P C was partially purified from C. roseus cells by the fractionation w ith am m onium sulphate and chrom atography on Q -Seph arose as described elsew h ere (N agan o et al., 1994). The en zym e was purified about 8-fold , as com pared to the lev el in the supernatant o f the h om o g en a te with 40% re covery o f total activity. N o P E P phosphatase ac tivity, which m ay interfere w ith accurate kinetic studies o f PEPC , was present in the preparation o f PEPC . The specific activity o f the final prep aration was approxim ately 95 nkat m g -1 protein. Purification o f cy to so lic P K The cytosolic form o f PK (PK c) was partially purified by the m eth od o f Plaxton (1988) with m odifications. Freshly harvested cells (25 g fresh w eight) w ere h o m ogen ized w ith 2 volu m es o f e x traction buffer, w hich con sisted o f 50 mM p otas sium phosphate buffer (pH 7.6), 2 m M E D T A , 2.5 m M M gC l2, 2 m M DTT, 50 m M NaF, 0.1 m M PEP, 1 m M PMSF, 20% (v/v) glycerol and 2.5% (w /v) PVP. The h om o g en a te w as centrifuged at 17 ,000x g for 20 m in, and the resultant supernatant was filtered through M iracloth (C albiochem , La Jolla, C A , U .S .A .). To rem ove plastid-associated form PK, the supernatant ob tain ed was divided equally b etw een tw o 500 ml flasks and h eated at 60 °C for 5 min. The extract was then co o led on crushed ice to 4 °C and centrifuged as above. P oly eth ylen e glycol (PE G ; m olecular m ass 8000; Sigma C hem . Co., St. Louis, MO, U .S .A .) was added to

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the resultant supernatant fraction to bring the final concentration o f P E G to 3% (w/v) and the m ix ture was stirred for 20 m in. A fter centrifugation as above, the p ellets w ere discarded, and the super natant was adjusted to 8.5% (w/v) with PEG , and stirred for 60 m in, and then it was centrifuged as described above. The surface o f p ellets that co n tained PKe w as briefly w ashed with the extraction buffer to rem ove PEG , and then p ellets w ere dis solved in 4 ml o f elution buffer, which con sisted of 10 m M potassium phosp hate buffer (pH 7.1), 5 m M M gC l2, 1 mM E D T A , 2 mM D T T and 20% (v/v) glycerol. The en zym e fraction was centrifuged at 2 7 ,0 0 0 x g for 20 m in, and the resultant supernatant was applied to a colum n o f Q -Seph arose (15 mm i.d. x 200 m m ) which had b een equilibrated with the elution buffer. The colum n was w ashed with the sam e buffer, at 2.5 ml m in -1 , until the ab sorbance at 280 nm (A 280) decreased to 0.05. PK was eluted with a 300 ml linear gradient o f KC1 ( 0 - 7 0 0 m M ) and fractions (3.5 ml each) w ere co l lected. The active fraction which was eluted by approxim ately 200 m M KC1, was used as the prep aration o f enzym e. The en zym e was purified about 65-fold and the overall recovery was about 10%. The specific activity o f the final preparation was approxim ately 100 nkat m g -1 protein.

N A D - M D H (H atch et al., 1982): 50 m M (N -[2hydroxyethyl]piperazine-N '-[2-ethanesulfonic acid]) (H ep es)-N a O H buffer (pH 7.2), 0.2 mM N A D H , 1 m M oxaloacetate. N A D -M E (Fathi and Schnarrenberger, 1990): 50 m M H ep es-N a O H buffer (pH 7.2), 5 m M m alate, 2 m M N A D +, 5 m M D TT, 0.1 m M sodium E D T A , 0.05 mM C o A and 2 mM M nC l2.

A ssays o f en zy m a tic activities

M etabolism o f N a H 14C 0 3

The assays o f activities o f individual enzym es w ere based on the published m ethods, as d e scribed in the references cited below . C on cen trations o f reagents and pH values for the various assays w ere op tim ized for assays o f the activities o f enzym es from C. roseus. The total volu m e o f each reaction m ixture was 1 ml. R eactions were perform ed at 30 °C. A ctivities o f PEPC , PK, N A D -M D H and N A D M E w ere determ in ed spectrophotom etrically. The com p osition o f reaction m ixtures was as follow s, with references cited in parentheses: PEPC (Z ip fel et al., 1990): 25 m M Tris(hydroxym ethyl)am inom eth ane (Tris)-H C l buffer (pH 8.0), 2 m M PEP, 1 m M K H C 0 3, 5 m M M gC l2, 2 m M DTT, 0.2 mM N A D H and 33 nkat M D H . PK (Plaxton, 1988): 50 m M Tris-HCl buffer (pH 7.5), 1 m M P E P 2 m M A D P, 75 m M KC1, 10 m M M gC l2, 2 m M DTT, 0.2 m M N A D H , 33 nkat lactate dehydrogenase.

C ells (100 mg fresh w eight) w ere su spend ed in 2 ml o f 25 mM H ep es-N a O H buffer (p H 7.2) that contained 0.5 m M N a H 14C 0 3 (specific activity 74 kBq [„imol"1, IC N Biom edicals, Inc., Irvine, C A , U .S.A .) in the main com partm ent o f a 30 ml E rlenm eyer flask with a centre w ell. Filter-paper w etted with 0.1 ml o f 20% K O H was inserted in the cen tre w ell to trap 14C 0 2. A fter incubation at 27 °C for 30 min, cells w ere collected on a layer on Miracloth by filtration under vacuum and w ashed briefly with distilled water. W ashed cells w ere left in a flask so that radioactivity could be “ch a sed ”. H arvested cells w ere h om ogenized w ith 80% eth a nol and treated with 6% PC A to rem ove any re m aining 14C 0 2. The radioactivity o f the ethanolsoluble and ethanol-insoluble fractions w as m ea s ured separately. The am ount o f radioactivity in each fraction was sum m ed and the total was re garded as the radioactivity that had b een fixed by the cells.

N ative electrophoresis o f P E P C N on-denaturing gels containing 7.5% polyacryl am ide (Ishida and A shihara, 1993) w ere used. G els w ere stained for PEPC activity by the m ethod o f Q ueiroz-C laret and Q u eiroz (1992).

Q uantitation o f m etabolites M etabolites w ere extracted with 6% perchloric acid (P C A ) and quantitated enzym atically (K ubota and A shihara, 1990, 1993). E xp erim en ts to assess recovery w ere perform ed in parallel w ith assays, as described elsew h ere (K ubota and A sh i hara, 1993). R ecoveries w ere ab ove 85% in all cases.


M etabolism o f [4 -14CJm alate [4-14C ]M alate w as synthesized enzym atically from com m ercially purchased N a H 14C 0 3 (specific activity, 2.1 G B q m m o l-1) by the m ethod d e scribed by A m in o (1992). M ethods for the adm in istration o f radiochem icals, extraction o f m etab o lites and analysis o f labelled com p oun ds w ere the sam e as th ose described in a previous paper (N agan o and A shihara, 1993) excep t that [U -14C]glutam ine was replaced by [4-14C]m alate.

Results and Discussion In the con ven tion al glycolic pathway, conversion o f P E P to pyruvate is catalyzed by PK. In addition, this con version can also be perform ed by seq u en tial reactions that are catalyzed by PEPC, N A D d ep en d en t m alate d eh ydrogen ase (N A D -M D H ) and N A D -d e p e n d en t m alic en zym e (M E ), re spectively, in plant cells (W iskich and Dry, 1985; B ryce and ap R ees, 1985). O xaloacetate, a product o f the reaction catalyzed by PEPC , is converted to m alate by cytosolic N A D -M D H . In cells o f C. roseus, som e m alate, after transport into m ito chondria, was m etab olized directly by the T C A cycle, but the rest w as converted to pyruvate by m itochondrial N A D -M E , with the release of C 0 2 (Fig. 1). In the present study, w e exam ined the coarse and fine control o f the key enzym es in these tw o pathways. From our results, we exam ined w hether the alternative pathway, catalyzed by

745

PEPC , is the predom inant pathw ay in Pi-starved cells.

C oarse co n tro l o f the activities o f various en zy m es Fig. 2 show s the m axim um catalytic activities of PK, PEPC , M D H and M E in P i-fed (+ P i) and Pistarved ( - P i ) cells. The lev el o f the extractable PEPC activity increased 2.3- to 2.6-fold w hen cells that had b een cultured in co m p lete LS m edium for 7 days w ere transferred to the fresh P i-deficient LS m edium . Since the increase in the activity of P E PC in Pi-starved cells was suppressed com p letely by cycloh exim id e (25 ^ig m l-1 ), it seem s that PE P C w as synthesized de n o vo during the 24 h after transfer o f the cells to the P i-deficient m edium . B y contrast, the level o f PK activity was higher in P i-fed cells than in Pi-starved cells. Sim i lar results w ere also ob tain ed in an earlier study (Li and A shihara, 1990). H ow ever, the activity o f PK reported in our previous paper was signifi cantly low er than that ob tain ed in this study. The difference is m ainly due to a d ifferen ce b etw een the extraction m ed ia used. In the present study, we found that P laxton ’s extraction m edium (Plaxton, 1988), which contains 50 mM phosphate buffer and 20% glycerol, is very suitable for stabilization of the activity o f PK. The activities o f M D H and M E in the Pi-starved cells w ere slightly low er than or sim ilar to those in the P i-fed cells. Our previous analysis indicated that the flux o f glycolysis in

Fig. 1. Reactions catalyzed by pyruvate kinase and phosphoenolpyruvate carboxylase and possible pathways for conversion of phosphoenolpyruvate to pyruvate in Catharanthus roseus cells. MDH, NAD-malate dehydrogenase; ME, NAD-malic enzyme; PEPC, phosphoenolpyruvate carboxylase; PK, pyruvate kinase; PEP, phosphoenolpyru vate; OAA, oxaloacetate.

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Fine co n tro l o f the activities o f P K a n d P E P C

i tbi

« i >> > X

0

1 2

4 Age of culture (days)

Fig. 2. Maximum activities of phosphoenolpyruvate car boxylase (a), pyruvate kinase (b), NAD-malate dehydro genase (c) and NAD-malic enzyme (d) in suspensioncultured Catharanthus roseus cells grown in complete (heavily shaded columns) or in Pi-depleted (open col umns) LS medium. Values are means ± s.d. of results from four separate experiments. Initial enzymatic activi ties in cells in the inoculum are shown by lightly shaded columns.

C. roseus cells is eq u ivalen t to less than 1.7 nm ol P E P consum ed per sec per fr. wt. (K ubota and A shihara, 1990). Thus, m axim um catalytic activi ties o f PEPC and PK seem ed to be at least 6 tim es higher than that exp ected from the flux o f g lyco lysis in vivo. It is also n otew orth y that the m axi m um activity o f M E was alm ost the sam e as that o f PK, but the activity o f M D H was 5 0 - 1 0 0 tim es higher than those o f the other three enzym es, PEPC, PK and N A D -M E . T herefore, it is difficult to conclude that the increased level o f PE P C ac tivity (protein), i.e., coarse control o f PEPC , leads directly to the increase in the contribution o f the alternative pathw ay to glycolysis in Pi-starved cells. T hese results sim ply suggest that both PK and PEPC are functional as the final en zym es o f glycolysis in Pi-starved cells. The activities o f both enzym es in vivo are con trolled by the co n cen trations o f the substrates and effectors for these enzym es, which m ay change greatly in Pi-fed and Pi-starved cells. We can exp ect that the fine co n trol o f the activities o f PK and PEPC is im portant in an analysis o f the role o f the bypass in Pistarved cells.

In order to exam ine the fine control o f the ac tivities o f PK and PEPC , cytosolic PK and PEPC w ere partially purified for kinetic studies. A l though PEPC seem ed to be syn th esized de n o vo in Pi-starved cells, activities o f P E P C from both Pi-fed and Pi-starved cells w ere elu ted as a single peak from a colum n o f Q -Sepharose by approxi m ately 0.3 m KC1 (Fig. 3). The PE P C in each frac tion was then analyzed by PA G E on native gels. A clear stained band o f activity that corresp ond ed to PE P C was detected at the sam e p osition in each case (insets in Fig. 3). Furtherm ore, the kinetic properties o f PEPC from Pi-starved cells w ere very similar to those from Pi-fed cells. It has b een argued that the affinity for P E P of PEPC is at least on e order of m agnitude low er than that o f PK in higher plants (C o p ela n d and Turner, 1987; D avies, 1979; O ’Leary, 1982). H o w ever, the K m of PEP for PEPC from C. roseus was sm aller than that for PKc at p hysiological pH (Table I). Therefore, in C. roseus cells, the parti-

Elution volume (ml)

Fig. 3. Profiles of elution of phosphoenolpyruvate car boxylase (PEPC) after column chromatography on Q-Sepharose. Insets show the distribution of activity of PEPC on 7.5% polyacrylamide gels, (a) PEPC from 1-day-old Pi-fed cells, (b) PEPC from 1-day-old Pistarved cells. (Age refers to days after transfer to fresh medium in this and other legends.)

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M. Nagano et al. ■ Glycolytic Bypass Catalyzed by PEPC Table I. Comparison of K m values of substrates for PKc and PEPC that had been partially purified from Catha ranthus roseus cells. The enzymatic activities were meas ured at physiological pH (7.5). Km values were deter mined from double-reciprocal plots. Kinetic data were subjected to linear regression analysis and the correlation coefficient in each case was greater than 0.98. Substrate

PKc

PEP ADP

100

90

[[i m ]

PEPC

[ jam ]

45 -

tioning o f P E P b etw een PE P C and PKc is likely to favor P E P C if inhibitors and/or activators of these tw o en zym es are absent. A sim ilar low K m (50 [xm) for P E P C w as also reported recently in the case o f PE P C from soyb ean nodule and it was determ in ed in the p resen ce o f 15% glycerol at pH 7 (Schuller et al., 1990b ). The K m o f A D P for PKc from C. roseus cells was 90 [am. This result also suggests that P E P C has an advantage over PK w hen the availability o f A D P is lim ited. The effects o f various effectors at different con centrations o f on the activities o f PE P C and PKc,

purified from P i-fed cells, w ere exam in ed (Fig. 4). M alate inhibited the activity o f PE P C but did not greatly in fluence the activity o f PK w ithin the range o f con centration s sh ow n in Fig. 4. Citrate and aspartate also inhibited P E P C but to a lesser extent. A TP inhibited both enzym atic activities. The con centration o f A T P for 50% inhibitions w ere 1.9 mM (P E P C ) and 3.1 mM (P K c), resp ec tively. A D P and A M P w ere w eak inhibitors of PEPC.

L evels o f m eta b o lites in P i-fed an d P i-sta rved cells In order to estim ate the activity o f P E P C and PKc in vivo, cellular levels o f the substrates and effectors o f th ese en zym es w ere m easured. U ntil tw o days after the transfer o f cells to co m p lete and P i-depleted m edium , the lev el o f P E P in the Pifed and Pi-starved cells w as similar. H ow ever, after 4 days, the lev el in the Pi-fed cells b ecom e low er than in the Pi-starved cells. T hese results are consistent w ith the ob servation that the level of P E P was low w hen the rate o f respiration was high (K ubota and A shihara, 1993). E stim ated cytoplasm ic con centration s o f PEP varied from 90 to 210 [am, if w e assum e that the cytoplasm represents 10% o f the total volu m e o f the cell and that P E P is p resen t only in the cyto plasm . T hese valu es w ere sim ilar to or slightly higher than the K m values o f P E P for P E P C and PKc. L evels o f m alate and ATP, p oten t inhibitors of PEPC , w ere significantly reduced in Pi-starved cells (Table II and Li and A shihara, 1990). If m alate and A T P are exclu sively located in the cytosol, the activity o f P E P C w ould seem to be

Table II. Levels of PEP, malate and pyruvate in suspension-cultured Catharanthus roseus cells grown in com plete (+Pi) and Pi-deficient (-P i) medium. The levels are expressed as nmol g fr. wt.-1. Metabolite

Age of culture [days] 0

E ffe cto r or substrate (mM)

Fig. 4. Effects of malate (a), citrate (b), aspartate (c), ATP (d). A D P (e) and AMP (f) on the activities of phos phoenolpyruvate carboxylase (---- • ---- ) and pyruvate kinase ( — O — ) from 1-day-old Pi-fed Catharanthus roseus cells.

PEP Malate Pyruvate

12 ± 1

1 + Pi -P i

2

20 ± 6 20 ± 4

21 ± 5 18 ± 4

1391 ± 17 + Pi -P i

513 ± 22 212 ± 34

2215 ± 128 176 ± 19

65 ± 21 + Pi

55 ± 7 165 ± 12

80 ± 16 75 ± 7

4 9 ± 1 18 ± 2 1265 ± 79 119 ± 16 124 ± 4 69 ± 10

M. Nagano et al. ■ Glycolytic Bypass Catalyzed by PEPC

748

'0

10

20

30

Duration of chase (min)

AO Duration of incubation (min)

inhibited com p letely in Pi-fed cells. H ow ever, sig nificant am ounts o f m alate m ay be present in vacuoles. N everth eless, it is ob vious that inhibition o f PE P C by th ese effectors is relieved to a greater or lesser extent in Pi-starved cells.

M etabolism o f N a H 14C O 3 an d [4 -,4C ]m alate in P i-starved cells The enzym atic studies m en tion ed ab ove only provided and indication that the bypass by PEPC , N A D -M D H and N A D -M E could occur in Pistarved C. roseus cells. Bryce and ap R ee s (1985) concluded that the bypass op erates in roots o f P isu m and P lan tago from p ulse-ch ase experim ents w ith 14C 0 2. Sim ilar exp erim en ts w ere perform ed using N a H 14C 0 3 and Pi-starved C. roseus cells (Fig. 5 a). C onsistent with the results o f Bryce and ap R ee s (1985), there was appreciable fixation o f 14C at the end o f the pulse, and su bsequ en tly sub stantial loss o f 14C from the cells during the chase. This rapid d ecarboxylation o f the products o f dark fixation o f H C 0 3~ in Pi-starved cells suggests that P E P from glycolysis was con verted to pyruvate w ith the release o f 14C 0 2. We also ob served that 5 m M 2-n-butylm alonate, an inhibitor o f the trans port o f m alate into m itochondria (W iskish, 1975) caused 40% inhibition o f the fixation o f H C 0 3~. This result is com p atib le w ith the results from pea roots, in which 36% inhibition o f fixation was o b served. Bryce and ap R ee s (1985) su ggested that 2-/7-butylm alonate inhibits the transport o f m alate

Fig. 5. (a) Loss of 14C from components fixed by the 1-day-old Pi-starved Catharanthus roseus cells after administration of N aH 1 4 C 0 3 for 30 min. (b) Metabolism of [4-1 4 C]malate by 1-day-old Pi-starved Catharanthus roseus cells. Total uptake of 14C by the cells (A); incor poration into C 0 2 (A), perchloric acid-soluble fraction ( • ) and perchloric acid-insoluble frac tion (O).

into m itochondria and that the resultant accum u lation o f m alate in the cytosol w ould be ex p ected to inhibit PEPC. To exam ine the m etabolism o f m alate in further detail, the fate o f radioactivity from [4-14C ]m alate in Pi-starved cells was in vestigated (Fig. 5 b). R apid release o f large am ounts o f 14C 0 2 from [4-14C]m alate was observed. In addition, approxi m ately 20% o f the radioactivity that was taken up was distributed in the soluble fraction and the rest was incorporated into the P C A -in solu b le fraction (possibly as protein). A fter a 60 m in incubation, 79.5% , 7.8% , 10.3% and 2.5% o f the total radioac tivity taken up by the cells was recovered as C O z, organic acids, free am ino acids and protein, re spectively. T hese results indicate that ex o g en o u sly supplied m alate was alm ost exclu sively m etab o lized and m ost o f it seem ed to be con verted to pyruvate with the release of C 0 2. T h ese results support the hypothesis that the bypass for co n v er sion o f PEP to pyruvate is functional in C. roseus cells. Similar results have b een ob tain ed in suspension-cultured p hotoautotroph ic cells o f C h en o p o d iu m rubrum (A m in o, 1992). In darkness, 96% o f radioactivity from [4-14C ]m alate that had b een taken up by C h en opodiu m cells was m etab olized , and m ore than 66% o f the radioactivity was re leased as 14C 0 2. The rapid release o f 14C 0 2 from recently fixed H C 0 3_ and from [4-14C ]m alate w as also ob served in Pi-fed cells (data not show n). Thus, the bypass is probably functional as a relief pathw ay in both

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M. Nagano et al. ■ Glycolytic Bypass Catalyzed by PEPC P i-fed and Pi-starved cells, although the extent o f its contribution m ay be controlled by several factors, as m en tion ed above. R o le o f the P E P C bypass in P i-starved cells R esu lts o f the presen t study suggest that the ac tivity o f P E P C relative to that o f PK increased in Pi-starved C. roseus cells in vivo since the m axi m um catalytic activity o f PE P C increased and the con centration s o f negative effectors o f PEPC d e creased in Pi-starved cells. W hen the cellular con centration o f Pi is extrem ely low, the production o f PE P C p rotein is increased, as is that o f other Pi-recycling enzym es, such as several phosphatases and R N a ses (U e k i and Sato, 1971; D u ff et al., 1991; N ürnberger et al., 1990). Thus, one o f the functions o f P E P C in Pi-starved cells seem s to be the production o f Pi from PEP. A second function o f P E P C is the conversion o f PE P to pyruvate w ithout ad en ine nucleotides. The levels o f adenine n u cleotid es are greatly reduced in Pi-starved cells (U kaji and A shihara, 1986; 1987; A shihara et al.,

Amino S. (1992), Intracellular conversion of malate and localization of enzymes involved in the metabolism of malate in photoautotrophic cell cultures of Cheno podium rubrum. Z. Naturforsch. 47c, 545-552. Ashihara H., Horikosi T., Li X.-N., Sagishima K. and Yamashita Y. (1988 a), Profiles of enzymes involved in glycolysis in Catharanthus roseus cells in batch sus pension culture. J. Plant Physiol. 133, 38-45. Ashihara H., Li X.-N. and Ukaji T. (1988b), Effect of inorganic phosphate on the biosynthesis of purine and pyrimidine nucleotides in suspension-cultured cells of Catharanthus roseus. Ann. Bot. 61, 225-232. Bryce J. H. and ap Rees T. (1985), Rapid decar boxylation of the products of dark fixation of C 0 2 in roots of Pisum and Plantago. Phytochemistry 24, 1635-1638. Copeland L. and Turner J. F. (1987), The regulation of glycolysis and the pentose phosphate pathway. In: The Biochemistry of Plants, Vol. 11 (D. D. Davies, ed.). Academic Press, San Diego, pp. 107-128. Davies D. D. (1979), The central role of phosphoenolpy ruvate in plant metabolism. Annu. Rev. Plant Physiol. 30, 131-158. Duff S. M. G., Moorhead G. B. G., Lefebvre D. D. and Plaxton W. C. (1989), Phosphate starvation inducible “bypasses” of adenylate- and phosphate-dependent glycolytic enzymes in Brassica nigra suspension cells. Plant Physiol. 90, 1275-1278. Duff S. M. G„ Plaxton W. C. and Lefebvre D. D. (1991), Phosphate starvation response in plant cells: de novo synthesis and degradation of acid phosphatase. Proc. Natl. Acad. Sei. U.S.A. 8 8 , 9538-9542.

1988 b; K ubota et al., 1989). Thus, P E P C appears to be a m em ber o f a group o f glycolytic bypass enzym es w hose activities are stim ulated by Pi star vation and which include PFP and non-phosphorylating N A D P -g ly cera ld eh y d e 3-phosphate d eh ydrogen ase (D u ff et al., 1989; T h eod orou and Plaxton, 1993). The anaplerotic role o f PEPC is also considerable, but it d o es seem to be lim ited since m ost o f the carbon fixed by P E P C in Pistarved cells is im m ediately released as C 0 2. H o w ever, com pared with the reaction catalyzed by PK, the reaction catalyzed by P E P C is energetically w asteful b ecau se A TP is not gen erated during the conversion o f P E P to pyruvate. T herefore, in nor m ally grow ing cells, the activity o f P E P C seem s to be suppressed by effectors, such as A T P and m alate, and PK functions p redom inantly as the enzym e that catalyzes the final step in glycolysis. A c k n o w led g em en t The authors thank Dr. S. A m ino, D ep artm ent of Botany, U n iversity o f Tokyo, for his helpful com m ents about the synthesis o f [4-14C]m alate.

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