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Plant Physiol. (1983) 72, 134-138 0032-0889/83/72/0134/05/$00.50/0 Synthesis of Oxalic Acid by Enzymes from Lettuce Leaves Received for publication September 8, 1982 and in revised form December 17, 1982 DAVID D. DAVIES1 AND HASSAN ASKER1 Station de Physiologie Vegeitale, Institut National de la Recherche Agronomique, La Grande Ferrade, 33140 Pont-de-la-Maye, Bordeaux, France ABSTRACT local market. Purification of Glycolate Oxidase and Lactate Dehydrogenase. A rapid purification oflactate dehydrogenase and glycolate oxidase from All operations were carried out at 20C. Washed and deveined lettuce (Lactuca saliva) leaves is described. The kinetics of both enzymes lettuce leaves (400 g) were homogenized with 40 g of insoluble are reported in relation to their possible roles in the production of oxalate. Polyclar and 400 ml of K-phosphate (pH 7.4, 0.1 M) containing Lettuce lactate dehydrogenase behaves like mammalian dehydrogenase, fl-mercaptoethanol (5 mM). Prior to homogenization, the mixture catalyzing the dismutation of glyoxylate to glycolate and oxalate. A model was deoxygenated by bubbling N2. After homogenization, the is proposed in which glycolate oxidase in the peroxisomes and lactate extract was squeezed through cheese cloth and centrifuged at dehydrogenase in the cytosol are involved in the production of oxalate. 23,000g for 25 min. The supernatant was fractionated with The effect of pH on the balance between oxalate and glycolate produced (NH4)2SO4, the fraction precipitating between 25 and 55% satu- from glyoxylate suggests that in leaves lactate dehydrogenase may function ration was collected by centrifuging (23,000g for 20 min), dissolved as part of an oxalate-based biochemical, pH-stat. in 50 ml of Tris-HCl buffer (pH 8.0, 5 mM), and dialyzed against 5 L ofthe same buffer. The buffer was changed until no (NH4)2SO4 could be detected by Nesslers reagent. Any precipitate was re- moved by centrifuging at 32,000g for 10 min and the clear solution poured on to a DEAE-cellulose column (2.5 x 30 1m) previously equilibrated with the same buffer. When the extract had passed through the column, the column was washed with 50 ml of the The enzyme glycolate oxidase (EC 1.1.3.1), which catalyzes the buffer used for equilibration. The enzymes were eluted with a oxidation of short chain a-hydroxyacids, was first demonstrated linear gradient of KCl (0-0.5 M) in 500 ml of the same buffer, and in green leaves by Tolbert et al. (28) and shown to be a flavoprotein 5-ml fractions were collected. Glycolate oxidase emerged as a by Zelitch and Ochoa (31). Originally, the enzyme was thought single peak followed by a single peak of lactate dehydrogenase. not to be capable of oxidizing glyoxylate, but Richardson and Purification of Glycolate Oxidase. The peak fractions were Tolbert (22) demonstrated that purified glycolate oxidase from a combined and dialyzed for 4 h against K-phosphate (pH 6.3, 50 number of plants catalyzed the oxidation of glyoxylate to oxalate. mM) and any precipitate which formed was removed by centrifug- It is assumed that the substrate is the hydrated form of glyoxylate ing at 32,000g for 10 min. The clear solution was applied to a CH(0H2)COOH, and, as such, it is an a-hydroxyacid analog. The column (1.5 x 12 cm) of Sepharose-aminohexyl oxamate previ- implication that glycolate oxidase is responsible for the in vivo ously equilibrated with K-phosphate (pH 6.0, 50 mM). When the synthesis of oxalate from glyoxylate is paralleled by similar con- extract had passed through the column, the column was washed siderations in the case of rat liver (15). On the other hand, with a solution of KC1 (0.5 M) in the same buffer until pigments mammalian lactate dehydrogenase (EC 1.1.1.27), which can cat- present in the extract had reached the bottom of the column. The alyze the dismutation of glyoxylate to glycolate and oxalate (23), pigments (and a small amount ofglycolate oxidase) were removed has been considered the main enzyme catalyzing the oxidation of by washing the column with 3 bed volumes ofbuffer without KCl. glyoxylate to oxalate in leucocytes and erythrocytes (25) as well as The enzyme was then eluted with glycine buffer (pH 8.5, 0.1 M). in the cytosol of human liver and heart (11). Purification ofLactate Dehydrogenase. The peak fractions from The demonstration by Betsche et al. (2) that some green leaves the DEAE-column were combined and dialyzed for 5 h against contain lactate dehydrogenase raises the possibility that it plays a K-phosphate (pH 6.8, 50 mM). The solution was clarified by part in the production of oxalate in leaves. In this paper, we centrifuging (32,000g for 10 min) and NADH (25 mg/100 ml) was examine this possibility and suggest that the oxidation of glyox- added. The solution was then applied to a column (1.5 x 12 cm) ylate to oxalate involves the interaction of lactate dehydrogenase of Sepharose aminohexyl oxamate, previously equilibrated with and glycolate oxidase. K-phosphate (pH 6.8, 50 mM) containing KC1 (0.5 M) and NADH (1 16 jtM). When 10 ml of extract had passed into the column, the MATERIALS AND METHODS column was washed with 30 ml of the equilibrating buffer. This procedure was repeated until all the extract had been applied to Reagents. Reagents for polyacrylamide gel electrophoresis as the column. After the final wash, the enzyme was eluted by well as glyoxylate, oxamate, oxalate, glycolate, and nucleotides omitting NADH from the buffer. were obtained from Sigma. CNBr-activated Sepharose, PBE 94, Affinity Gel. Immobilized Sepharose aminohexyl oxamate was and poly buffer 96 were obtained from Pharmacia, and [I-14C] prepared by the method of O'Carra and Barry (18). glyoxylate from the Radiochemical Centre, Amersham. All other Protein Measurement. Protein was measured by the method of reagents were the highest purity available. Chiappelli et al. (4). Plant Material. Lettuce (Lactuca sativa) was bought from a Enzyme Assays. Glycolate Oxidase. Glycolate oxidase was assayed by a slight 'Present address: School of Biological Sciences, University of East modification of the method of Zelitch (30). A stock solution Anglia, Norwich NR4 7TJ U.K. containing Na glycolate (2 mM), 2,6-dichlorophenolindophenol 134 Downloaded from on May 4, 2019 - Published by www.plantphysiol.org Copyright © 1983 American Society of Plant Biologists. All rights reserved. LACTATE DEHYDROGENASE AND OXALIC ACID SYNTHESIS 135 scribed by Dietz and Lubrano (8). Gels were stained for proteins and lactate dehydrogenase activity as described by Poerio and 2 Davies (21). For detection of lactate dehydrogenase, the solution E contained L(+) lactate (50 mM), NAD+ (1.25 mM), N-methylphen- azine methosulfate (0.1 mM), 3-(3,5-dimethylthiazol-2-yl)-2,5 di- z D phenyltetrazolium bromide (1 mM), and 73 mm glycine/NaOH buffer (pH 8.5). For detection ofglycolate oxidase, the lactate and NAD+ were replaced by glycolate (2 mM). 1 Chromatofocusing. Chromatofocusing was performed with u polybuffer exchanger PBE-94, using Tris-HCl buffer (pH 8.5, 25 mM) as the starter buffer and polybuffer 96 for elution, as described by Pharmacia. This method is particularly effective for the lettuce NJ z glycolate oxidase, but because ofthe high isoelectric point reported LLJ b. for the enzyme from peas (13), the method may require modifi- "a cation when applied to peas and possibly to other plant material. L I I 20 40 60 80 Determination of Oxalate. The formation of ['4CJoxalate from was and FRACTION NUMBER [1-'4C]glyoxylate measured after adding carrier oxalate precipitating as the calcium salt as described by Hodgkinson and FIG. 1. DEAE-cellulose chromatography of glycolate oxidase and lac- Wilkinson (12). Radioactivity was determined in an Intertech- tate dehydrogenase from lettuce leaves, following (NH4)2SO4 fractionation nique scintillation counter. (30-55% saturation). (0), glycolate oxidase; (O), lactate dehydrogenase. RESULTS (35 mg/l) and Bicine buffer (pH 8.5, 80 mM) was incubated at 25°C and deoxygenated by the constant bubbling of N2. Enzyme Purification of Glycolate Oxidase and Lactate Dehydrogenase. (50 1d) was mixed with 950 pi of the stock solution in a l-ml The two enzyme activities were separated on DEAE-cellulose cuvette (light-path, 1 cm) and the decrease in A at 620 nm was (Fig. 1). The best separation was achieved when the fraction measured in a Perkin Elmer 550 S spectrophotometer. precipitating between 30 and 50%o saturation of (NH4)2SO4 was Glyoxylate Oxidase. Glyoxylate oxidase activity of glycolate used, but a higher recovery oflactate dehydrogenase was achieved oxidase was measured in the same way as glycolate oxidase except using a 25 to 55% (NH4)2SO4 fractionation. A high degree of that glyoxylate (25 mM) replaced the glycolate. further purification (36-fold for glycolate oxidase and 1,295-fold Lactate Dehydrogenase. Lactate dehydrogenase with pyruvate for lactate dehydrogenase) was achieved by affinity chromatog- and NADH as the substrates were measured according to Davies raphy on Sepharose aminohexyl oxamate. We have previously and Davies (5). used this material to purify lactate dehydrogenase from plants Glyoxylic Reductase. The glyoxylic reductase activity of lactate (21), and its use for the purification of glycolate oxidase suggested dehydrogenase was measured in the same way as lactate dehydro- itself from the report that N-octyloxamate is a strong inhibitor of genase, except that Bicine buffer (pH 8.5, 100 mM) was used and mammalian glycolate oxidase (24). The overall purification for glyoxylate (50 mM) replaced pyruvate. both enzymes is given in Table I. Glyoxylate Dehydrogenase. The glyoxylate dehydrogenase activ- Gel electrophoresis ofboth purified enzymes indicated that they ity of lactate dehydrogenase was measured by incubating Na were homogeneous and no evidence for the presence ofisoenzymes glyoxylate (50 mM), NAD (2.75 mg), Bicine buffer (pH 8.5, 100 was obtained.

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