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Enzymes associated with β-carboxylation in Ectocarpussiliculosus (Phaeophyceae): are they involved in netcarbon acquisition?Sylvia Busch & Rainer Schmida Fachbereich Biologie/Botanik, Philipps-Universität, Lahnberge, 35032 Marburg, GermanyPublished online: 03 Jun 2010.

To cite this article: Sylvia Busch & Rainer Schmid (2001) Enzymes associated with β-carboxylation in Ectocarpus siliculosus(Phaeophyceae): are they involved in net carbon acquisition?, European Journal of Phycology, 36:1, 61-70

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Eur. J. Phycol. (2001), 36: 61–70. Printed in the United Kingdom 61

Enzymes associated with β-carboxylation in Ectocarpus

siliculosus (Phaeophyceae) : Are they involved in net carbon

acquisition?

SYLVIA BUSCH AND RAINER SCHMID

Fachbereich Biologie}Botanik, Philipps-UniversitaX t, Lahnberge, 35032 Marburg, Germany

(Received 21 July 2000 ; accepted 10 November 2000)

The hypothesis that carbon acquisition in phaeophytes of lower organization is controlled by a blue-light-regulated C%mechanism was

tested by various approaches with the filamentous species Ectocarpus siliculosus. Here we report analyses at the enzymatic level. Enzymes

potentially participating in a C%cycle were assayed in extracts, and activities of a phosphoenolpyruvate carboxykinase (PEPCK), of NAD-

and NADP-malate dehydrogenases, and of a NADP-malic enzyme were detected. No activity could be measured of phosphoenolpyruvate

carboxylase or of pyruvate-Pi-dikinase, which is essential to complete the cycle by regenerating phosphoenolpyruvate from pyruvate. The

kinetic properties of all enzymes were determined. Malic enzyme and PEPCK were purified to homogeneity, as tested by gel filtration.

The molecular mass of native malic enzyme was determined to be 440 kDa. In SDS gel electrophoreses it produced a single band with a

relative molecular mass of 53 kDa, which suggested that the native molecule might be a homo-octamer. The molecular mass of PEPCK

was 90 kDa and separation on SDS gels produced two major bands, one of 56 kDa and one of 18 kDa, which appeared not to be a

degradation product of the larger polypeptide. The composition of the native enzyme is unclear. In extracts, the activities of malic

enzyme and PEPCK followed a circadian rhythm, but none of the enzymes tested was found to be blue-light-dependent. The above

results neither prove nor dismiss the possibility that a C%mechanism operates in Ectocarpus.

Key words : blue light, carbon acquisition, circadian rhythm, Ectocarpus, malic enzyme, phosphoenolpyruvate carboxykinase,

photosynthesis

Introduction

Previous investigations of the stimulation of red-light-saturated photosynthesis by blue light in brown algae(Dring, 1989 ; Forster & Dring, 1992 ; Schmid et al., 1992,1994) have shown that blue light increases the affinity forinorganic carbon, at least in those species that have beenanalysed for this effect (Forster & Dring, 1992 ; Schmid &Dring, 1996a, b). In the absence of Ci (inorganic carbon) inthe bulk medium, pulses of blue light were able tostimulate photosynthetic oxygen evolution in Ectocarpus

(Schmid&Dring, 1996a) and Laminaria saccharina (Schmid,unpublished). Under normal Ci conditions, they alsostimulated oxygen evolution in Laminaria without sim-ultaneous stimulation of CO

#consumption (Schmid et al.,

1996). These observations have been taken as a sign of theexistence of an internal carbon pool and it has beensuggested that, in the Phaeophyceae (Heterokontophyta),CO

#is fixed into an intermediate that is stored under red

light conditions. Oxygen evolution was suggested toincrease as a consequence of the blue light activation of anenzyme catalysing the release of CO

#from this in-

termediate (Schmid & Dring, 1996a, b). It appeared most

Correspondence to : R. Schmid. Tel : ­49 6421 28 6571. Fax : ­49

6421 28 2057. e-mail : schmid!mailer.uni-marburg.de.

attractive to suggest a C%-like pathway for such a process,

by analogy with the situation in CAM plants, with malateor aspartate functioning as a stable intermediate (cf.Schmid & Dring, 1996b). A functional C

%or CAM

metabolism in brown algae of higher organization hadbeen proposed by several investigators ( Joshi & Karekar,1973 ; Kerby & Raven, 1985 ; Axelsson et al., 1991), but itsexistence was dismissed after some scrutiny (Kremer &Ku$ ppers, 1977 ; Kremer, 1981a, b ; Johnston, 1991). How-ever, the revival of the C

%}CAM-like metabolism in our

model was based on the belief that a number ofphysiological or morphological characteristics of thehigher brown algae had not been accounted for adequately(Schmid & Dring, 1996b). Also, more recent studies implythat, in Fucus vesiculosus, CO

#is stored partly in inorganic

and partly in organic form (Kawamitsu & Boyer, 1999), acondition which might have confused matters.

The working hypothesis of a blue-light-activated C%

pathway has been followed up using Ectocarpus as a modelsystem for the brown algae of lower organization byanalysis of metabolic pools and "%C fixation patterns(Hillrichs & Schmid, 2001) and by measuring inorganiccarbon uptake and accumulation (Schmid & Hillrichs,2001). These studies revealed that an inorganic and not anorganic carbon pool was present within the cells. How-

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62S. Busch and R. Schmid

ever, malate appeared among the very early labellingproducts together with phosphoglycerate. This would beexpected if efficient C

%carboxylation was operating, in

addition to the C$carboxylation in the reductive pentose

phosphate cycle, but the observations on metabolite poolsand of their blue-light-dependent changes, as well as oftheir behaviour in radioactive chase experiments, stronglycontradicted the idea of a C

%mechanism in Ectocarpus.

In the present investigation, we analysed enzymes thatwould be expected to participate in a C

%metabolic

pathway. We also tried to characterize the key enzymesphosphoenolpyruvate carboxykinase (PEPCK) andNADP-dependent malic enzyme in order to understandtheir function in the metabolism of Ectocarpus.

Materials and methods

Cultivation

Unialgal cultures of Ectocarpus siliculosus (Dillwyn) Lyngb.were grown in glass dishes in Provasoli’s enrichedseawater medium (PES ; Provasoli, 1968) under a light-dark cycle of 12 :12 h and at a temperature of 15 °C. Thewhite culture light (1±5 W m−#) was a mixture of cold andwarm fluorescent tubes. Two culture strains were used forthe experiments. One strain (Es PAr 1027 Sp.) wassporophytic and had been obtained from Prof. D. Mu$ ller(Konstanz ; cf. Mu$ ller, 1979). The other one (Es BHI 293)was gametophytic (parthenosporophytic) and had beenisolated from plants collected in Northern Ireland(Ballyhenry Island, Co. Down). The results with bothstrains were essentially the same. Maize for the extractionof PEP carboxylase and of pyruvate-Pi-dikinase wasgrown in the greenhouse and obtained from the BotanicalGarden of the University of Marburg.

Irradiation conditions

For the experiments with red or blue light, plants wereirradiated using projectors (Pradovit, Leitz, Germany)equipped with either a red Plexiglas filter (No. 502, Ro$ hm,Darmstadt, Germany) or a blue glass filter (BG12, Schott,MainzGermany). Blue light (12 µmol m−# s−") was alwaysgiven simultaneously with the photosynthetically satu-rating red background light (120 µmol m−# s−").

Enzyme assays

All enzyme activities were determined by readingabsorbance changes of NAD(P) or NAD(P)H in aspectrophotometer at 340 nm. The carboxylating activityof PEPCK was determined by linking the production ofoxaloacetic acid to NADH oxidation via malate de-hydrogenase (MDH). The assay (modified after Johnston& Raven, 1989) consisted of 100 mol m−$ Hepes-NaOH(pH 7), 40 mol m−$ NaHCO

$, 4 mol m−$ MnCl

#,

2 mol m−$ ADP, 5 mol m−$ phosphoenolpyruvate (PEP),0±25 mol m−$ NADH, 5 units of MDH (suspension inbuffered ammonium sulphate, Sigma) and 50 µl extract.

For the decarboxylating direction the assay of Hatch(1973) was used. Malic enzyme was determined by aslight modification of the method of Wedding (1989)in an assay containing 25 mol m−$ Tris}HCl (pH 8),0±5 mol m−$ EDTA, 5 mol m−$ MgCl

#, 2±5 mol m−$ mal-

ate (sodium salt), 0±25 mol m−$ NADP and 50 µl extractper millilitre assay. NAD-malate dehydrogenases as wellas pyruvate-Pi-dikinase were determined after Nakamoto& Edwards (1983). For the determination of NADP-malatedehydrogenase, this assay was modified by replacing theTris buffer with Hepes}NaOH (0±1 kmol m−$, pH 7) andNADH with NADPH (0±2 mol m−$).

Enzyme extraction

Filaments of Ectocarpus siliculosus were collected on a net,washed with tap water and blotted in order to determinethe fresh weight. They were then homogenized withmortar and pestle to a fine powder in liquid nitrogen. Thequality of the disruption was checked under the micro-scope. The cold powder was stirred into the homo-genization buffer (buffer A) containing 0±1 kmol m−$ Tris}HCl (pH 8), 2 mol m−$ Na

$-EDTA, 5 mol m−$ CaCl

#,

5 mol m−$ MgCl#, 50 mol m−$ sodium ascorbate,

500 mol m−$ sorbitol and 5 mol m−$ dithiothreitol (DTT).By the addition of sorbitol to the buffer the physodesremained largely undamaged and constituted the top layerof the pellet after the subsequent centrifugation (20 min,50000 g, 4 °C). The supernatant of this centrifugation wasthen subjected to fractionated ammonium sulphate pre-cipitation with a first precipitation step at 55% ammoniumsulphate saturation and a second step at 80% saturation.The precipitates were collected by centrifugation andthe resulting pellets were re-dissolved when neededin 20 mol m−$ Tris}HCl (pH 8), 1 mol m−$ MgCl

#,

1 mol m−$ DTT (buffer B). These concentrated crudepreparations were taken for the determinations of thekinetic properties of all enzymes (see Table 1). K

mvalues

were determined by fitting the substrate dependencies ofthe enzyme activities to Michaelis–Menten type kineticsusing double reciprocal plots.

After the various light treatments (including themeasurements of circadian activity), enzyme extractionswere performed under the light of a red fluorescent tube toavoid possible stimulation by blue light in vitro, andextracts were kept in dark boxes during their transport tothe centrifuges.

Purification of PEPCK

The fraction between 55% and 80% ammonium sulphatesaturation contained all of the activity of PEPCK. Theprecipitate was collected by centrifugation (20 min,50000 g, 4 °C) and re-dissolved in a small volume ofbuffer B. This was separated on Sephadex G-100 equi-librated with buffer B. The active fractions were collectedand immediately applied to a PBE 94 chromatofocusingcolumn (Pharmacia) that had been equilibrated with25 mol m−$ ethanolamine}acetic acid (pH 9±4), 1 mol m−$

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Enzymes of β-carboxylation in Ectocarpus 63

Table 1. Enzymes that may be connected with β-carboxylation and their properties

Remarks

PEP carboxylase – Not detected

PEP carboxykinase MW (native) 90000 ; MR

a 18000}56000 ;

Km

(ADP) 0±27 mM inverse reaction with about 1}10 of carboxylating activity ;

Km

(CO#) 4±67 mM pH optimum: " pH 8; isoelectric point : pH 6±8–7±1

Km

(PEP) 0±46 mM

K"/#

(Mn#+) 0±45 mM

NADP-malic enzyme MW (native) 440000 ; MR

a 53000 ;

Km

(malate) 0±58 mM reverse reaction : no activity ; pH optimum: pH 7±5 ;

Km

(NADP) 0±12 mM sigmoidal dependency on Mg#+

K"/#

(Mg#+) C 0±8 mM

NAD-malate dehydrogenaseb

Km

(oxaloacetate) ! 0±01 mM

Km

(NADH) 0±34 mM

NADP-malate dehydrogenaseb Substrate inhibition by NADP at high concentration

Km

(oxaloacetate) 0±05 mM

Km

(NADP) 0±05 mM

Pyruvate-Pi-dikinase Not detected

a After SDS gel electrophoresis.b Determined in crude concentrates after ammonium sulphate precipitation. Distinguished only by the use of NAD or NADP.

MgCl#, 1 mol m−$ DTT. Adsorbed proteins were eluted

with Polybuffer 96 (Pharmacia, pH 6, diluted 1 :5),1 mol m−$ MgCl

#, 1 mol m−$ DTT, and again the active

fractions were collected. The combined fractions wereapplied to a ‘Resource Q’ ion exchange-column (1 ml,Pharmacia) and separated with a gradient of0–0±4 kmol m−$ KCl in buffer B using an HPLC system(Kontron). Active fractions were pooled and concentratedin micro-concentrators (Centricon, Amicon). The concen-trated sample was subjected to a final gel filtration stepin buffer B by HPLC on a Bio-Sil Sec-250 column(300 mm¬7±8 mm, BioRad). To determine the molecularweight, a mixture of standard proteins was used con-taining bovine thyroglobulin and gamma-globulin,chicken ovalbumin, horse myoglobin and vitamin B

"#

(BioRad). Purity was checked by SDS gel electrophoresis.

Purification of malic enzyme

The pellet from the 55% saturated ammonium sulphateprecipitation was re-dissolved in a small volume of20 mol m−$ Tris}HCl (pH 7±5), 1 mol m−$ MgCl

#,

1 mol m−$ DTT (buffer C) and applied to a Sephadex G-100 column that had been equilibrated with the samebuffer. Active fractions were combined and bound to asmall column (approx. 3 ml) of Red Sepharose Cl-6B(Pharmacia). The column was then washed with40 mol m−$ of KCl in buffer C and bound proteins wereeluted with a gradient of 40–350 mol m−$ KCl in the samebuffer. Fractions with malic enzyme activity were dialysedagainst buffer C and applied to a ‘Resource Q’ ionexchange column (1 ml, Pharmacia). They were elutedwith a gradient of 0–0±4 kmol m−$ KCl in buffer C usingthe HPLC system as above. Fractions with activity were

again combined, concentrated and subjected to a BioSilSec-250 gel filtration column (see above). Molecularweight determinations were done as described for PEPCK.

Gel electrophoresis

Proteins were separated by SDS gel electrophoresis in aslab gel apparatus on 12% polyacrylamide with the gelsystem of Laemmli (1970) and stained with the silverstaining procedure according to Blum et al. (1987).

Protein determination

Proteins were determined with a modified Lowry assay(Peterson, 1977).

Results

Initially, attempts to extract enzymes from Ectocarpus

siliculosus yielded highly variable activities, and activitiestended to be higher in cultures that had been freshlytransferred into new culture medium (not shown). Thisseemed to be mostly due to the release of phenoliccompounds from the physodes during extraction, becausethe addition of sorbitol or sucrose at final concentrationsof 0±5 kmol m−$ invariably produced good results. In thiscase, the physodes remained mostly intact and were foundas a dark brown layer on top of the pellet after clearing thehomogenate by centrifugation. Nevertheless, the activitiesin the crude extracts were relatively low. Therefore, weincluded steps of ammonium sulphate precipitation in thestandard extraction protocol.

The extracts were tested for any enzyme activities thatmight play a role in a C

%carboxylic acid cycle, except for

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64S. Busch and R. Schmid

Table 2. Purification of PEPCK from Ectocarpus siliculosus

Total protein

(mg)

Total activity

(µmol min−")

Specific activity

(µmol mg−" min−")

Yield

(%) Factor

Crude extract 155 3±5 0±023 100 1

55–80% (NH%)#SO

%48±5 2±5 0±052 71 2±3

Sephadex G-200 25±8 0±8 0±031 23 1±4Chromatofocusing 0±89 0±5 0±56 14±2 24

Resource Q (HPLC) 0±18 0±13 0±71 3±7 31

Gel filtration (HPLC) 0±007 0±028 4 0±8 174

About 20 g of fresh weight were used for the extraction.

activities associated with the formation and mobilizationof aspartate, because aspartate had not shown significantlabelling in "%C inorganic carbon fixation experiments(Hillrichs & Schmid, 2001). The activities tested werethose of PEP carboxylase, PEPCK, malic enzyme, malatedehydrogenases (MDH) and pyruvate-Pi-dikinase (Table1). No activity was detected for PEP carboxylase or forpyruvate-Pi-dikinase, although extracts of maize, whichwere prepared for comparison, showed activities thatcorresponded to reported values (e.g. Nakamoto &Edwards, 1983) when assayed under the same conditions.

As indicated in Table 1, the activities detected werethose of PEPCK, NADP-malic enzyme (NAD-malic en-zyme activity was not found) and NAD- as well as NADP-dependent MDHs. The MDH activities represented atleast three different enzymes because NADH- andNADPH-dependent activities clearly differed in theiraffinities for oxaloacetate (Table 1) and, in separations onvarious columns, there were at least three distinct peaks(not shown), one of which had high affinity for NADPHwhereas the other two had normal affinity for NADH butnone forNADPH. TheK

mofNAD-MDH for oxaloacetate

was so low that it could not be determined with theresolution of our equipment. As the lowest concentrationof oxaloacetate used was 0±01 mol m−$ (giving a verysmall final absorption difference of NADH in the assay)and the activity was still as high as at high oxaloacetateconcentrations, the K

mmust have been lower than

0±01 mM (Table 1).To determine the activity of PEPCK, a fractionated

precipitation with ammonium sulphate had to be intro-duced because, in crude extracts and in precipitates of totalprotein (0–80% ammonium sulphate), pyruvate kinaseand lactate dehydrogenase interfered with the coupledassay. In this case, the presence of PEP also causedoxidation of NADH but this activity was CO

#-inde-

pendent. Both interfering activities, however, were com-pletely removed by a first precipitation step at 55%ammonium sulphate saturation. This step also precipitatedmalic enzyme (see below).

On all columns used, the activity of PEPCK eluted as asingle peak. However, separations on columns thatinteracted with the enzyme by reversible binding (e.g. ionexchange columns) rendered the enzyme highly unstable,whereas its activity in the crude extracts or in the

subsequent ammonium sulphate precipitation steps couldbe maintained over long periods (not shown). In thedesign of the purification procedures, therefore, thecolumns interacting with the enzyme were used for thelater steps (Table 2). The purification protocol consisted offractionated ammonium sulphate precipitation, a crudedesalting and molecular sieving step on Sephadex G-100,chromatofocusing, and a final high-resolution molecularsieving step by HPLC (Table 2). The most efficientpurification step was chromatofocusing with a gradientdescending from pH 9±4 to pH 6. Here, the enzyme elutedbefore the bulk of the other proteins at an apparentisoelectric point of pH 6±8 (Table 1). It should bementioned, however, that part of the enzyme activity didnot bind to the column when applied at pH 8±5 and thispart of the activity could not be bound when it wasapplied again to a freshly equilibrated column. In SDS gels,this non-binding fraction showed large amounts of an18 kDa protein (Fig. 1) which was usually found to beassociated with the purified PEPCK preparation (seebelow). The native molecular mass (M

r) of the enzyme

was determined in the gel filtration step to be 90 kDa. Re-application of the active fractions to the same columnproduced lower molecular mass peaks (around 30 and18 kDa) lacking activity as well as the now diminishedpeak at 90 kDa. When the native 90 kDa enzyme wasseparated by SDS gel electrophoresis at least two bandswere found, one with an M

rof 56 kDa and a second one

with an Mrof 18 kDa (Fig. 1). Occasionally, faint bands

were visible in the range of 30 kDa. The ratio of the largerto the smaller band was not constant in the differentpreparations and the 18 kDa band generally prevailed.

Kinetic properties of PEPCK were determined with there-dissolved precipitations after the 55–80% ammoniumsulphate step (see above). Later steps of the purificationprotocol proved to be too unstable for this kind ofanalysis. The three substrates used by PEPCK for thecarboxylating direction (CO

#, PEP, ADP) all produced

normal Michaelis–Menten kinetics with Km

values asgiven in Table 1. The pH dependence of the carboxylatingactivity showed a relatively broad optimum at aboutpH 6±7 (Table 1). The enzyme also operated in thedecarboxylating direction, but maximum rates were onlyabout one-tenth of those of PEP carboxylation. Manga-nese was required for PEPCK activity (Table 1).

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Enzymes of β-carboxylation in Ectocarpus 65

malic enzyme PEPCK

Fig. 1. Gel electrophoresis of crude fractions and of purified

phosphoenolpyruvate carboxykinase (PEPCK) and malic enzyme

of Ectocarpus siliculosus. Proteins were separated by SDS gel

electrophoresis and bands were silver stained. The figure

combines lanes from more than one gel. They were mounted in a

size that gave the same migration distances for the reference

proteins. Lane 1, reference proteins ; lane 2, precipitate at 0–55%

ammonium sulphate saturation containing malic enzyme activity ;

lane 3, purified malic enzyme ; lane 4, precipitate at 55–80%

ammonium sulphate saturation containing PEPCK activity ; lane 5,

polypeptides not binding to the chromatofocusing column, note

the extremely strong band at 18 kDa ; lane 6, purified PEPCK.

The complete activity of malic enzyme was found in thelower concentrated precipitation step after ammoniumsulphate fractionation (55% saturation). Its further puri-fication protocol consisted of desalting and crude gelfiltration on a Sephadex G-100 column followed byaffinity chromatography on Red Sepharose (bindingenzymes that use NADP), desalting, ion exchangechromatography and gel filtration by HPLC (Table 3). Thebinding to Red Sepharose could not be competed for with

Table 3. Purification of NADP-malic enzyme from Ectocarpus siliculosus

Total protein

(mg)

Total activity

(µmol min−")

Specific activity

(µmol mg−" min−")

Yield

(%) Factor

Crude extract 164 1±5 0±0091 100 1

0–55% (NH%)#SO

%70±4 0±6 0±011 40a 1±2

Sephadex G-200 54±8 1±0 0±018 67 2

Red Sepharose 1±8 0±49 0±27 33 30

Resource Q (HPLC) 0±048 0±094 1±97 25±5 216

Gel filtration (HPLC) 0±007 0±041 5±65 2±75 620

About 20 g of fresh weight were used for the extraction.a Activity in assay somewhat lower because of the presence of ammonium sulphate.

Fig. 2. Dependence of the activity of malic enzyme from

Ectocarpus siliculosus on the concentration of Mg#+ in the assay.

Malic enzyme was extracted from the filaments and concentrated

by precipitation with 55% ammonium sulphate saturation.

Standard errors are smaller than the symbols.

NADP in various combinations with salt, pyruvate ormalate. Therefore, the enzyme was recovered from thecolumn by a KCl gradient. In the ion exchangechromatography, most of the malic enzyme activity elutedbefore the bulk of the rest of the proteins and the activefractions from the following gel filtration produced ahomogeneous peak after re-chromatography on the samecolumn. The relative molecular mass of the native enzymewas determined to be 440 kDa. Separation of the peakfractions by SDS polyacrylamide-gels (Fig. 1) produced asingle band with a M

rof 53 kDa.

The properties of malic enzyme were determined withthe ammonium sulphate precipitate as well as with thepurified enzyme. With malate as well as with NADP,normal Michaelis–Menten kinetics were observed and thepartially purified enzyme and the pure preparation had thesame K

mvalues (Table 1). The enzyme required divalent

cations (Mg#+, Mn#+) to be active and the dependence ontheir concentration was sigmoidal (Fig. 2) with no activityat Mg#+ concentrations of 0±3 mol m−$ and lower. Thecooperativity, on the other hand, was high and concen-trations as low as 1 mol m−$ were almost fully saturating.Half-saturation was achieved with 0±7–0±8 mol m−$

MgCl#. The pH optimum (around pH 7±5) for the reaction

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66S. Busch and R. Schmid

Fig. 3. Circadian changes in the extractable activities of PEPCK

and of malic enzyme. Data from a representative experiment out

of three are shown. The algae were treated with continuous red

light of 120 µmol m−# s−" and samples were extracted at different

times and subjected to fractionated ammonium sulphate

precipitation. The activity of malic enzyme was determined in the

fraction of 0–55% saturation and that of PEPCK in the fraction of

55–80% saturation.

was sharper than that of PEPCK and the activities 0±3 pHunits before or after the maximum were only about two-thirds of those at the optimum pH. Attempts to measurethe reverse (carboxylating direction) reaction failed.

Photosynthesis of Ectocarpus siliculosus and other brownalgae of lower organization showed clear circadianrhythmicity in continuous red light, and the stimulationafter blue light pulses compensated for this rhythm(Schmid & Dring, 1992 ; Schmid et al., 1994). Therefore,both PEPCK and malic enzyme were tested for therhythmicity of their activities. In continuous red light, thetwo enzymes showed a clear circadian rhythm afterextraction, and their phase relation coincided with that ofphotosynthesis, with peaks in the middle of the pro-spective light period and troughs in the prospective night(Fig. 3). The maximal activities were about 3–4 timeshigher than the minimal activities. However, in contrast tophotosynthesis, a circadian rhythm of the enzymes wasalso detected under white light conditions, as tested byextractions at noon and at midnight (see the absence ofstimulation, below).

Assuming that the operation of a C%cycle is responsible

for the CO#import into the plastids in the presence of blue

light, enzyme activities were calculated that would allowa five-fold stimulation of the photosynthetic oxygen evol-ution found in red light (0±04 mmol O

#h−" g−" protein ;

based on the rates of oxygen evolution in red lightreported by Forster & Dring, 1994, and a determinedprotein to fresh weight ratio of 4±6³0±1 mg g−"). Theextracted activities exceeded the calculated ones in allcases (Fig. 4 for algae extracted at noon) by a factor of 4for NADP-MDH up to a factor of 40 for NAD-MDH.

Furthermore, the potential activating action of bluelight on the enzymes was tested after pre-irradiating thealgae with red light for a period of 5 h followed by anadditional 30 min of blue light. After extraction, there wasno significant difference between the activities of algae

Fig. 4. Activities of the four enzymes that could contribute to a

C%cycle in red light (RL) and in red plus blue light (BL). Plants

were irradiated from 0700 hours with red light

(120 µmol m−# s−") for 5 h and then some were irradiated with

additional blue light (12 µmol m−# s−") for 30 min. Thereafter, the

plants were extracted and the activities of the enzymes were

determined after fractionation and concentration with ammonium

sulphate. The dotted lines represent the activities required to

allow a five-fold stimulation of photosynthesis, if a functional C%

mechanism is assumed. Error bars represent standard errors

(n¯ 4).

from the red light controls and those after the blue lighttreatment (shown for the situation at noon in Fig. 4). Thiswas independent of whether the algae were extracted atthe circadian time of their peak activity during the day(Fig. 4) or of their minimal activity at night (not shown).

Discussion

Enzymes that catalyse carboxylating (PEPCK) anddecarboxylating (malic enzyme) activities were purified tohomogeneity (for problems with PEPCK see below). Thepurified preparation of NADP-malic enzyme showed onlya single band on SDS polyacrylamide gels at 53 kDa. Sincethe molecular mass of the native enzyme was 440 kDa, anoctameric organization appeared to be the most likelyconformation. Other NADP-malic enzymes from varioushigher plants consist of between four and six identicalsubunits (Edward & Andreo,1992 ; Saitou et al., 1992 ;

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Enzymes of β-carboxylation in Ectocarpus 67

Colombo et al., 1997). In maize, two isoforms exist withdifferent monomer sizes (72 kDa, 62 kDa) that are con-nected with different functions (Pupillo & Bossi, 1979 ;Scagliarini et al., 1988). Octameric NADP-malic enzymeshave not been reported from higher plants. This is alsosupported by the lower molecular mass of the monomericform of the enzyme from Ectocarpus. The role of NADP-malic enzyme in higher plants depends on its location.There are two forms : one in the cytosol, and one in thechloroplasts. In the cytosol, a participation in intracellularpH regulation has been proposed (Pupillo & Bossi, 1979 ;Edwards & Andreo, 1992), and more recent suggestionsfor internal pH regulation that include alternative pathwayoxidation (Sakano, 1998) strengthen the idea of theinvolvement of malic enzyme (and PEP carboxylase andPEPCK) in the pH-stabilizing mechanism. Malic enzymeparticipating in pH stabilization requires a pH optimum ofits activity more in the acidic pH range (cf. Sakano, 1998).With a determined optimum of pH 7±5 (Table 1) forEctocarpus its role in the pH-stat mechanism seems lesslikely. Other suggestions are that malic enzyme providesNADPH for cytosolic processes (Edwards & Huber, 1981).In CAM plants, NADP-malic enzyme occurs togetherwith the NAD-specific counterpart (Winter, 1985) and, inthe inducible CAM plant Mesembryanthemum crystallinum,the structure of the NADP-specific enzyme appears to bedifferent according to whether the plants are operating inC$

or C%

mode (Saitou et al., 1994). In NADP-malicenzyme-type C

%plants, NADP-malic enzyme is specifi-

cally located in the chloroplast and is responsible for thedecarboxylating activity in the bundle sheath cells of theseplants. Further investigations are required to establishwhether the NADP-malic enzyme in Ectocarpus is alsolocated in the plastids.

The situation was more complex for PEPCK. Afterelectrophoresis, a larger protein band (56 kDa) invariablyoccurred together with a usually intensely stained band at18 kDa. The two bands may represent subunits or thesmall peptide could have been a degradation product.However, the 18 kDa band may not have been part of theenzyme itself since the relative proportions of the bands inthe gels varied considerably, indicating the absence of astrict stoichiometry. On the other hand, the molecularmass of the native (active) enzyme (90 kDa) also couldhave resulted from the combination of one of the largerand two of the small polypeptides to form a heterotrimer.If the 18 kDa polypeptide was not part of the enzymeitself but was associated with it in a complex, the nativemolecular weight as determined by gel filtration should belower than 90 kDa and should vary with the degree ofassociation with the small polypeptide. An enzymecomplex seems to be the most likely scenario at presentbecause re-chromatography of the native enzyme byHPLC on the gel filtration column produced a peak of thenative enzyme and of smaller inactive products whosemolecular masses were in the range of the bands evidentafter electrophoresis. The 18 kDa protein seems unlikelyto have been a degradation product because it was also

found in large amounts in an active fraction that could notbe bound to the chromatofocusing column (Fig. 1, lane 5).Another argument comes from preliminary evidence(Busch & Schmid, unpublished) which indicates thatantibodies against the 18 kDa protein do not cross-reactwith the 56 kDa polypeptide. It is quite possible that thefinal purification product of PEPCK from Ectocarpus doesnot yet represent the native enzyme. In order to stabilizethe enzyme activity we had to extract at pH 8. At this pHthe enzyme from higher plants is prone to lose its N-terminal end, although this does not affect the maximalactivity (Walker & Leegood, 1995 ; Walker et al., 1997).Whether this applies to Ectocarpus too will be addressedwhen antibodies against the 56 kDa polypeptide com-ponent are available.

PEPCK from brown algae has been studied repeatedlyin the past (Akagawa et al., 1972 ; Kerby & Evans, 1983a,b ; Johnston & Raven, 1989). The molecular mass of theenzyme from Ascophyllum nodosum, as determined bygel filtration, was 60 kDa (Kerby & Evans, 1983b).In acrylamide gradient-gel separations of anotherpreparation from A. nodosum, all protein bands hadmolecular masses higher than 60 kDa ( Johnston & Raven,1989) and it was argued that the enzyme forms a higher-molecular-weight complex. Only one of the molecularmasses of proteins found in the native preparation from A.nodosum coincided with that of the final purificationproduct from Ectocarpus (the smallest protein band infigure 7 in Johnston & Raven, 1989, with an extrapolatedM

rof about 90 kDa). In higher plants, PEPCK seems to be

a monomer of 74 kDa in gluconeogenetic and most CAMplants and of 67–71 kDa in C

%grasses (Walker & Leegood,

1996 ; Walker et al., 1997). In the former, it contains an N-terminal sequence of 12 kDa that seems to be involved inits light-dependent regulation via phosphorylation}dephosphorylation (Walker et al., 1997). The N-terminalend of the enzyme from C

%plants is rapidly cleaved during

isolation (Walker et al., 1997). None of these featuresseems to match the characteristics of PEPCK fromEctocarpus.

Since the activity of PEPCK in the carboxylatingdirection is roughly 10 times greater than that in thedecarboxylating direction, the function of the enzyme in

vivo seems to be to provide oxaloacetate. However, as theenzyme operates in both directions, involvement inintracellular pH stabilization may be another function. Theenzyme from Ascophyllum nodosum appears to be activatedby the presence of Mg#+ and substrate ( Johnston &Raven, 1989). This was not observed with PEPCK fromEctocarpus (data not shown). But, as a rule, the buffers weused for extraction and for purification always containedMg#+ ions. Furthermore, in our purification protocols,destabilization of PEPCK regularly occurred after it wasbound to a column. Therefore, we suggest that thefunction of Mg#+ is to stabilize the enzyme by enablingassociation with a protein factor that is required for itsactivity (e.g. the 18 kDa protein?). Re-introduction ofMg#+ after its removal during a preparation would lead to

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68S. Busch and R. Schmid

reconstitution and the presence of substrates wouldmodify the speed of this reconstitution.

Carbon acquisition in Ectocarpus has been suggested tooperate via an intracellular C

%mechanism similar to that

reported for Udotea (Reiskind et al., 1989 ; Reiskind &Bowes, 1991) and the decarboxylating enzyme com-ponent was expected to require blue light for its activation(Schmid & Dring, 1996a, b). It is evident that the operationof such a metabolic pathway requires the presence of acomplete set of enzymes. In agreement with other authors(Akagawa et al., 1972 ; Kremer & Ku$ ppers, 1977 ; Kremer,1981b ; Johnston, 1991), there is no evidence for theexistence of a PEP carboxylase, and functions that areassociated with its operation in other plants have to becarried out by PEPCK. This includes its suggested role inthe C

%cycle. However, our measurements indicate that

not all the enzymes required were present in Ectocarpus

and, because of the apparent absence of pyruvate-Pi-dikinase, which would be needed to regenerate PEP frompyruvate, the operation of a C

%pathway becomes unlikely.

There are more arguments against the presence of a C%

mechanism in brown algae (reviewed by Johnston, 1991 ;see also Kremer & Ku$ ppers, 1977). They are based onobservations on phaeophytes of higher organization(mainly fucoids) and their validity has been questionedbecause their morphological characteristics had not beentaken into account (see Schmid & Dring, 1996a ; Schmid et

al., 1996). However, a major argument against theoperation of a C

%pathway in brown algae had been the

low affinity of PEPCK for CO#. To allow efficient CO

#

fixation in such a pathway the Km

should be in the rangeof that of Rubisco. The K

mfor CO

#of PEPCK of Ectocarpus

(4±6 mol m−$) is far too high (even higher than determinedfor other phaeophytes so far ; cf. Johnston, 1991) tocompete with Rubisco (K

mfor other brown algae is

between 12 and 60mmolm−$ ; Johnston, 1991) for theavailable CO

#. However, a K

mdetermined under physio-

logical conditions rather than under optimal conditionsmay reveal a more realistic picture of the properties of theenzyme.

The results of these investigations taken alone do notnecessarily disprove the presence of a C

%cycle in

Ectocarpus, because the evidence is circumstantial. Ourinability to detect pyruvate-Pi-dikinase activity does notmean that the enzyme never occurs in this species, and theabsence of blue light effects on the activity of the extracted(and concentrated) enzymes does not mean that suchregulation does not occur in vivo. Our original suggestion(Schmid & Dring, 1996a, b) was that the decarboxylatingactivity would be blue-light-activated. The kinetic datasuggest that PEPCK operates in the carboxylating ratherthan the decarboxylating direction (see above) and thatmalic enzyme has only decarboxylating activity. Thissuggests that the regulated enzyme should be malicenzyme and not PEPCK. Like the other enzymes, malicenzyme did not show differences in its extractableactivities. However, one possible mechanism for regu-lating malic enzyme in vivo would be via the concentration

of divalent cations such as Mg#+. If Mg#+ concentrationswere directly or indirectly regulated by blue light, theactivity of the enzyme could be changed from zero to100% merely by raising the Mg#+ concentrations from 0±3to 1 mol m−$ (see Fig. 2). There is an absolute requirementfor Mg#+ ions in the stroma of isolated chloroplasts tooperate the reductive pentose phosphate cycle (Heldt,1979), and this has been used to suggest that Mg#+ mightbe a means to regulate the activity of this cycle. A similarhypothesis could be constructed for the operation of malicenzyme in Ectocarpus.

Another argument against an operational C%mechanism

is that all measured enzyme activities were far higher thanthose needed to drive the pathway in red plus blue light,and also in red light alone. Regulation of the cycle by bluelight, as suggested, would have required rate-limitingactivities of at least malic enzyme in red light, whenphotosynthesis is carbon-limited. However, again, themeasured activities do not necessarily represent theactivities of the same enzymes in vivo, and ionic conditions(including pH) as well as substrate concentrations could befar less favourable than those providing maximum activityin the assays.

In essence, the investigations of the enzymes alone donot indicate conclusively whether a C

%metabolic cycle is

active in Ectocarpus, although clear evidence against itsexistence comes from investigations at the metabolitelevel. But given the absence of a C

%syndrome, we face

new problems connected with the function of PEPCK andmalic enzyme. Whereas no immediate function is evidentfor malic enzyme in brown algae, different roles have beensuggested for PEPCK. In laminarians, its main role in thedark was proposed to be associated with the mobilizationof mannitol (Kremer, 1981a). However, if its main functionwere associated with its activity at night in Ectocarpus,the circadian pattern which shows highest activities atnoon (Fig. 3) would be hard to explain. Furthermore,mobilization of mannitol is a seasonal activity (Davison &Reed, 1985) in the laminarians, and it is doubtful whethersuch a seasonality could be relevant for Ectocarpus wherethe storage capacity is likely to be low and where it isdoubtful whether the growing tips rely on transportprocesses out of the mature parts of the filaments. AsPEPCK appears to constitute the only β-carboxylatingenzyme in the Phaeophyceae (Table 1 ; Akagawa et al.,1972 ; Kremer & Ku$ ppers, 1977 ; Kremer, 1981b for areview), at least one of its functions has to be anaplerotic,in compensating for the loss of metabolites from the TCAcycle. Participation, in combination with malic enzyme, inpH stabilization in the cytosol seems possible, but thefunction of such a mechanism (see review by Sakano,1998) would require that the pH optimum of malicenzyme should be more acidic, as pointed out above. Inthe presence of PEPCK, both PEPCK and malic enzymerequire strict regulation of their activities to prevent ametabolic loop occurring, which would bypass pyruvatekinase in the glycolytic pathway (Plaxton, 1996) – unlessthe operation of the bypass is needed for pH regulation

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Enzymes of β-carboxylation in Ectocarpus 69

(Sakano, 1998). Clearly, further characterization of bothmalic enzyme and PEPCK is required to establish theirfunction in the filamentous brown alga Ectocarpus siliculosus

as well as in brown algae of higher organization.

Acknowledgements

This work was supported by a grant from the DeutscheForschungsgemeinschaft (Schm 557}3). We are indebtedto Professor Paul Galland for his moral support and forproviding laboratory facilities. We also thank ProfessorM.J. Dring for his detailed criticism and help with themanuscript and Dr Gareth Pearson for freezing some ofthe samples at night, while we were slumbering, partlyworried (S.B.) and partly peacefully trusting (R.S.), athome.

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