Planta (1986)167:119-128

P l a n t a 9 Springer-Verlag 1986

Enzymes of serine and glycine metabolism in leaves and non-photosynthetic tissues of Pisum sativum L. N.J. Walton* and H.W. Woolhouse Agricultural and Food Research Council Photosynthesis Group, John Innes Institute, Colney Lane, Norwich NR4 7UH, UK

Abstract. A comparative study is presented of the activities of enzymes of glycine and serine metabolism in leaves, germinated cotyledons and root apices of pea (Pisum sativum L.). Data are given for aminotransferase activities with glyoxylate, hydroxypyruvate and pyruvate, for enzymes associated with serine synthesis from 3-phosphoglycerate and for glycine decarboxylase and serine hydroxymethyltransferase. Aminotransferase activities differ between the tissues in that, firstly, appreciable transamination of serine, hydroxypyruvate and asparagine occurs only in leaf extracts and, secondly, glyoxylate is transaminated more actively than pyruvate in leaf extracts, whereas the converse is true of extracts of cotyledons and root apices. Alanine is the most active amino-group donor to both glyoxylate and hydroxypyruvate. 3Phosphoglycerate dehydrogenase and glutamate :O-phosphohydroxypyruvate aminotransferase have comparable activities in all three tissues, except germinated cotyledons, in which the aminotransferase appears to be undetectable. Glycollate oxidase is virtually undetectable in the non-photosynthetic tissues and in these tissues the activity of glycerate dehydrogenase is much lower than that of 3-phosphoglycerate dehydrogenase. Glycine decarboxylase activity in leaves, measured in the presence of oxaloacetate, is equal to about 30-40% of the measured rate of CO2 fixation and is therefore adequate to account for the expected rate of photorespiration. The activity of glycine decarboxylase in the non-photosynthetic tissues is calculated to be about 2-5% of the activity in leaves and has the characteristics of a pyridoxalAgricultural and Food Research Council Food Research Institute - Norwich, Colney Lane, Norwich NR4 7UA, UK

* P r e s e n t address:



GOGAT=L-Glutamine:2-oxoglutarate amino-

and tetrahydrofolate-dependent mitochondrial reaction; it is stimulated by oxaloacetate, although not by ADP. In leaves, the measured activity of serine hydroxymethyltransferase is somewhat lower than that of glycine decarboxylase, whereas in root apices it is substantially higher. Differential centrifugation of extracts of root apices suggests that an appreciable proportion of serine hydroxymethyltransferase activity is associated with the plastids. Key words: Serine - Glycine - Photorespiratory metabolism - Pisum (photorespiration).


Glycine and serine are intermediates in the photorespiratory metabolism of glycollate to 3-phosphoglycerate (Tolbert 1980; Keys 1980). However, they are also important in the biosynthesis of purines (glycine), in the generation of cysteine and tryptophan (serine) and as sources of one-carbon units. They are also, of course, constituents of proteins. In non-photosynthetic tissues, serine is derived from 3-phosphoglycerate via O-phosphohydroxypyruvate and O-phosphoserine, although the enzymes of this pathway are also present in leaves (Cheung et al. 1968). The activity of this pathway is much lower than that of the photorespiratory pathway from glycollate. The quantitative importance of photorespiration as a loss of photosynthetically-fixed carbon has tended to dominate considerations of glycine and serine metabolism in plants. Consequently, there has been less interest in the extent to which enzymes related to photorespiration are present in non-photosynthetic tissues and few comparisons have been made between functionally related but


metabolically distinct enzymes in photosynthetic and non-photosynthetic tissues. This paper presents a survey of enzyme activities in leaves, germinated cotyledons and root apices of Pisum sativum L. with particular reference to (i), aminotransferases, (ii), enzymes of the "phosphorylated" and "non-phosphorylated" pathways from 3-phosphoglycerate and, (iii) glycine decarboxylase and serine hydroxymethyltransferase. The data provide a broad basis using a single plant species for more detailed comparative enzymology on important related enzymes from the different tissues. Materials and methods Chemicals. All radioisotopes were obtained from Amersham International, Amersham, Bucks., UK, with the exception of [3-14C]hydroxypyruvic acid (see below). Phosphohydroxypyruvic acid was purchased from Sigma (London) Chemical Co., Poole, Dorset, U K as the tricyclohexylammonium salt of the dimethylketal and the free acid was isolated according to the supplier's recommendations (see Ballou and Hesse 1956). Tetrahydrofolic acid was also purchased from Sigma and reassayed spectrophotometrically before use (Zakrewski and Sansone 1971). Other reagents were of the highest purity available commercially from BDH Chemicals, Poole, Dorset, UK, or from Sigma.

[3-14C]Hydroxypyruvate was generated from L-[3-14C]serine. A pea leaf extract was prepared and passed through SephadexG25 as described below and 1.5 ml of the extract was incubated in 2 ml of solution containing 2 gmol of L-[3-I4C]serine (specific activity 185GBq.mol-1), 1.6gmol of Na glyoxylate, 0.2 gmol of pyridoxal phosphate and 5 gl of catalase suspension (Boehringer, Mannheim, F R G ; 3 250 U- tool- 2). Prior to initiating the reaction, the solution was gassed thoroughly with N2 ; the reaction was then started by addition of the Na glyoxylate through a self-sealing rubber stopper. The reaction mixture was incubated, with continued N2 sparging, for 30 min at 30 ~ C and then acidified with 0.5 ml of 0.1 M HC1. (The reaction was performed under Nz to minimise the oxidation of glyoxylate by glycollate oxidase). The acidified mixture was then applied to a column (1 ml) of AG50W-X8, 100-200 mesh, cationexchange resin (Bio Rad Laboratories, Watford, UK), in the H § form, to remove unreacted L-[3-14C] serine. The column was eluted with a further 10 ml water and then the total eluate (pale yellow-green) was freeze-dried and the residue redissolved in approx. 0.5 ml of 10 mM HC1. This solution was then divided between four columns of Sephadex-G25 held in 0.2 ml vol. (approx.) disposable pipette tips inserted into microcentrifuge tubes. The columns were initially centrifuged, and then further eluted and centrifuged using successive aliquots (firstly 0.1 ml; then 0.3 ml) of 10 m M HC1; the two initial pale green proteincontaining eluates were discarded and the four subsequent colourless, radioactive eluates were retained. These were pooled and freeze-dried, redissolved in about 1.5 ml of 5 m M HC1, divided into 0.2-ml aliquots and stored at - 2 0 ~ C. After reaction with an excess of 2,4-dinitrophenylhydrazone in HCI, 90-95% of the radioactivity was extractable into ethyl acetate; cellulose thin-layer chromatography of the 2,4-dinitrophenylhydrazone in propan-2-ol/water/ammonia, S.G., 0.88 (20:2 : 1, by vol., Smith and Smith 1969) revealed the radioactivity to cochromatograph with authentic hydroxypyruvate 2,4-dinitrophenylhydrazone.

N.J. Walton and H.W. Woolhouse: Serine and glyeine metabolism

Plant material. Leaves of peas (Pisum sativum L. cv. Birte) were harvested from seedlings 15 d old. Seeds were surface-sterilised by soaking for 15 min in a 2% solution (approx.) o f N a hypochlorite and were then thoroughly washed prior to being soaked overnight (approx. /8 h) under running tap water. The seeds were then placed between several sheets of absorbent paper thoroughly moistened with tap water and kept in a seed tray in a polyethylene bag for 3 d at 16-20 ~ C. The germinated seedlings were then transferred to plastic trays containing holes through which pea roots could be inserted into a trough of continuously aerated tap water. The seedlings were allowed to grow for a further 11 d under a 14-h photoperiod with a photon flux density of 250 gmol quanta-m e.s-1 (photosynthetically active radiation) and a temperature of 20-25 ~ C. Leaves at the fifth node (counting from the base of the plant) were harvested immediately before use. Root apices (apical 6 mm) and cotyledons were harvested fi'om dark-grown seedlings 3 d old. Seeds were treated as described above, but germinated in moist absorbent paper at 20-25 ~ C for 50-53 h. Preparation of extracts. Leaf extracts were prepared by grinding about twenty leaves thoroughly with 3-4 vol. of chilled 20 m M K phosphate buffer, pH 8, containing 0.05% (v/v) Triton X-]00 and 2 mM 2-mercaptoethanol, at 0-4 ~ C with a pestle and mortar and a little acid-washed sand. Extracts of root apices (0.6 g) and cotyledons (10, from 10 seeds) were prepared in the same manner. Extracts to be used for the assay of phosphatase activities, and also concurrently for the assay of 3-phosphoglycerate dehydrogenase, glycerate dehydrogenase and hydroxypyruvate reductase, were prepared using 20 m M 4-(2-hydroxyethyl)-lpiperazineethanesulfonic acid (Hepes)-NaOH buffer, pH 8, containing 0.05% (v/v) Triton X-100 and 2 m M 2-mercaptoethanol. Extracts were then cleared by centrifugation for 15 min at 15000g and ( ~ 4 ~ and, except when used for the assay of serine hydroxymethyltransferase, passed through a column (25 cm long, 0.7 cm internal diameter) of Sephadex G-25 equilibrated with the extraction buffer; the protein-containing eluate was retained and used immediately for enzyme assays.

Preparation of leaf protoplasts and lysates. Pea leaf protoplasts were prepared as described by Walton and Woolhouse (1983), except that purification of the protoplasts was achieved by centrifuging the aqueous dextran-polyethyleneglycol two-phase systems for 5 min at 200 g instead of 500 g and the protoplasts were finally resuspended in 3-4 ml of a solution (protoplast incubation medium) containing 0.5 M sorbitol, 1 mM CaCI2, 5 m M KHzPO4and 30 m M Hepes-NaOH, pH 7.6. Protoplasts were lysed by forcibly ejecting the suspension twice from a syringe through a steel needle (23G); breakage of the protoplasts was virtually 100% as judged by microscopic examination. Crude mitochondrial preparations from root apices and cotyledons. Root apices (about 1.6 g) or cotyledons (:tO) were ground gently at 0-4 ~ C , using a pestle and mortar, in three volumes of chilled protoplast incubation medium, containing 0.1% (w/v) bovine serum albumin and 6 m M 2-mercaptoethanol. The mortar was rinsed with a further 4 ml of this medium and the extract was then centrifuged for 10 min at 2000 g (in later experiments, 5 min at 1000 g) and 0 - 4 ~ and the precipitated debris discarded. The supernatant solution was centrifuged for 20 min at 12000 g and 0 - 4 ~ and the precipitate was resuspended in 1-3 ml of the grinding medium. This preparation was used for the assay of glycine decarboxylase activity. The yield of mitochondria in this preparation, relative to the total content in the tissue, was calculated from the recovery of fumar-

N.J. Walton and H.W. Woolhouse: Serine and glycine metabolism ase, determined spectrophotometrically at 240 nm (Woo and Osmond 1976). An aliquot of the initial extract was ground further using sand and Triton X-100 (approx. 0.2%, v/v) and a sample of the mitochondrial preparation was solubilised with Triton X-100 (approx. 0.2%, v/v); fumarase in both preparations was then measured after brief centrifugation in a microfuge (Beckman, High Wycombe, Bucks, UK). (The recovery of fumarase in the mitochondrial preparation was 25-50% using an initial centrifugation of 5 rain at 1000 g).

Assays. Except for glycine decarboxylase, enzymes were assayed in tissue extracts prepared in the presence of Triton X-100 as described above. Enzyme assays were performed at 30 ~ C except where otherwise stated. Glycollate oxidase (EC was assayed radiometrically essentially as indicated by Walton (1983), with 5 mM glycollate in 70 m M glycylglycine-NaOH buffer, pH 8.8, using an incubation period of 10 min. Glycoxylate and pyruvate aminotransferases (EC;; were assayed radiometrically essentially as described by Walton (1983) for the determination of [1-14C]glycine formed from [l-l~C]glyoxylate, by oxidation of the [1-~C]glyoxylate substrate with KMnO4. Preliminary experiments established that [1J4C]alanine (in addition to [1-~4C]gly cine) was not oxidized under the conditions used. The assay conditions were those previously described for transamination reactions, using 20 m M L-amino acid and 5 mM 2-oxo acid (Walton 1983), except that with glutamate as the amino-group donor, K phosphate buffer, pH 7.1, was employed and that reactions with other amino-group donors were performed in boric acid-NaOH buffer, pH 8.3. Reaction mixtures were incubated for 10 min. The routine control reaction mixtures contained extract in the absence of amino-group donors; non-enzymic transaminations in the absence of extract were found to be negligible. Commercial preparations of [1-14C]pyruvate were repurifled by thin-layer chromatography before use, using the solvent system described by Goldfine (1960). The purified radiochemical was stored at - 2 0 ~ in 5 mM HC1, without unlabelled carrier, for no longer than one month and made up to a neutralized 50 mM stock solution, approx. 0.75 G B q . m o l - 1 , immediately before use. [1-~4C]Glyoxylate was stored in 5 mM glyoxylic acid and made up to a 50 mM solution of Na is salt, specific activity approx. 0.75 G B q . m o l - 1 , immediately prior to use. Hydroxypyruvate aminotransferase was assayed radiometrically using [3-14C]hydroxypyruvate prepared in the laboratory from L-[3-14C]serine (see above). Assays were performed as described for glyoxylate and pyruvate aminotransferases, using [3-~4C]hydroxypyruvate (specific activity in the assay approx. 0.15 GBq-mol-~), except that transamination was determined by assaying the radioactivity in the amino-acid fraction after stopping the reactions with 0.2 ml of 0.2 M H2804. Acidified reaction mixtures were applied to columns (0.5 ml) of Bio-Rad AG50W-X8 cation-exchange resin, 100-200 mesh, in the H +form. Unreacted [3-14C]hydroxypyruvate was eluted by washing the columns with 5 ml of H20. The amino-acid fraction was then eluted in 5 ml of 6 M NH4OH, evaporated to dryness at 30 ~ C and redissolved in 0.5 ml of H 2 0 ; 0.35 ml was removed for scintillation counting.

Glutamate :O-phosphohydroxypyruvate


(EC was assayed at pH 8.3 in boric acid-NaOH buffer in 0.25 ml of the reaction mixture used for the other aminotransferases, with the substitution of 5 mM phosphohydroxypyruvate. Reactions were carried out in 10-ml glass centrifuge tubes. To remove any hydroxypyruvate, either present as an impurity in the phosphohydroxypyruvate substrate or generated by phosphatase action during the assay, reaction mixtures

12l contained 1.8 mM N A D H and 0.5 U glyoxylate reductase (Sigma), treated with Sephadex-G25 before use. Reaction mixtures were preincubated for 10 min in the absence of extract. After incubation for 20 rain, reactions were stopped by adding 0.02 ml of 0.2 M H2SO4. To each reaction mixture, 0.15 g of AG50W-X8, cation-exchange resin, 100-200 mesh, moisture content approx. 50%, in the H+-form, was then added to remove unreacted glutamate. The reaction mixture and resin were thoroughly mixed; 0.4 ml of water was then added again with thorough mixing. The tube was centrifuged and 0.4 ml of the superuatant solution was pipetted into a scintillation vial and mixed with 1.5 ml of 100 m M imidazole-HC1 buffer, pH 7.0, 0.05 ml of 1 m M Na L-[U-14C]glutamate (specific activity approx. 3.7 GBq.mo1-1) and 0.05 ml of (Boehringer) glutamate:oxaloacetate aminotransferase (prepared by centrifuging 50 pl of the suspension in 3.2 M (NH,~)2SO4, containing 20 U, and resuspending the precipitate in 2.5 ml of 100 m M imidazole-HC1 buffer, pH 7.0). The reaction mixture was incubated for 90 rain at 30 ~ C and stopped by the addition of 20 pl of 2.5 M H2SO4. A plastic tube containing 70 pl of 20% (w/v) K O H was then inserted, the vial was closed with a self-sealing rubber stopper and 1 ml of 30% (w/v) HzO2 was added, with thorough mixing, to decarboxylate [U-14C]2-oxoglutarate generated by enzyme-catalysed exchange. After 2 h, 50 pl of the KOH was taken for the determination of radioactivity by scintillation counting. Reaction mixtures were routinely accompanied by controls acidified prior to the addition of extract and by a range of 2-oxoglutarate standards, from 0 to 20 nmol, from which phosphohydroxypyruvate was omitted. Controls incubated without glutamate or to which extract was added after incubation gave identical results. Phosphohydroxypyruvate did not affect the exchange reaction catalysed by glutamate:oxaloacetare aminotransferase. The production of 2-oxoglutarate by phosphohydroxypyruvate aminotransferase was calculated from the radioactivity recovered in the 2-oxoglutarate standards. Serine hydroxymethyltransferase (EC was assayed as described by Taylor and Weissbach (1965), using an incubation period of 10 min, except that reaction mixtures contained 5 mM L-[3-14C] serine (specific activity 1.85 GBq.mol i) and the reaction was carried out in boric acid-NaOH buffer, pH 8.8. Glycine decarboxylase (EC was assayed using a standard incubation period of 20 min and a basic reaction mixture, with variations indicated with the individual experiments, similar to that of Woo and Osmond (1976) and containing pea-leaf protoplast incubation medium (0.5 M sorbitol, 1 mM CaC12, 5 mM KH2PO4 and 30 m M Hepes-NaOH, pH 7.6) with the addition of 2 m M MgCI2, 0.25 mM pyridoxal phosphate, 5 mM oxaloacetate and 20 mM [1-14C]glycine (specific activity 0.19 GBq-mol-1). The reactions were carried out in scintillation vials fitted with small plastic tubes containing 70 gl of 20% (w/v) K O H and closed with self-sealing rubber stoppers and were stopped by injecting 0.1 ml of 2.5 M H2SO,~. After 2 h, samples (50 gl) were removed and the absorbed 14CO2 was determined by scintillation counting. Reaction mixtures were accompanied by controls containing boiled protoplast or mitochondrial preparations. Hydroxypyruvate reductase (EC was assayed spectrophotometrically as described by Stabenau (1974). D-3-Phosphoglycerate dehydrogenase (EC and Dglycerate dehydrogenase (EC were both assayed spectrophotometrically as described by Larsson and Albertsson (1979). D-3-Phosphoglyceratephosphatase was assayed as described by Randall et al. (1971), with the substitution of 2-(N-morpho-


N.J. Walton and H.W. Woolhouse: Serine and glycine metabolism

Table 1, Aminotransferase activities with glyoxylate, hydroxypyruvate and pyruvate in pea leaves. Activities were determined

as described in the text, at 30~ and pH 8.3 (pH 7.1 for glutamate) with 20 mM amino acid and 5 mM oxoacid. Values are gmol- rain - t- g - t FW and are means • SDs of the number of determinations in parentheses Oxoacid

Glyoxylate Hydroxypyruvate Pyruvate

Amino acid Serine




6.64 • 0.92(4) 5.07 • 0.54(4) 0.10 • 0.03(4)

5.96 • 0.78(4) 0.32 • 0.05(4) 3.79 • 0.72(4)

8.40 _+1.29(4) 2.59 _ 0 A4(4) 4.21 • 0.88(4)

1.55 • 0.31 (4) 1.66 • 0.06(4) 0.18 _+0.05(4)

Table 2. Aminotransferase activities with glyoxylate, hydroxypyruvate and pyruvate in germinated pea cotyledons. Legend as

for Table 1 Oxoacid

Glyoxylate Hydroxypyruvate Pyruvate

Amino acid Serine




0.04_ 0.02(4) 0.05 • 0.02(3) 0.02 +_0.03(4)

0.15 • 0.03(4) 0.03 • 0.03(3) 1.15 • 0.08(4)

0.52_+ 0.12(4) 0.05 + 0.02(3) 1.97 • 0.18(4)

0.03 +_0.03(4) 0.02 • 0.01(3) 0.04_+ 0.03(4)

Table 3. Aminotransferase activities with glyoxylate, hydroxypyruvate and pyruvate in pea root apices. Legend as for Table 1 Oxoacid

Glyoxylate Hydroxypyruvate Pyruvate

Amino acid Serine




0.07 • 0.03(4) 0.10 • 0.05(3) 0.00 • 0.00(4)

0.18 • 0.03(4) 0.03 • 0.04(3) 1.90 • 0.43(4)

0.43 • 0.08(4) 0.13 _ 0.03(3) 2.70 • 0.44(4)

0.04 • 0.01 (4) 0.07 • 0.06(3) 0.02 • 0.01(4)

lino)ethanesulfonic acid (Mes)-NaOH buffer, pH 6.5, for Na cacodylate, pH 5.9. O-Phospho-L-serine phosphatase (EC was assayed by the method of Larsson and Albertsson (1979). Fumarase (EC was assayed at 25~ as described by Woo and Osmond (1976). Catalase (EC was measured at 25~ essentially by the method of Liick (1963) L-Glutamine :2-oxoglutarate aminotransferase (GOGAT; EC was measured spectrophotometrically at 25 ~ C by the method of Emes and Fowler (1979), in the presence of N A D H and against a control cuvette lacking glutamine.

Measurements of gross photosynthesis by individual intact, attached leaves under growth-room conditions were made using a portable two-gas field system allowing exposure to a pulse of 14CO2 under steady-state conditions (Incoll 1977); determinations were made at a COs concentration in air of 411 gl. 1-~, and with a flow rate of 200 ml.min -1, a t4CO2 pulse volume of 10 ml and a specific radioactivity (1~CO2 pulse) of 93 GBqt o o l - 1 After 30 s of photosynthesis, the leaf was ground in liquid N2 with 2 M chilled perchloric acid and the acid-stable ~4C incorporation was determined by liquid scintillation counting.

Chlorophyll and pheophytin were measured by the methods of Arnon (1949) and Vernon (1960), respectively.


Aminotransferases. In Tables 1-3 are compared the activities of aminotransferases using glyoxylate, hydroxypyruvate and pyruvate in leaves, root apices and germinated cotyledons. Serine and hydroxypyruvate are transaminated substantially only in leaves and, although glyoxylate is transaminated in both root apices and cotyledons, the activity in these tissues is low compared with pyruvate transamination, whereas the converse is true of leaves; serine, hydroxypyruvate and glyoxylate are, of course, established intermediates of the photorespiratory pathway. In leaves, the most active amino-group donor to glyoxylate is alanine, followed by serine and glutamate; appreciable but considerably lower activity is also observed with asparagine. Apart from an enzymic exchange reaction with hydroxypyruvate, serine is transaminated actively only with glyoxylate; in none of the tissues is there appreciable transamination with 2-oxoglutarate or oxaloacetate (data not shown).

N.J. Walton and H.W. Woolhouse: Serine and glycine metabolism


Table 4. Activities of glycollate oxidase, hydroxypyruvate reductase and enzymes of serine synthesis from 3-phosphoglycerate and glycerate. Activities were determined at 30 ~ C as described in the text. Values are g m o l - m i n - t . g - 1 FW and are means • SDs of the number of determinations in parentheses Enzyme

Tissue Leaves

Germinated cotyledons

Root apices

2.30 +_0.89(4) 16.6 +1.7 (3) 0.49 • 0.03(3) 0.10+_0.01(3) 6.1 +1.2 (3) 4.9 _+0.4 (3) 0.13 • 0.03(5)

0.08 _ 0.05(4) 0.46+0.09(4) 0.012(1) 0.12_+0.02(4) 2.8 +1.0 (3) 1.8 _+0.2 (3) 0.00 + 0.00(3)

0.08 _+0.07(4) 1.00_+0.10(4) 0.015(1) 0.13+0.05(4) 3.8 +0.6 (3) 2.1 _+0.4 (3) 0.12 _+0.05(4)

Glycollate oxidase Hydroxypyruvate reductase Glycerate dehydrogenase 3-Phosphoglycerate dehydrogenase 3-Phosphoglycerate phosphatase O-Phosphoserine phosphatase Glutamate: O-phosphohydroxypyruvate aminotransferase

Enzymes of the glycerate and phosphoglycerate pathways. The activities in the three tissues of five enzymes involved or potentially involved in serine synthesis from 3-phosphoglycerate are given in Table 4, together with the activities of glycollate oxidase and hydroxypyruvate reductase. The photorespiratory pathway enzymes, glycollate oxidase and hydroxypyruvate reductase, are present in very substantial activities only in leaves, although some hydroxypyruvate reductase activity, attributable possibly to lactate dehydrogenase, is present in the two non-photosynthetic tissues. When assayed in the reverse direction, however, the activity of the enzyme measured as glycerate dehydrogenase is very much lower than that of 3-phosphoglycerate dehydrogenase in cotyledons and root apices, in contrast to the ratio of activities in leaves. All three tissues contain high activities of phosphatases capable of hydrolysing 3-phosphoglycerate and O-phosphoserine. Glutamate: O-phosphohydroxypyruvate aminotransferase, assayed at pH 8.3 (optimal) and with the same substrate concentrations as the hydroxypyruvate aminotransferases reported in Tables 1-3, is very much less active than alanine: hydroxypyruvate aminotransferase in leaves (see Table 1); and in both leaves and root apices the activity measured is comparable to that of 3-phosphoglycerate dehydrogenase, the enzyme which generates O-phosphohydroxypyruvate. Surprisingly, the O-phosphohydroxypyruvate aminotransferase is undetectable in germinated cotyledons. This apparent lack of activity appears not to be the result of any inhibitor in the cotyledon extracts since co-extraction of cotyledons and root apices leads to a measured activity predicted from the extraction of root apices alone (data not shown).

Glycine decarboxylase and serine hydroxymethyltransferase. Some characteristics of the glycine de-

5. Some characteristics of glycine decarboxylation in crude mitochondrial preparations from pea seedling root apices and cotyledons. Incubations were performed in the standard reaction mixture described in Materials and methods, with the additions and omissions indicated, and the values given are nmol of 14CO2 released in 20 min at 30 ~ C. Based on the recovery of fumarase in the mitochondrial preparations, the activities (gmol. m i n - 1. g - 1 FW) of glycine decarboxylase for each preparation in the standard mixture, with the addition of 2 mM NAD, 5 mM dithiothreitol (DTT) and 0.2 mM tetrahydropteroylmonoglutamate (tetrahydrofolate; H4PteGlu), are: Root 1, 0.058; Root 2, 0.059; Cotyledon 1, 0.044; Cotyledon 2, 0.038. n.d., not determined Table

Addition to standard mixture

Omission from standard mixture

Tissue preparation Root apices












ADP, 4 mM











NAD, 2 mM






NAD, 2 mM

Pyridoxal phosphate





NAD, 2 mM; DTT, 5 mM










NAD, 2 raM; DTT, 5 mM H4PteGlu, 0.2 mM NAD, 2 raM; Triton X-100, 0.05%, v/v






NAD, 2 mM; isonicotinyl hydrazide, 10 m M










NAD, 2 mM; semicarbazide, 10 mM


N.J. Walton and H.W. Woolhouse: Serine and glycine metabolism

Table 6. Glycine decarboxylation and serine hydroxymethyltransferase activity in leaves, root apices and cotyledons of pea seedlings9 Values were determined at 30 ~ C, except where indicated, and are means • SDs of the number of determinations in parentheses9 Glycine decarboxylation was determined in the standard reaction mixture with the addition of 2 m M N A D and 5 m M dithiothreitol and, except in the case of leaf protoplast lysates where no stimulation was demonstrable, 09 mM tetrahydrofolate. Glycinedecarboxylase activities have been calculated on the basis of fumarase recovery or chlorophyll content as described in the text. Gross rates of 14CO2 fixation by intact, attached leaves were determined as indicated in Materials and methods. Chl, chlorophyll; Pheo, pheophytin; n.d. not determined Enzyme or reaction

Tissue Leaves

Glycine decarboxylation (oxaloacetate-supported) Serine hydroxymethyltransferase Gross photosynthetic 14CO2 fixation

(gmol-mg 1 Chl or Pheo- h - l)

(gmol 9m i n - 1

31.5(25.0)~ •

1.3 (calculated mean) 0.86 • 0.09(3) n.d.

n.d. 67.3 • 15.2(8)


Root apices (gmol- m i n - 1 .g-1 FW)

Cotyledons (gmol. rain- 1 .g-1 FW)

0.05• 0.01(3)

0.03 • 0.01(4)

0.21 ___0.05(4) n.d.

0.07 • 0.01(4) n.d.

1 FW)

(25 c) a Calculated mean value for 25 ~ C , from a temperature-variation experiment

carboxylation reaction in crude mitochondrial preparations from root apices and germinated cotyledons are shown in Table 5. The reaction in these preparations is not stimulated by ADP. On the other hand, there is a substantial stimulation by oxaloacetate. This appears not to be enhanced further by the addition of NAD, although this cofactor has been added in most reaction mixtures. Dithiothreitol and tetrahydrofolate both stimulate the reaction to a variable extent. The omission of pyridoxal phosphate causes a slight and variable inhibition. The reaction is appreciably inhibited by isonicotinyl hydrazide and much more severely by Triton X-100 or semicarbazide. These are characteristics consistent with the presence of a pyridoxal-dependent reaction associated with the mitochondrial inner membrane. The activities of glycine decarboxylase (oxaloacetate-supported) and serine hydroxymethyltransferase in leaves, root apices and germinated cotyledons are compared in Table 6. In the case of leaves, the glycine decarboxylase activities have been determined on a chlorophyll basis in protoplast lysates and the activity on a fresh-weight basis has been calcuated using an experimentally determined value of 2.5 m g . g - 1 fresh weight for the chlorophyll content of the leaves. For the root apices and cotyledons, the glycine decarboxylase activities have been calculated on a fresh-weight basis using fumarase as a mitochondrial marker (see Materials and methods). The activity of glycine decarboxylase in the non-photosynthetic tissues is about 2-5% of that in leaves. A direct comparison is made in Table 6 between the rate of glycine decarboxylation (oxaloacetate-supported) in leaves and

the fixation of 14CO2 under growth-room conditions; glycine decarboxylation is about 30-40% of the rate of CO2 fixation. In leaves, the activity of serine hydroxymethyltransferase, measured at the experimentally determined optimal pH, 8.8, and in the direction of serine breakdown, is comparable to, although rather less than, that of oxaloacetate-supported glycine decarboxylation; however, in germinated cotyledons and especially in root apices, the activity of serine hydroxymethyltransferase appreciably exceeds that of glycine decarboxylation. This implies that the functional relationship between these two enzymes differs between the leaves and the non-photosynthetic tissues.

Subcellular location of serine hydroxymethyltransferase. This aspect has been examined using differential centrifugation and comparison of the distribution of serine hydroxymethyltransferase activity in tissue fractions with that of the marker enzymes, fumarase, catalase and L-glutamine:2-oxoglutarate aminotransferase (GOGAT). The distribution of these enzymes between a 1 000-g precipitate, a 12000-g precipitate and a 12000-g supernatant fraction is shown in Table 7; similar results were obtained in two other experiments under slightly different conditions. Serine hydroxymethyltransferase activity is about equally distributed between the 1000-g precipitate and 12000-g precipitate fractions with about half the total activity remaining in the 12000-g supernatant fraction. In contrast, fumarase is predominantly in the 12000-g precipitate, whilst the highest activity of GOGAT is recovered very largely in the 12000-g superna-

N.J. Walton and H.W. Woolhouse: Serine and glycine metabolism Table 7. Distribution of serine hydroxymethyltransferase activity between fractions of an extract of pea root apices prepared by differential centrifugation. 1 g of root apices was ground as described in Materials and methods for the extraction of crude mitochondrial preparations and the extract was filtered through two layers of Miracloth moistened with the extraction medium, using a further 3 vol. of extraction medium to wash. The resulting extract was centrifuged at 1000 g for 1 min and then at 12000 g for 20 rain at 0 - 4 ~ in a fixed-angle rotor. Activities in the 1000-g precipitate, 12000-g precipitate and 12000-g supernatant fractions are presented as percentages of the activity in the uncentrifuged extract. The activities (gmolrain 1) in this extract (vol. 3.5 ml) were: serine hydroxymethyltransferase, 0.031; G O G A T , 0.15; fumarase, 0.65; catalase, 58 (at 7.5 mM H202) Fraction

Enzyme Serine hydroxymethyltransferase

1000-g ppt. 12000-g ppt. 12000-g supt. Total




32 27 55

41 12 30

30 65 19

0 24 65





tant fraction and is essentially absent from the 1 000-g precipitate. These results indicate that serine hydroxymethyltransferase does not follow the distribution of the mitochondrial marker enzyme, fumarase and indicate strongly that an appreciable amount of activity may be associated with the plastids, where GOGAT is located (Emes and Fowler 1979). It is also possible that some of the activity is cytosolic since a higher proportion of the enzyme is recovered in the 12000-g supernatant fraction than is the case with either fumarase or GOGAT. Discussion

The data presented here indicate the extent to which enzyme activities associated with photorespiration are also present or absent in non-photosynthetic tissues. The low activities of glycollate oxidase and hydroxypyruvate reductase in root apices and germinated cotyledons are reflected in the spectrum of aminotransferase activities in the tissues. Thus the non-photosynthetic tissues show, amongst the reactions examined, substantial transamination reactions only between glutamate and pyruvate and, to a much lesser extent, alanine and glyoxylate (neglecting exchange transamination between alanine and pyruvate); reactions with serine and hydroxypyruvate are virtually absent. It is now known that in leaves glutamate:glyoxylate aminotransferase and glutamate:pyruvate amino-


transferase are functions of the same protein (Noguchi and Hayashi 1981; Biekmann and Feierabend 1982) which also possesses alanine:glyoxylate aminotransferase activity (Noguchi and Hayashi 1981; Nakamura and Tolbert 1983). In contrast, glutamate :pyruvate aminotransferase purified from tomato fruit has been found to have no activity with glyoxylate (Rech and Crouzet 1974). The observations on glyoxylate and pyruvate transamination made here are, therefore, fully consistent with the presence in the non-photosynthetic tissues of a distinct isoenzyme (or isoenzymes) of glutamate: pyruvate aminotransferase having a lower affinity for glyoxylate than the leaf enzyme. Turning to the transamination reactions apparently specific to leaves or photosynthetic tissues, the activities with serine, hydroxypyruvate and asparagine are almost certain to be catalysed by a single enzyme (see Ireland and Joy 1983) which probably also has alanine-glyoxylate, in addition to alanine-hydroxypyruvate, aminotransferase activity (see Noguchi and Hayashi 1980). The observations on hydroxypyruvate transamination reported here are generally consistent with those of Liang et al. (1984), using spinach leaf peroxisomes, who observed very active transamination with alanine and lesser activities with glycine and asparagine; however, the physiological significance of these reactions, especially with asparagine, must remain questionable. So must the role of alanine in photorespiration since, presumably, it is both generated and utilised by the glutamate:pyruvate (glyoxylate) aminotransferase. It is not established whether the synthesis of serine from 3-phosphoglycerate in leaves ever occurs to an important extent by way of a "nonphosphorylated" pathway with glycerate and hydroxypyruvate as intermediates (Fig. 1). The data presented here are broadly in agreement with those of Cheung et al. (1968) and emphasise that two of the enzymes of this sequence (Nos. 2 and 6 in Fig. 1) are present simply on account of the photorespiratory activity of the tissue, during which the enzymes catalyse the reverse reactions as part of the conversion of serine to 3-phosphoglycerate. Furthermore, phosphatases (probably non-specific) capable of hydrolysing 3-phosphoglycerate are very active in all three tissues examined, despite the low activities of glycerate dehydrogenase and hydroxypyruvate aminotransferase in root apices and cotyledons. Conversely, it has not been possible to establish with any certainty the physiological role of particular, relatively substrate-specific, 3phosphoglycerate phosphatases, even though purified preparations of such enzymes have been stud-


N.J. Walton and H.W. Woolhouse: Serine and glycine metabolism 8 P D-Glycerate

3-Phosphoglycerate q







C?unlt 5 Serine

O-Phosphoserine Ii

P Glycine

/ C02,NH 3


Fig. 1. Summary of pathways of serine metabolism. (1) Glycollate oxidase. (2) Aminotransferase with the following activities : serine-glyoxylate; alanine-glyoxylate; alanine-hydroxypyruvate; asparagine-glyoxylate; asparagine-hydroxypyruvate. (3) Aminotransferase with the following activities: glutamateglyoxylate; alanine-glyoxylate; glutamate-pyruvate. (4) Glycine decarboxylase complex, with associated serine hydroxymethyltransferase. (5) Serine hydroxymethyltransferase (unassociated with glycine decarboxylase). (6) Hydroxypyruvate reductase/ glycerate dehydrogenase. (7) D-Glycerate kinase. (8) 3-Phosphoglycerate phosphatase. (9) 3-Phosphoglycerate dehydrogenase. (10) Glutamate: O-phosphophohydroxypyruvate aminotransferase. (11) O-Phosphoserine phosphatase. The photorespiratory pathway involves reactions of (1), (2), (3), (4), (6) and (7). The "non-phosphorylated" pathway of serine synthesis involves reactions of (8), (6) and (2). The "phosphorylated" pathway of serine synthesis consists of reactions catalysed by (9), (10) and (11).

ied in some detail (Randall and Tolbert 1971a, b; Mulligan and Tolbert 1980). Finally, although glycerate is known to be an early carboxyl-labelled product of 14CO2 fixation (Mortimer 1961; Hess and Tolbert 1966) it is unknown to what extent it can be further converted to serine. In contrast, the "phosphorylated" pathway from 3-phosphoglycerate via O-phosphohydroxypyruvate (Fig. 1, reactions 9-11) is well established as the apparent principal route to serine in nonphotosynthetic tissues (though the apparent absence here of O-phosphohydroxypyruvate aminotransferase in germinated cotyledons is surprising). However, comparable enzyme activities are also present in leaves (Table 4). It is possible that in leaves the pathway generates serine during darkness when photorespiration is not occurring (though a "non-phosphorylated" pathway via glycerate and hydroxypyruvate might also function under these conditions). On the other hand, there are reports that O-phosphoserine is a product of photosynthesis (Chapman and Leech 1976; Daley and Bidwell 1977) so the pathway may also be active in the light, for reasons which are unknown at present. Larsson and Albertsson (1979) have indicated, incidentally, that a large proportion of both 3-phosphoglycerate dehydrogenase and Ophosphoserine-phosphatase activity is in the chlo-

roplasts, whereas the O-phosphohydroxypyruvate aminotransferase is predominantly extrachloroplastic and they have suggested that the "phosphorylated" pathway might occur both within and outside the chloroplasts. The characteristics of glycine decarboxylation in root apices and germinated cotyledons (Table 5) are similar to those reported previously for nonphotosynthetic tissues (Prather and Sisler 1972; see also Clandinin and Cossins 1975). Like the leaf glycine decarboxylase (data not shown; Woo and Osmond 1976), the reaction in these tissues is stimulated by oxaloacetate, which presumably acts here also as an acceptor of reducing equivalents. As indicated in Table 6, the activity of glycine decarboxylase in these non-photosynthetic tissues, measured in the presence of oxaloacetate, is about 2-5% of that in leaves. The recent data of Singh and Naik (1984) using intact rice tissues indicate a rather higher figure of about 14% ; on the other hand, glycine decarboxylase has been undetectable in mitochondria prepared from non-green tissues of spinach (Gardestr6m et al. 1980; Oliver 1981). The metabolic function of this activity is obscure but it may be responsible for the supply of onecarbon units to the mitochondrion if such units are unable to pass directly into the mitochondrial matrix (see Cybulski and Fisher 1976) and, further, if the matrix does not contain appreciable activity of free serine hydroxymethyltransferase (i.e. unassociated with glycine decarboxylase) which could generate one-carbon units from serine (see Fig. 1). The extent to which glycine decarboxylase in nonphotosynthetic tissues may be linked obligatorily to the formation of serine and also to electron transport and ATP synthesis requires further investigation. The activity of glycine decarboxylase in leaves, 3 0 4 0 % of the rate of CO2 fixation, is about adequate to account for the rate of flux through glycine if it is assumed that this flux is equal to about 50% of the rate of gross photosynthesis under normal atmospheric conditions (Somerville and Somerville 1983). Theoretically, the activity of serine hydroxymethyltransferase in leaves should be at least equal to this, though the enzyme is determined by measuring serine breakdown and not serine synthesis and, as pointed out by Keys (1980), the enzyme may not necessarily function optimally when dissociated from the glycine decarboxylase complex. The ratio of activities of serine hydroxymethyltransferase to glycine decarboxylase and the differential-centrifugation data of Table 7 indicate that much of the serine hydroxymethyltransferase of

N.J. Walton and H.W. Woolhouse: Serine and glycine metabolism

root apices is outside the mitochondrion, probably in the plastids, and not associated with glycine decarboxylation. This enzyme very probably differs from the mitochondrial enzyme of leaves, which accounts for about 90% of the activity measurable in leaves (Woo 1979), in catalysing the breakdown of serine to glycine, rather than the synthesis of serine from glycine in conjunction with glycine decarboxylation (see Fig. 1). So far, very detailed kinetic data are available only for serine hydroxymethyltransferase from mung-bean seedlings - a non-photosynthetic source (Rao and Rao 1981). This enzyme has complex kinetics; it is competitively inhibited by glycine and shows a sigmoid saturation curve for tetrahydrofolate which is abolished by N A D H and enhanced by N A D § and adenine nucleotides. The physiological significance of these properties is not clear but it seems likely that a comparative study of the enzyme from photosynthetic and non-photosynthetic tissues of the same plant would be very revealing. We thank D.A. Jones for valuable assistance with the measurements of photosynthesis and Dr. Alison Smith for helpful discussions.

References Arnon, D.I. (1949) Copper enzymes in isolated chloroplasts. Polyphenol oxidase in Beta vulgaris. Plant Physiol. 24, 1-15 Ballou, C.E., Hesse, R.(1956) The synthesis and properties of hydroxypyruvic acid phosphate. J. Am. Chem. Soc. 78, 3718 3720 Biekmann, S., Feierabend, J. (1982) Subcellular distribution, multiple forms and development of glutamate-pyruvate (glyoxylate)aminotransferase in plant tissues. Biochim. Biophys. Acta 721,268-279 Chapman, D.J., Leech, R.M. (1976) Phosphoserine as an early product of photosynthesis in isolated chloroplasts and in leaves of Zea mays seedlings. FEBS Lett. 68, 160-164 Cheung, G.P., Rosenblum, I.Y., Sallach, H.J. (1968) Comparative studies of enzymes related to serine metabolism in higher plants. Plant Physiol. 43, 1813-1820 Clandinin, M.T., Cossins, E.A. (1975) Regulation of mitochondrial glycine decarboxylase from pea mitochondria. Phytochemistry 14, 387-391 Cybulski, R.L., Fisher, R.R. (1976) Intramitochondrial localization and proposed metabolic significance of serine transhydroxymethylase. Biochemistry 15, 3183-3187 Daley, L.S., Bidwell, R.G.S. (1977) Phosphoserine and phosphohydroxypyruvic acid. Evidence for their role as early intermediates in photosynthesis. Plant Physiol. 60, 109-114 Emes, M.J., Fowler, M.W. (1979) The intracellular location of the enzymes of nitrate assimilation in the apices of seedling pea roots. Planta 144, 249 253 Gardestr6m, P., Bergrnan, A., Ericson, I. (1980) Oxidation of glycine via the respiratory chain in mitochondria from different parts of spinach. Plant Physiol. 65, 389 391 Goldfine, H. (1960) The formation of ~-hydroxy-)p-methylglutamic acid from a common impurity in pyruvic acid. Biochim. Biophys. Acta 40, 557-559

127 Hess, J.L., Tolbert, N.E. (1966) Glycolate, glycine, serine and glycerate formation during photosynthesis by tobacco leaves. J. Biol. Chem. 241, 5705-5711 Incoll, L. (1977) Field studies of photosynthesis - monitoring with 14CO2. In: Environmental effects on crop physiology, pp. 137-155, Landsberg, J.J., Cutting, C.V., eds. Academic Press, London New York Ireland, R.J., Joy, K.W. (1983) Purification and properties of an asparagine aminotransferase from Pisum sativum leaves. Arch. Biochem. Biophys. 223, 291-296 Keys, A.J. (1980) Synthesis and interconversion of glycine and serine. In: The biochemistry of plants, vol. 5: Amino acids and derivatives, pp. 359-374, Miflin, B.J., ed. Academic Press, New York London Larsson, C., Albertsson, E. (1979) Enzymes related to serine synthesis in spinach chloroplasts. Physiol. Plant. 45, 7-10 Liang, Z., Yu, C., Huang, A.H.C. (1984) Conversion of glycerate to serine in intact spinach leaf peroxisomes. Arch. Biochem. Biophys. 233, 393-401 Liick, H. (1963) Catalase. In: Methods of enzymatic analysis, pp. 886-888, Bergmeyer, H.-U., ed. Academic Press, New York London Mortimer, D.C. (1961) Some observations on the formation of glyceric acid during photosynthesis experiments. Can. J. Bot. 39, 1-5 Mulligan, R.M., Tolbert, N.E. (1980) Properties of a membrane-bound phosphatase from the thylakoids of spinach chloroplasts. Plant Physiol. 66, 1169 1173 Nakamura, Y., Tolbert, N.E. (1983) Serine: glyoxylate, alanine: glyoxylate and glutamate :glyoxylate aminotransferase reactions in peroxisomes from spinach leaves. J. Biol. Chem. 258, 7631 7638 Noguchi, T., Hayashi, S. (1980) Peroxisomal localization and properties of tryptophan aminotransferase in plant leaves. J. Biol. Chem. 255, 2267-2269 Noguchi, T., Hayashi, S. (1981) Plant leaf alanine:2-oxoglutarate aminotransferase. Biochem. J. 195, 235-239 Oliver, D.J. (1981) Formate oxidation and oxygen reduction by leaf mitochondria. Plant Physiol. 68, 703-705 Prather, C.W., Sisler, E.C. (1972) Glycine and glyoxylate decarboxylation in Nicotiana rustica roots. Phytochemistry 11, 1637-1647 Randall, D.D., Tolbert, N.E. (1971 a) 3-Phosphoglycerate phosphatase in leaves. I. Purification and characterization. J. Biol. Chem. 246, 5510-5517 Randall, D.D., Tolbert, N.E. (1971b) 3-Phosphoglycerate phosphatase in plants. III. Activity associated with starch particles. Plant Physiol. 48, 488 492 Randall, D.D., Tolbert, N.E., Gremel, D. (1971) 3-Phosphoglycerate phosphatase in plants. II. Distribution, physiological considerations and comparison with P-glycolate phosphatase. Plant Physiol. 48, 480M87 Rao, D.N., Rao, N.A. (1982) Purification and regulatory properties of mung bean (Vigna radiata L.) serine hydroxymethyltransferase. Plant Physiol. 69, 11-18 Rech, J., Crouzet, J. (1974) Partial purification and initial studies of the tomato L-atanine:2-oxoglutarate aminotransferase. Biochim. Biophys. Acta 350, 39~399 Singh, P., Naik, M.S. (1984) Metabolism of glycine in roots of wheat and rice seedlings. J. Plant Physiol. 117, 1 6 Smith, I., Smith, M.J. (1969) Ketoacids. In: Chromatographic and electrophoretic techniques, 3rd edn., vol. 1, pp. 330341, Smith, I., ed. Heinemann, London Somerville, S.C., Somerville, C.R. (1983) Effect of oxygen and carbon dioxide on photorespiratory flux determined from glycine accumulation in a mutant of Arabidopsis thaliana. J. Exp. Bot. 34, 415~424

128 Stabenau, H. (1974) Verteilung von Microbody-Enzymen aus Chlamydomonas in Dichtegradienten. Planta 118, 35-42 Taylor, R.T., Weissbach, H. (1965) Radioactive assay for serine transhydroxmethylase. Anal. Biochem. 13, 80-84 Tolbert, N.E. (1980) Photorespiration. In: The biochemistry of plants, vol. 2: Metabolism and respiration, pp. 488-525, Davies, D.D., ed. Academic Press, New York London Vernon, L.P. (1960) Spectrophotometric determination of chlorophylls and pheophytins in plant extracts. Anal. Chem. 32, t144-1150 Walton, N.J. (1983) Glyoxylate amination by pea leaf extracts. Plant Sci. Lett. 30, 203-209 Walton, N.J., Woolhouse, H.W. (1983) Effects of illumination

N.J. Walton and H.W. Woolhouse: Serine and glycine metabolism and glycollate oxidation in promoting glyoxylate decarboxylation by pea leaf protoplasts. Planta 158, 469-471 Woo, K.C. (1979) Properties and intramitochondrial localization of serine hydroxymethyltransferase in leaves of higher plants. Plant Physiol. 63, 783-787 Woo, K.C., Osmond, C.B. (1976) Glycine decarboxylation in mitochondria isolated from spinach leaves. Aust. J. Plant Physiol. 3, 771-785 Zakrewski, S.F., Sansone, A.M. (1971) A new preparation of tetrahydrofolic acid. Methods Enzymol. 18, 728-731 Received 23 July; accepted 10 September 1985

Enzymes of serine and glycine metabolism in leaves and non-photosynthetic tissues of Pisum sativum L.

A comparative study is presented of the activities of enzymes of glycine and serine metabolism in leaves, germinated cotyledons and root apices of pea...
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