identification and characterization of glycolate oxidase and related

Plant Physiol. (1992) 98, 887-8930032-0889/92/98/0887/07/$01.00/0

Received for publication June 24, 1991Accepted August 30, 1991

Identification and Characterization of GlycolateOxidase and Related Enzymes from the EndocyanoticAlga Cyanophora paradoxa and from Pea Leaves1

Thomas Betsche*, Dietmar Schaller, and Michael MelkonianD6partement de Physiologie Vegetale et Ecosystemes, Centre de Cadarache, F 13108 Saint Paul lez Durance,

France (T.B.); Botanisches Institut der Universitat Munster, Schlossgarten 3, D 4400 Monster, Germany (D.S.); andBotanisches Institut der Universitat Koin, Gyrhofstr. 15, D 5000 KoIn, Germany (M.M.)

ABSTRACT

Glycolate oxidase (GO) has been identified in the endocyanomCyanophora paradoxa which has peroxisome-like organelles andcyanelles instead of chloroplasts. The enzyme used or formedequimolar amounts of 02 or H202 and glyoxylate, respectively.Aerobically, the enzyme did not reduce the artificial electronacceptor dichlorophenol indophenol. However, after an inhibitorof glycolate dehydrogenase, KCN (2 millimolar), was added tothe assay medium, considerable aerobic glycolate:dichlorophenolindophenol reductase activity was detectable. The leaf GO inhib-itor 2-hydroxybutynoate (30 micromolar), which binds irreversiblyto the flavin moiety of the active site of leaf GO, inhibited Cyano-phora GO and pea (Pisum sativum L.) GO to the same extent.This suggests that the active sites of both enzymes are similar.Cyanophora GO and pea GO cannot oxidize D-lactate. In contrastto GO from pea or other organisms, the affinity of CyanophoraGO for L-lactate is very low (Km 25 millimolar). Another importantdifference is that Cyanophora GO produced sigmoidal kineticswith 02 as varied substrate, whereas pea GO produced normalMichaelis-Menten kinetics. It is concluded that there is consider-able inhomogeneity among the glycolate-oxidizing enzymes fromCyanophora, pea, and other organisms. The specific catalaseactivity in Cyanophora was only one-tenth of that in leaves. NADH-and NADPH-dependent hydroxypyruvate reductase (HPR) andglyoxylate reductase activities were detected in Cyanophora.NADH-HPR was markedly inhibited by hydroxypyruvate above 0.5millimolar. Variable substrate inhibition was observed with gly-oxylate in homogenates from different algal cultures. It is pro-posed that Cyanophora has multiple forms of HPR and glyoxylatereductase, but no enzyme clearly resembling leaf peroxisomalHPR was identified in these homogenates. Moreover, noserine:glyoxylate aminotransferase activity was detected. Theseresults collectively indicate the possibility that the glycolate me-tabolism in Cyanophora deviates from that in leaves.

Cyanophora paradoxa is an obligate photoautotrophic, un-icellular organism (alga) that has cyanelles instead of chloro-plasts (22). Cyanelles are morphologically similar to cyano-bacteria, and it was therefore proposed that Cyanophora

A substantial portion of this work was done in Munster whenT.B was Priv.-Doz. at the Botanical Institute.

887

would constitute an endosymbiosis. This proposal has beensupported by the findings that, in contrast to leafchloroplasts,cyanelles have a rudimentary cell wall that is composed ofseveral layers and contains peptidoglucans (1). Convincingevidence is now manifold, however, that cyanelles are organ-elles rather than endosymbiotic prokaryotes. Cyanelles haveno respiratory electron transport chain (30), and the size ofthe genome of cyanelles is 5 to 10% of that of free-livingcyanobacteria. This is about the genome size of chloroplasts(7). Nevertheless, there are important differences betweenchloroplasts and cyanelles. For example, the cyanelle genomecodes for both Rubisco subunits (18), whereas the chloroplastgenome encodes only for the large subunit. Moreover, cy-anelles have other transport mechanisms than chloroplastsand probably export glucose instead of triose phosphates (30).

Mitochondria and microbodies (peroxisome-like organ-elles) attached to the cyanelles were observed by EM inCyanophora (28). In leaf cells, a similar spatial relationshipexists between peroxisomes and chloroplasts which is thoughtto facilitate the exchange of photorespiratory metabolites.Because Cyanophora has ribulose bisphosphate carboxylasebut no C02 concentration mechanism (A. Goyal and N.E.Tolbert, personal communication), photorespiration is likelyto occur. To date, however, there is no information about thephotorespiratory pathway in this particular organism. Mostunicellular eukaryotic algae, and possibly all cyanobacteria,oxidize glycolate by GD2. This enzyme is associated withmitochondrial membranes or the lamellae (10, 27). In Char-ophyceae and leaves, glycolate is oxidized by peroxisomal GO(15, 20, 32), and recently, GO was reported to occur in otheralgal groups also (16, 17, 31). GO has been distinguished fromGD on the basis of the following criteria: (a) GD is inhibitedby CN- (2 mM), (b) GD can convert D-lactate; whereas GO isnot inhibited by CN- and can convert L-lactate (15); (c)isolated GO can directly use 02 as an electron acceptor andforms H202; whereas GD requires an intermediate electronacceptor (15, 27) and does not produce H202; (d) GD in cell

2 Abbreviations: GD, glycolate dehydrogenase; GO, glycolate oxi-dase; DCPIP, 2,6-dichlorophenol indophenol; HBA, 2-hydroxybutyn-oic acid; HPMS, 2-pyridil-hydroxymethanesulfonic acid; GAPDH,glyceraldehyde phosphate dehydrogenase.

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homogenates can readily reduce the artificial electron acceptorDCPIP in the presence of 02; whereas the DCPIP reductaseactivity of isolated GO is inhibited by 02 (13, 15).

It has been shown, however, that the inhibitory effect of02on the DCPIP reductase activity ofGO is variable and speciesdependent (4, 8, 13, 15). Moreover, a serological study sug-gested some immunological similarity between GO and GD(9). Nevertheless, the above-mentioned criteria taken togetherallow us to distinguish two basic groups of glycolate-oxidizingenzymes, GO and GD, and phylogenetic significance has beenattributed to the distribution of GO and GD in plants andalgae (8, 13-15, 29, 32).

In view of the particular intracellular organization of Cy-anophora having cyanobacteria-resembling chloroplasts andperoxisome-like organelles, we examined this organism forthe presence of photorespiratory (leaf peroxisomal) enzymeswith emphasis on the identification and characterization ofthe enzyme for glycolate oxidation.

MATERIALS AND METHODS

Plant Materials

Cyanophora paradoxa Korshikov (strain 29.80, algae col-lection, Gottingen, Germany) was cultivated in a modifiedWaris culture medium (22) at 14C and 50 ,uE m-2 s-' PPFDand a light/dark regimen of 14 h/10 h, respectively. Thegrowth medium was continuously aerated. The algae wereharvested when the cell density was 5 x I07 cells mL-'. Pisumsativum L. seeds (pea dwarf variety Kleine Rheinlanderin)were soaked in water overnight and sown in vermiculite.Growth conditions were: 20°C, 250 AE m-2 s-' PPFD, 14 h/10 h light/dark period (respectively), and 70% RH. Commer-cial fertilizer was used.

Enzyme Extraction

The pellet obtained by centrifugation (300g, 5 min) of algalsuspension (between 150 mL and 3 L) was resuspended inwater and centrifuged at 300g for 5 min. The followingextensive differential extraction procedure was used to removesoluble compounds and to solubilize possible membrane-associated glycolate-oxidizing enzymes. Moreover, the specificactivity ofGO after differential extraction was higher than ina simple homogenate (see below for definition). The algalpellet was resuspended in 50 mm Mops (10 mL L' algasuspension), pH 6.0, and the cells were disrupted by sonica-tion (20 W, 90 s, vial cooled in ice/water). The supernatantobtained by centrifugation at 26,000g for 15 min (4°C) con-tained little GO and no glycolate:DCPIP reductase activity.This step was repeated, and the pellet was resuspended in asmall volume of200 mm Tris/HCl, pH 8.2, with 100 mm KCI(5 mL L` original algal suspension) and centrifuged at26,000g for 15 min (4°C). The resulting supernatant (denoted"alkaline fraction") was used to characterize GO. No substan-tial GO activity and no GD activity (aerobic glycolate:DCPIPreductase activity was measured) was eluted from the pelletby buffers containing up to 1 M KCI or 0.1% (v/v) Triton X-100. The resuspended pellet itself (without centrifugation andbefore treatment) was also enzymatically inactive.To extract other enzymes (and GO), water-washed algae

(see above) were resuspended in 200 mm Tris/HCl, pH 8.2,with 100 mm KC1, then sonicated, and centrifuged at 26,000gfor 15 min (4C). The supernatant was denoted "homogenate"and used for enzyme determinations.To isolate GO from pea, 2 g of leaves was ground in a

mortar with sand in 10 mL of 50 mm Mops, pH 6.0. Thefurther differential extraction was done as described for Cy-anophora after sonication. To extract other enzymes (andGO), pea leaves were ground with sand in 200 mm Tris/HCl,pH 8.2, with 100 mm KCI, and homogenate was prepared asdescribed in the preceding paragraph.

Purification of GO

GO was purified from pea leaves and Cyanophora by amethod (6) similar to that described by Kerr and Groves (21).All steps were carried out in the cold. A differential extractionstep was introduced in our method (see "Enzyme Extrac-tion"). A 20-mL aliquot of the alkaline fraction was desaltedby Sephadex G-25 (30- x 1.5-cm column) equilibrated in 50mM Tris/HCl, pH 8.5. The peak fractions were pooled andsubjected to anion exchange chromatography using SephadexQAE-50 (30- x 3.5-cm column) equilibrated in 50 mm Tris/HCI, pH 8.5. GO did not bind and was eluted using the samemedium. The peak fractions were subjected to cation ex-change chromatography using Sephadex CM-50 equilibratedin 50 mM Bicine/Na+, pH 8.2 (30- x 1.0-cm column). Elutionwas done by an exponential salt gradient (0-700 mM KCIover 300 mL of 50 mM Bicine/Na+, pH 8.2). The peakfractions were pooled.

Enzyme Determinations

The standard assay medium was 50 mM Tris/HCl, pH 8.2,and 5 mM glycolate. To assay GO, the phenylhydrazinemethod which detects the glyoxylate formed (4) was usedunless otherwise noted. Glycolate-dependent DCPIP reduc-tion in the presence of 02 (considered as GD activity) wasdetermined spectrophotometrically with 0.14 mm DCPIP inthe above medium without phenylhydrazine. Enzyme-cata-lyzed H202 formation was determined by a coupled assayusing commercial peroxidase with aminoantipyrine as a sub-strate (19). Enzyme-catalyzed 02 consumption, with glycolateand L-lactate or D-lactate as substrates, was determined by an02 electrode in 50 mm Tris/HCl, pH 8.2, without phenylhy-drazine. To record enzyme kinetics with 02 as varied sub-strate, air-saturated and N2-bubbled buffer (50 mm Tris/HCl,pH 8.2) was mixed, and the resulting O2 concentration andglycolate-dependent 02 consumption by enzyme catalysiswere measured with an O2 electrode.

Catalase was determined spectrophotometrically at A230 in50 mM phosphate/K+, pH 7.0, with 13.4 mM H202.NAD(P)H-glyoxylate reductase activities and hydroxypy-

ruvate reductase activities were determined spectrophotomet-rically in 50 mM phosphate/K+, pH 6.4, at various substrateconcentrations (20).GAPDH dehydrogenase was determined as described in

ref. 3.NAD-lactate dehydrogenase was assayed following NADH

oxidation (reverse reaction) with pyruvate as a substrate (5).

888 BETSCHE ET AL.



GLYCOLATE OXIDASE IN CYANOPHORA

Serine:glyoxylate aminotransferase was estimated by a cou-

pled spectrophotometric assay (20) using NADH-hydroxypy-ruvate reductase which reduces the hydroxypyruvate formedfrom serine. A high-affinity glyoxylate reductase in Cyano-phora (see "Results") produced considerable background(NADH oxidation), and no stimulation by serine of NADHoxidation was observed. To assess the reliability of the assay

method for Cyanophora, a control experiment with a mixtureof Cyanophora and leaf extracts was conducted. Serine:gly-oxylate aminotransferase activity was detected in the mixture.

RESULTS AND DISCUSSION

Identification of GO

To probe Cyanophora for the presence of GD, glycolate-dependent DCPIP reduction was determined after extractionwith media of different pHs and stringencies (wide range ofconcentrations of KCI and Triton X-100). Glycolate:DCPIPreductase activity was not detected aerobically in any extract.By contrast, rapid glycolate-dependent O2 uptake was ob-served in homogenate prepared with alkaline medium (pH8.2), and equimolar amounts of H202 and glyoxylate were

formed by the alkaline fraction (see "Materials and Meth-ods"). Thus, the enzymes fulfills the requirements for classi-fication as GO.Concerning the intracellular localization of Cyanophora

GO, it was observed that GO activity did not increase, andspecific GO activity declined, in homogenates after extensivesonication (300 s instead of 60-120 s) which physically dam-aged the cyanelles. This suggests that GO is located in thecytosol (eukaryotic portion) of Cyanophora.

Purification of Cyanophora GO was attempted using a

method developed for pea GO. This method includes an

acidic extraction step, and it was observed that both pea GO(6) and Cyanophora GO are insoluble when acidic extractionmedium of low ionic strength was used. GOs from Cyano-phora or pea were found to be soluble in alkaline medium ofhigh ionic strength (Table I). The extracts were desalted, andaliquots were subjected to ion exchange chromatography.Neither GO bound to QAE-cellulose (anion exchanger) at pH8.5, whereas some other protein was retained. This suggeststhat both Cyanophora GO and pea GO are highly alkalineproteins (isoelectric point, 9.6 for pea GO; ref. 21 and our

own observation). LeafGO was bound by, and could then be

eluted from, the following CM-Sephadex column (cation ex-

changer). By contrast, no active Cyanophora GO was elutedfrom this column, or the enzyme was bound irreversibly(Table I). This suggests that there is some substantial differ-ence between Cyanophora and pea GO.The QAE chromatography step resulted in only a fivefold

enrichment and considerable loss ofCyanophora GO. Becausesoluble small molecular weight compounds, which can influ-ence enzyme kinetics, were already removed by the acidicextraction step (see "Materials and Methods"), the alkalinefraction was used in many experiments.

First, the effects of GD and GO inhibitors on the Cyano-phora enzyme were determined. The GD inhibitor CN- (2mM) stimulated glycolate-dependent O2 uptake, and this effectdecreased during purification (results not shown). This can

easily be explained by the inhibition of contaminant catalasewhich would dissipate H202 produced by GO. The effect ofKCN on glyoxylate formation could not be determined be-cause CN- reacts with glyoxylate. Commercial catalase (noCN-) added to the assay medium inhibited O2 uptake (resultsnot shown). Glycolate DCPIP reductase activity was notdetected aerobically in any fraction (no catalase added). Afteraddition of CN- (2 mM), however, considerable glycolateDCPIP reductase activity was observed in all fractions. Similarresults were obtained with pea GO (4). The latter experimentsshowed that inhibition by CN- of contaminant peroxidase,which could possibly reoxidize reduced DCPIP using enzyme-formed H202, cannot fully explain the stimulation by CN- ofglycolate-dependent DCPIP reduction (ref. 4; T. Betsche, D.Schaller, and M. Melkonian, unpublished results). HBA (30,uM) and HPMS (5 ,uM) both similarly inhibited GO fromCyanophora and pea in all fractions. In the alkaline fraction,inhibition by HBA was 86% when glyoxylate formation was

measured and 100% when 02 uptake was measured. Inhibi-tion by HBA of glycolate DCPIP reductase activity (with 2mM CN-) was 82%. HPMS inhibited glycolate-dependent 02

uptake by 55%. No values for the effect of HPMS on glyox-ylate formation by GO and glycolate DCPIP reductase activityare available because HPMS interfered with both assays. Themode of action of HBA and HPMS as inhibitors of theflavoproteins leafGO and bacterial lactate oxidase is different(12). HBA is a 2-hydroxy acid substrate analog and suicidesubstrate that binds to the active site of leafGO having flavinmononucleotide as a prosthetic group (12). In contrast, HPMS

Table I. Extraction and Purification of GO from C. paradoxa and P. sativum

Activities were determined aerobically by the phenylhydrazine method which detects the glyoxylateformed. The alkaline fraction was set as reference to calculate purification and yield. Note that thedifferential extraction itself produced a threefold enrichment of GO with homogenate as reference (seeTable ll). Values in parentheses are for P. sativum.

Fraction Activity Specific activity Purification Yield

nkat nkat mg-' relative units %

Acidic fraction, pH 6.0 0.01 0.01 (0.01) 1 (5)Alkaline fraction, pH 8.0; 0.5 M KCI 1.22 1.51 (0.45) 1.0 (1.0) 100 (100)Anion exchange chromatography 0.38 1.90 (3.80) 4.8 (8.4) 32 (22)(Sephadex QAE-50)

Cation exchange chromatography <0.003 (25.7) (57) <0.3 (19)(Sephadex CM-50)

889




(11) forms a inhibitory ketoacid adduct. The strong andsimilar inhibitions by HBA and HPMS of Cyanophora GOand pea GO suggest that their active sites are similar.

Enzyme Kinetics

pH Dependency

The pH optimum for glyoxylate formation (determined asglyoxylate phenylhydrazone using the alkaline fraction) byCyanophora GO was observed at pH 8.2 or 7.9 in Tris/Cl orTes/K+ buffer, respectively (Fig. 1, Tes not shown). SimilarpH dependency was observed with pea GO (results notshown). The activity of Cyanophora GO declined rapidly inthe acidic range, and a second weak optimum of variableheight was observed at pH 6.6 in phosphate buffer with sixenzyme preparations. This optimum in the acidic pH rangewas also observed with some preparations (alkaline fractions)from pea leaves. The significance of the acidic pH optimumis unclear. Mops strongly inhibited Cyanophora GO at acidicand alkaline pHs (Fig. 1), whereas Mes and Tes inhibited little(results not shown). These buffers are sulfonic acid derivativesand share this structure with a number of known potentinhibitors of GO, e.g. HPMS (11). The pH optimum ofglycolate-dependent 02 uptake was at pH 9.0 (Tris/Cl1) andthe curve was flatter than that observed for glyoxylate for-mation (2 mM CN- was added to avoid interference bycatalase). This was also seen with pea GO (T. Betsche, D.Schaller, and M. Melkonian, unpublished results). These re-sults suggest that the ratio of glyoxylate formation to 02uptake is pH dependent. However, a thorough substrate-product analysis at different pH values is required to substan-tiate this proposal.

100

80

4-

0 60

5<0._

u 40_.c0>1

""A

uI *6 7 8 9 I 11

pHI

Figure 1. pH dependency of GO from C. paradoxa in different buffers.The alkaline fraction was used in these measurements. Assay con-ditions: glyoxylate formation was determined by the phenylhydrazineassay with 5 mm glycolate in air-saturated Tris/HCI (50 mM; *),phosphate/K+ (50 mM; O), and Mops/Na+ (50 mM; U). Oxygen uptakewas determined with 2 mm KCN in air-saturated Tris/HCI (50 mM; A)and phosphate/K+ (50 mM; A).

Table II. Affinity for Different Substrates of GO Isolated fromCyanophora and from Pea Leaves

Partially purfied enzyme (the alkaline fraction; see "Materials andMethods") was used in these aerobic measurements without DCPIP

Km or Ks)o.5) values (*)

C. paradoxa P. sativum

Mm

Glycolate (glyoxylate formation) 266 220Glycolate(02 uptake) 267 236L-Lactate (02 uptake) 25,000 3,2008Oxygen, with glycolate (3 mM) 1398 571

(02 uptake)Oxygen, with L-lactate (15 mM) 571a 284

(02 uptake)a Sigmoidal kinetics were observed and Ks(O.5) values are presented.

Substrate Affinity

Kinetics were recorded using the alkaline fraction preparedfrom Cyanophora and, for comparisons, from pea leaves.Both GOs were found to have high and similar affinity forglycolate (Table II). No activity was detected with D-lactatewith either enzyme. GOs isolated from leaves (e.g. ref. 15;Table II) or green algae (8, 15, 31), can use L-lactate as asubstrate. In contrast, the affinity of Cyanophora GO for L-lactate is very low (Table II). With Cyanophora GO, sigmoidalkinetics were observed for oxygen as varied substrate, and aHill number of 3.8 was calculated with glycolate as 2-hydroxyacid substrate (Fig. 2). Thus, the kinetic behavior of Cyano-phora GO toward 02 corresponds to that of allostericallyregulated enzymes. However, mixtures of enzymes havingsimilar substrate specificity can also produce a sigmoidalkinetic response. Normal Michaelis-Menten kinetics wererecorded with glycolate as varied substrate. This means thatsuch isozymes, if present in Cyanophora, would producenormal and identical Michaelis-Menten kinetics with glyco-late as a varied substrate but different kinetics with oxygen asvaried substrate. To date, however, there have been only tworeports that two glycolate-oxidizing enzymes, GO and GD,would occur together in the same organism (25, 26). Thus, itis more probable that Cyanophora has only one enzyme thatcan bind oxygen cooperatively.The finding that there are important differences in the

kinetic properties ofGOs from Cyanophora, other algae (15-17, 31), and leaves (ref. 21 and this paper) and that, like GD,GOs can reduce DCPIP aerobically at variable and often highrates (depending on the species and isolation or assay condi-tions; refs. 4, 8, and 14 and this paper) suggest that thegenerally accepted classification system, GO or GD, under-estimates the inhomogeneity among glycolate-oxidizing en-zymes from different organisms.

Other Photorespiratory Enzymes

During photorespiration in leaves, GO produces largeamounts of glyoxylate and H202 in the peroxisomes. Catalasedecomposes the H202, and peroxisomal aminotransferasessuch as serine:glyoxylate aminotransferase convert most of

890 BETSCHE ET AL.

4




0 50 100 150

1/ (02)4 ((L tiinol 10-10)-4)

20'

(kinetics not shown, see Table III for values). In anotherpreparation from a different algal culture, a second maximumat 7 mm glyoxylate and moderate substrate inhibition abovethis concentration were observed with NADH as the cosub-strate (Fig. 3B). It is possible to suggest based on these obser-vations that multiple forms ofhydroxypyruvate reductase andglyoxylate reductase are present in Cyanophora. One enzyme,

or type of enzymes, is inhibited by rather low substrateconcentrations. Cyanophora possibly has also a NADPH-hydroxypyruvate reductase that has a very high affinity forhydroxypyruvate and is insensitive to substrate inhibition(Fig. 3A) and a glyoxylate reductase that requires rather highglyoxylate concentrations (millimolar range) and prefersNADH (Fig. 3B). It was not possible to identify, withoutpurification, an enzyme closely resembling leaf peroxisomalhydroxypyruvate reductase that preferentially uses NADH,has a low affinity for glyoxylate (Km 5-20 mM for glyoxylate;Tolbert, Yamazaki, and Oeser 1970, see ref. 20), and is notinhibited by high glyoxylate concentrations. The resultsshown in Figure 3 suggest, however, that Cyanophora has an

enzyme similar to the recently identified cytoplasmicNADPH/NADH hydroxypyruvate reductase in spinachleaves (23). This leaf enzyme is also prone to substrate inhi-bition and is more active with hydroxypyruvate than withglyoxylate (see Fig. 3 for comparisons). The function of con-verting hydroxypyruvate leaked from the peroxisomes to glyc-erate has been attributed to this hydroxypyruvate reductase(23).Cyanidium caldarium can also be considered as a cyanome

and GO was identified in this alga. The catalytic properties ofCyanidium toward hydroxypyruvate and glyoxylate are, how-ever, clearly different from those of Cyanophora (16).No serine:glyoxylate aminotransferase was detected in Cy-

0O

Figure 2. Kinetics of GO from C. paradoxa with oxygen as variedsubstrate. Inset, Hill plot. The alkaline fraction was used in thesemeasurements. Assay conditions: glyoxylate formation was deter-mined by the phenylhydrazine assay in 50 mm Tris/HCI, pH 8.2, with5 mM glycolate.

the glyoxylate to glycine. Finally, glycerate is formed fromhydroxypyruvate (for a complete description of the photores-piratory cycles, see ref. 20). In spite of the abundance ofcatalase in leaf peroxisomes, the steady-state H202 concentra-tion is high enough to allow the formation of some formateand CO2 by nonenzymatic reaction of H202 with glyoxylate(for review, see ref. 20). In Cyanophora, the specific activityof catalase is one order ofmagnitude lower than in pea leaves,whereas the specific GO activities and the ratios of GO toNADPH-GAPDH dehydrogenase, a marker enzyme of pho-tosynthetic CO2 fixation, are similar (Table III). BecauseCyanophora has no CO2 concentration mechanism (A. Goyaland N.E. Tolbert, personal communication), it is probablethat the turnover of glycolate and, thus, the rate of H202formation (relative to the rate of photosynthetic CO2 fixation)are high in Cyanophora. One possible explanation for the lowcatalase activity is that the rate of H202 formation by GO insitu can be less than expected from the results of experimentswith isolated enzyme. This hypothesis is supported by thefindings that (a) leaf peroxisomes can rapidly catalyze glyco-late-dependent DCPIP reduction aerobically, whereas isolatedGO or purified GO cannot (ref. 4, this paper, and T. Betsche,D. Schaller, and M. Melkonian, unpublished results), and (b)natural phenolic compounds can serve as electron acceptorsfor leafGO (24).Hydroxypyruvate reductase and glyoxylate reductase were

detected in Cyanophora (Table III). Unlike leaf peroxisomalNADH-hydroxypyruvate reductase, the Cyanophora enzymewas markedly inhibited by hydroxypyruvate concentrationsabove 0.5 mM with NADH as a cosubstrate (Fig. 3A), whereassubstrate inhibition was weak with NADPH. In one prepara-tion, glyoxylate reductase was markedly inhibited by glyox-ylate above 0.4 mM with NADPH and NADH as cosubstrates

Table Ill. Enzymes of Photorespiration and Photosynthesis inHomogenates from Cyanophora and from Pea Leaves

Note that the acidic extraction step (see Table I and "Materials andMethods") was omitted in these experiments. For Cyanophora, someof the results obtained with two preparations from different algaecultures are presented separately. The preparation marked with anasterisk was used in Figure 3. The other values are averages frommeasurements with two preparations.

Specific Activity

C. paradoxa P. sativum

nkat mg-'GO (glyoxylate formation) 0.26 0.54Catalase 442 5800Serine: glyoxylate aminotransferase Not detected 1.5Glyoxylate reductase and hydroxypyruvate

reductase activities determined with0.22 mm NADH or NADPH and:

75 mm glyoxylate 0.28 3.42with NADH 0.74*0.5 mM glyoxylate 1.60/1.83 1.28/2.65with NADH/NADPH 1.07*/1.24*0.5 mM hydroxypyruvate 2.24*/1.59* 12.3/0.54with NADH/NADPHGAPDH-NADP 3.94 9.05

891




1-1

0.

._'.

Om0.2

0.1

4._

0

**0.4 0.6 0.8Hydroxypyruvate (mM)

NADH- Glyoxylate reductase

NADPH- Glyoxylate reductase o

0.2I 1/e

0.4 0.6Glyoxylate (mM)

Figure 3. NADH- and NADPH-dependent hydroxyFductase and glyoxylate reductase (B) activities at v

concentrations in homogenate from C. paradoxa. As50 mM phosphate/K+, pH 6.4, with 0.22 mm NADH o

anophora (see remarks in "Materials and Methbe worth noting in this context that, in free-liviteria, some glyoxylate is probably metabolized Iinstead of aminotransferase (2). In this light,results obtained with Cyanophora indicate the r

the cyanelles of Cyanophora have carboligase t(Like cyanobacteria (32), Cyanophora has no

dehydrogenase, and it was observed that Cyanessentially inactive with both D- and L-lactatethat no lactate shuttle is possible between lactatase (acting as pyruvate reductase) and GO (acoxidase) as has been suggested to occur in otl(32). It is important to mention in this resoccurrence ofGD or GO in an organism appearcoupled with the occurrence ofeither D-lactate dor L-lactate dehydrogenase, respectively (5, 32).

Remarks Concerning Evolution

The distribution of GO or GD among difgenerally regarded as a good phylogenetic ma

alga and higher plants (for review, see ref. 29). The Charophy-uctase - ceae share with higher plants, among other various structuralductase - and biochemical properties, the presence of GO. It appears

that all other unicellular green algae contain GD instead (8,13-15, 25). Because seven species of the Prasinophyceae as

A the presumptive ancestral group of green plants also haveGD, it has been concluded that the higher plant type GO (andpossibly the peroxisomes [32]) evolved later in evolution afterseparation of the Charophyceae from other green algae (13).Recently, however, GO was found in Cyanophora paradoxa,Bumilleriopsis filiformis (Tribophycaceae [ 17]), and Cyani-dium caldarium (Rhodophyta [16]). The basic kinetic prop-erties ofthese three algae and higher plants (leaves) are similar,although several marked differences were encountered (this

1 3 paper). The question of whether the GOs found in differentorganisms are homologous proteins can be addressed onlywhen nucleotide and/or amino acid sequence informationbecomes available.

BACKNOWLEDGMENTS

K. Heimann provided the algae for the work done during a researchvisit of T.B. in Koin. This is gratefully acknowleged. We thank G.Kreimer and A. Vermeglio for suggestions regarding the manuscript.

LITERATURE CITED

--0 1. Aitken A, Stanier RY (1979) Characterization of perptidoglucanfrom the cyanelles of Cyanophora paradoxa. J Gen Microbiol112: 219-223

2. Bergman B, Codd GA, Hallbom L (1984) Glycollate excretionby N2-fixing cyanobacteria treated with photorespiratory inhib-

10 30 itors. Z Pflanzenphysiol 113: 451-4603. Bergmeyer HU, Gawehn KK, Grassl M (1974) Enzyme als

biochemische Reagenzien. In HU Bergmeyer ed, Methodender enzymatischen Analyse. Verlag Chemie, Weinheim/Bergs-

)yruvate (A) re- trasse, pp 454-558faried substrate 4. Betsche T (1986) Glycolate:dichlorophenol indophenol reductase;say conditions: activity of glycolate oxidase in the presence of oxygen: experi-)r NADPH. ments with crude enzyme, purified enzyme, and peroxisomes

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