Characterization of a cDNA Encoding Lens culinaris Glycolate Oxidase and Developmental Expression of Glycolate Oxidase mRNA in Cotyledons and Leaves

mRNA obtained from green leaves of lentil (Lens culinaris) was used to construct a cDNA library in phage λgt11. The cDNA library was screened with antibodies raised against lentil glycolate oxidase and catalase. Clone CL 1 containing the full-length sequence complementary to glycolate oxidase mRNA was characterized and sequenced. In addition, a 800-base pair catalase cDNA clone was sequenced. To prove the correlation of cDNA insert in CL 1 with glycolate oxidase, the cDNA was transcribed in vitro. The mRNA was translated in vitro yielding a 43 kilodalton protein immunoprecipitable with anti-glycolate oxidase serum. Nucleotide sequences of lentil cDNA and spinach cDNA were 86% identical. Lentil glycolate oxidase was characterized by a C-terminal sequence -P-R-A-L-P-R-L. The expression of glycolate oxidase mRNA in cotyledons, leaves and roots was compared with that of catalase. In leaves, the relative amount of glycolate oxidase mRNA increased during the first 2 days of greening, but decreased later, and was hardly detectable during senescence. In cotyledons of germinating seeds, the level of glycolate oxidase mRNA was markedly lower than the catalase mRNA.     

Identification and Characterization of Glycolate Oxidase and Related Enzymes from the Endocyanotic Alga Cyanophora paradoxa and from Pea Leaves

Glycolate oxidase (GO) has been identified in the endocyanom Cyanophora paradoxa which has peroxisome-like organelles and cyanelles instead of chloroplasts. The enzyme used or formed equimolar amounts of O₂ or H₂O₂ and glyoxylate, respectively. Aerobically, the enzyme did not reduce the artificial electron acceptor dichlorophenol indophenol. However, after an inhibitor of glycolate dehydrogenase, KCN (2 millimolar), was added to the assay medium, considerable aerobic glycolate:dichlorophenol indophenol reductase activity was detectable. The leaf GO inhibitor 2-hydroxybutynoate (30 micromolar), which binds irreversibly to the flavin moiety of the active site of leaf GO, inhibited Cyanophora 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 contrast to GO from pea or other organisms, the affinity of Cyanophora GO for l-lactate is very low (K_m 25 millimolar). Another important difference is that Cyanophora GO produced sigmoidal kinetics with O₂ as varied substrate, whereas pea GO produced normal Michaelis-Menten kinetics. It is concluded that there is considerable inhomogeneity among the glycolate-oxidizing enzymes from Cyanophora, pea, and other organisms. The specific catalase activity in Cyanophora was only one-tenth of that in leaves. NADH-and NADPH-dependent hydroxypyruvate reductase (HPR) and glyoxylate reductase activities were detected in Cyanophora. NADH-HPR was markedly inhibited by hydroxypyruvate above 0.5 millimolar. Variable substrate inhibition was observed with glyoxylate in homogenates from different algal cultures. It is proposed that Cyanophora has multiple forms of HPR and glyoxylate reductase, but no enzyme clearly resembling leaf peroxisomal HPR was identified in these homogenates. Moreover, no serine:glyoxylate aminotransferase activity was detected. These results collectively indicate the possibility that the glycolate metabolism in Cyanophora deviates from that in leaves.     

Physical,chemical, and regulatory properties of glycolate oxidase in C<Subscript>3</Subscript> and C<Subscript>4</Subscript> plants

Physical, chemical, and regulatory properties of glycolate oxidase (GO) isolated from the leaves of C₄ and C₃ plants (Zea mays L., cv. Voronezhskaya 76 and Glycine max (L.) Merr., cv. Pripyat’, respectively) were studied. The homogenous preparations were obtained by multistage enzyme purification from soybean leaves and maize mesophyll and bundle sheath. The glycolate oxidase (GO) preparations obtained consisted of two types of subunits, 37 and 44 kD. The GO isolated from C₃ plant leaves had many in common with that extracted from C₄ plant bundle sheath as regards physical, chemical, and catalytic properties. The primary function of GO in both plant types is metabolism of glycolate, which is a product of ribulosebisphosphate oxalacetic acid oxidation and is used by plants for biosynthesis of hydrocarbons and amino acids.     

Effect of some reducing agents on water stress-induced oxidative and deteriorative processes ofVigna seedlings

Decreasing substrate osmotic potential produced in seedlings ofVigna catjang Endl. (cv. Pusa Barsati) proportional decrease in relative water content and leaf water potential, increase in respiration rate, proline content, H₂O₂ content, and the activities of indole acetic acid oxidase, ascorbic acid oxidase, peroxidase and glycolate oxidase but decrease in catalase activity and glycolate content. Pretreatment with reducing agents like L-cysteine or reduced glutathione (10?3 M) caused lower decrease in the relative water content, leaf water potential and glycolate content and reduced the rise of respiration rate, proline content and H₂O₂ content and also the activities of aforementioned oxidative enzymes, except catalase activity which was increased. Such treatments also maintained the chlorophyll and protein levels and decreased the tissue permeability. It was concluded that the treatment ofVigna seedlings with reducing agents reduced the deteriorative changes and oxidative processes which are characteristic of water stressed tissue.     

Molecular structure and subcellular localization of spinach leaf glycolate oxidase

Glycolate oxidase (E.C. 1.1.3.1) was purified from spinach leaves (Spinacia oleracea). The molecular weight of the native protein was determined by sucrose density gradient centrifugation to be 290,000 daltons (13S), whereas that of the monomeric form was 37,000 daltons. The quaternary structure of the holoenzyme is likely to be octameric, analogous to pumpkin cotyledon glycolate oxidase [Nishimura et al, 1982]. The subcellular localization of the enzyme was studied using linear sucrose density gradient centrifugation, and it was found that glycolate oxidase activity is detectable in both leaf peroxisomal and supernatant fractions, but not in chloroplasts and mitochondria; the activity distribution pattern is essentially similar to that for catalase, a known leaf peroxisomal enzyme. Ouchterlony double diffusion and immunotitration analyses, demontrated that the rabbit antiserum against purified spinach leaf glycolate oxidase cross-reacted, identically, with the enzyme molecules present in two different subcellular fractions, i.e, the leaf peroxisome and supernatant fractions. It is thus concluded that the enzyme present in the supernatant is due to the disruption of leaf peroxisomes during the isolation, and hence glycolate oxidase is exclusively localized in leaf peroxisomes in spinach leaves.     

Molecular structure and subcellular localization of spinach leaf glycolate oxidase

Glycolate oxidase (E.C. 1.1.3.1) was purified from spinach leaves (Spinacia oleracea). The molecular weight of the native protein was determined by sucrose density gradient centrifugation to be 290,000 daltons (13S), whereas that of the monomeric form was 37,000 daltons. The quaternary structure of the holoenzyme is likely to be octameric, analogous to pumpkin cotyledon glycolate oxidase [Nishimura et al, 1982]. The subcellular localization of the enzyme was studied using linear sucrose density gradient centrifugation, and it was found that glycolate oxidase activity is detectable in both leaf peroxisomal and supernatant fractions, but not in chloroplasts and mitochondria; the activity distribution pattern is essentially similar to that for catalase, a known leaf peroxisomal enzyme. Ouchterlony double diffusion and immunotitration analyses, demonstrated that the rabbit antiserum against purified spinach leaf glycolate oxidase cross-reacted, identically, with the enzyme molecules present in two different subcellular fractions, i.e, the leaf peroxisome and supernatant fractions. It is thus concluded that the enzyme present in the supernatant is due to the disruption of leaf peroxisomes during the isolation, and hence glycolate oxidase is exclusively localized in leaf peroxisomes in spinach leaves.     

Catalytic and redox properties of glycine oxidase from Bacillus subtilis

Glycine oxidase (GO) from Bacillus subtilis is a homotetrameric flavoprotein oxidase that catalyzes the oxidation of the amine functional group of sarcosine or glycine (and some d-amino acids) to yield the corresponding keto acids, ammonia/amine and H₂O₂. It shows optima at pH 7–8 for stability and pH 9–10 for activity, depending on the substrate. The tetrameric oligomeric state of the holoenzyme is not affected by pH in the 6.5–10 range. Free GO forms the anionic red semiquinone upon photoreduction. This species is thermodynamically stable, as indicated by the large separation of the two single-electron reduction potentials (ΔE ≥ 290 mV). The first potential is pH independent, while the second is dependent. The midpoint reduction potential exhibits a −23.4 mV/pH unit slope, which is consistent with an overall two-electrons/one-proton transfer in the reduction to yield anionic reduced flavin. In the presence of glycolate (a substrate analogue) and at pH 7.5 the potential for the semiquinone-reduced enzyme couple is shifted positively by ∼160 mV: this favors a two-electron transfer compared to the free enzyme. Binding of glycolate and sulfite is also affected by pH, showing dependencies that reflect the ionization of an active site residue with a pK_a ≈ 8.0. These results highlight substantial differences between GO and related flavoenzymes. This knowledge will facilitate biotechnological use of GO, e.g. as an innovative tool for the in vivo detection of the neurotransmitter glycine.     

菠菜中的乙醇酸氧化酶是一个同工酶

乙醇酸氧化酶（ＥＣ．１．１．３．１５．ＧＯ）是光呼吸途径的关键酶,降低其活性可提高Ｃ３植物如水稻的产量,在目前中国乃至世界人口不断增加和可耕种土地日益减少的情况下,对ＧＯ的研究具有重要的理论意义和实际应用价值．在光呼吸途径被提出后的几十年间,人们对如...     

Activities and subcellular localization of enzymes responsible for lipolysis and gluconeogenesis during the germination of Brassica campestris cv. esculenta seeds

During the growth of turnip seedlings, two new lipases have been demonstrated, one with a maximum activity at pH 4.5 (acid lipase) and the other with a maxima at pH 8.6 (alkaline lipase). Many different enzymes are involved in gluconeogenesis: catalase, isocitrate lyase, malate synthetase, malate dehydrogenase, aconitase, citrate synthetase, fumarase, glycolate oxidase, phosphoenol-pyruvate carboxykinase. All of these show maximum activity coinciding with the stage in which lipid hydrolysis is maximal and when the accumulation of soluble carbohydrates has also reached its peak. The alkaline lipase as found to be located mainly in the spherosomes, whereas the glyoxysomes contained the following main activities: catalase, isocitrate lyase, malate synthetase, malate dehydrogenase and citrate synthetase. Aconitase, together with cytochrome oxidase and fumarase showed their highest activity in the mitochondria, and the presence of malate dehydrogenase, citrate synthetase and glycolate oxidase was also observed in these organelles. In the membrane-bound fraction, the activities of cytochrome reductase, glycolate oxidase and phosphoenol-pyruvate kinase were marked, although the latter enzyme was even more active in the soluble fraction.     

The Effect of Light on the Structure and Organization of Lemna Peroxisomes

The effect of light on a number of Lemna minor enzyme activitieswas investigated. The levels of activity of glycolate oxidase,catalase and RuBPCase increased with increasing irradiance,paralleling the increase in Lemna growth rate. In contrast withresults obtained for other species, no glycolate oxidase activitycould be detected in etiolated Lemna fronds or when these weretreated with light or glycolate, in vivo or in vitro, for upto 24 h. The number of peroxisome profiles per cell section was determinedin Lemna grown under different light conditions. When frondswere grown under dim light, the number of peroxisome profilesper cell section appeared to increase with increasing irradiance,although no further increase in the peroxisome number was apparentwhen the fronds were grown under higher irradiances. The levelof glycolate oxidase activity per peroxisome was shown to increasewith increasing irradiance, whereas that of catalase remainedrelatively constant, indicating that differential addition ofenzymes to pre-existing peroxisomes is possible. Peroxisomes from Lemna grown under high irradiance were subjectedto serial sectioning and examined under the electron microscope.Some peroxisomes were found to have a three dimensional structuresuggesting either fission and/or fusion or branching of theseorganelles, supporting the hypothesis of a peroxisomal reticulum.The dynamic relationship between the various shapes is discussed. Key words: Peroxisomes, glycolate oxidase, catalase     

Exploring glycolate oxidase (GOX) as an antiurolithic drug target: molecular modeling and in vitro inhibitor study

Glycolate oxidase (GOX) is one of the principal enzymes involved in the pathway of oxalate synthesis. It converts glycolate to glyoxylate by oxidation and then glyoxylate is finally converted to oxalate. Therapeutic intervention of GOX in this consequence thus found potential in the treatment of calcium oxalate urolithiasis. In present investigation, we explored GOX in search of potential leads from traditional resources. Molecular modeling of the identified leads, quercetin and kaempherol, was performed by employing Glide 5.5.211 (SchrodingerTM suite). In the absence of pure human glycolate oxidase (hGOX) preparation, in vitro experiments were performed on spinach glycolate oxidase (sGOX) as both enzymes possess 57% identity and 76% similarity along with several conserved active site residues in common. We aimed to identify a possible mechanism of action for the anti-GOX leads from Tribuls terrestris, which can be attributed to anti-urolithic drug development. This study found promising in development of future GOX inhibitory leads.     

Effect of butyl 2-hydroxy-3-butynoate on sunflower leaf photosynthesis and photorespiration

Detached leaves and whole plants of sunflower were supplied with butyl 2-hydroxy-3-butynoate (BHB), a competitive inactivator of glycolate oxidase, to evaluate the possibility of inhibiting photorespiration and increasing photosynthetic efficiency. In all treatments in vivo and in vitro, BHB inhibited glycolate oxidase. With partially purified glycolate oxidase from spinach leaves, the apparent K_i for BHB was 13.2 micromolar.     

Purification and immuno-electron microscopical characterization of crystalline inclusions from plant peroxisomes

Summary This paper describes the first purification method for crystalline inclusions (cores) from plant peroxisomes and an ultrastructural characterization of these isolated cores. 5-day-old sunflower (Helianthus annuus L.) cotyledon fractions which were highly enriched in cores showed negligible activity of the matrix enzyme glycolate oxidase but high catalase activity. As proven by electron microscopy, crystalline particles were surrounded neither by matrix material nor by membranes. Their geometrical outlines and ultrastructure were identical to those of cores in tissue sections, as was their reactivity with three different polyclonal catalase antibodies in the immunogold technique. Three-dimensional reconstruction, based on the geometrical outlines and ultrastructure of sectioned isolated cores from sunflower, suggested that they were quadrangular blocks. Ultrastructural analysis revealed an even periodic arrangement of repeating units which are probably cubes with 20 nm long edges. Isolated peroxisomal cores from potato (Solanum tuberosum L.) tubers had outlines which suggested that they were even rhomboidal prisms. They showed a granular ultrastructure without any repeating units and contained catalase, demonstrated by immunogold labelling and enzyme activity measurement. The results presented here suggested the hypothesis that the structural elements in plant peroxisomal cores are made of enzymatically active catalase, although the substructure may vary from species to species.Abbreviations ACOx acyl-CoA oxidase - BSA bovine serum albumin - EDTA ethylenediamine-tetraacetate - GDH glutamate dehydrogenase - GOx glycolate oxidase - KPB potassium phosphate buffer     

Developmental Studies on Microbodies in Wheat Leaves : II. Ontogeny of Particulate Enzyme Associations

Crude particulate fractions from wheat leaves (Triticum vulgare L.) were separated on continuous sucrose density gradients, resulting in: broken chloroplasts, a mitochondrial fraction (indicated by cytochrome c oxidase), and microbodies. The visible band of the microbody fraction from adult leaves appears at a buoyant density of 1.25 grams per cm³ and contains most of the activities of catalase, glycolate oxidase, and hydroxypyruvate reductase on the gradient. In the shoots of freshly soaked seeds, catalase is already highly particulate. During further development in light or in darkness, 40 to 60% of the total activities of catalase and glycolate oxidase and 25 to 40% of the total activity of hydroxypyruvate reductase are always found in the particulate fractions of the leaves. In young developmental stages, the peaks of the activity profiles of the microbody enzymes appear on sucrose gradients at relatively low densities, first between 1.17 to 1.20 grams per cm³. During development in light, the buoyant density of the microbody fraction shifts to the final value of 1.25 grams per cm³. However, even after 1 week of growth in the dark, the microbody fraction from etiolated leaves was observed at buoyant densitites 1.17 to 1.24 grams per cm³ and did not appear as a defined visible band. A characteristic visible microbody band at a buoyant density 1.24 grams per cm³ was found when the dark-grown seedlings received only three separate 5-minute exposures to white light. A similar peak was also obtained from light-grown leaves in which chloroplast development had been blocked by 3-amino-1,2,4-triazole.     

Cytochemical demonstration of malate synthase and glycolate oxidase in microbodies of cucumber cotyledons

The cytochemical localizations of malate synthase (glyoxysomal marker) and glycolate oxidase (peroxisomal marker) have been examined in cotyledon segments and sucrose-gradient fractions from germinated cucumber (Cucumis sativus L.) seedlings. The seedlings were grown in the dark for 4 days, transferred to 4 hours of continuous light, then returned to the dark for 24 hours. Under these conditions, high specific activities for both glyoxysomal and peroxisomal enzymes are maintained in cotyledon homogenates and microbody-enriched fractions. Electron cytochemistry of the marker enzymes reveals that all or virtually all the microbodies observed in cotyledonary cells and sucrose-gradient fractions contain both enzymes. The staining in gradient fractions was determined from scoring a minimum of 600 photographed microbodies for each enzyme. After correcting for the number of particles stained for catalase reactivity (representing true microbodies), 94 and 97% of the microbodies were found stained for malate synthase and glycolate oxidase activity, respectively.     

Experimental Evidence for a Hydride Transfer Mechanism in Plant Glycolate Oxidase Catalysis

In plants, glycolate oxidase is involved in the photorespiratory cycle, one of the major fluxes at the global scale. To clarify both the nature of the mechanism and possible differences in glycolate oxidase enzyme chemistry from C₃ and C₄ plant species, we analyzed kinetic parameters of purified recombinant C₃ (Arabidopsis thaliana) and C₄ (Zea mays) plant enzymes and compared isotope effects using natural and deuterated glycolate in either natural or deuterated solvent. The ¹²C/¹³C isotope effect was also investigated for each plant glycolate oxidase protein by measuring the ¹³C natural abundance in glycolate using natural or deuterated glycolate as a substrate. Our results suggest that several elemental steps were associated with an hydrogen/deuterium isotope effect and that glycolate α-deprotonation itself was only partially rate-limiting. Calculations of commitment factors from observed kinetic isotope effect values support a hydride transfer mechanism. No significant differences were seen between C₃ and C₄ enzymes.     

Subcellular Distribution of Enzymes of Glycolate Metabolism in the Alga Cyanidium caldarium

The intracellular distribution of enzymes capable of catalyzing the reactions from phosphoglycolate to glycerate in the bluegreen colored eucaryotic alga Cyanidium caldarium has been studied. After separating the organelles from a crude homogenate on a linear flotation gradient, the enzymes glycolate oxidase and glutamate-glyoxylate aminotransferase along with catalase were present in the peroxisomal fraction (density: 1.23 grams per cubic centimeter). Serine hydroxymethyltransferase was found in the mitochondrial fraction (density: 1.18 grams per cubic centimeter). In contrast to the observations in green leaves of higher plants, the enzymes for the conversion of serine to glycerate (serine-glyoxylate aminotransferase and hydroxypyruvate reductase) were found only in the soluble fraction of the gradient. The partial characterization of enzymes from Cyanidium participating in glycolate metabolism revealed only slight differences from the corresponding enzymes from higher plants. The phylogenetic implications of the observed similarities between the enigmatic alga Cyanidium and higher plants are discussed.     

Glycine oxidase from Bacillus subtilis. Characterization of a new flavoprotein.

Glycine oxidase (GO) is a homotetrameric flavoenzyme that contains one molecule of non-covalently bound flavin adenine dinucleotide per 47 kDa protein monomer. GO is active on various amines (sarcosine, N-ethylglycine, glycine) and d-amino acids (d-alanine, d-proline). The products of GO reaction with various substrates have been determined, and it has been clearly shown that GO catalyzes the oxidative deamination of primary and secondary amines, a reaction similar to that of d-amino acid oxidase, although its sequence homology is higher with enzymes such as sarcosine oxidase and N-methyltryptophane oxidase. GO shows properties that are characteristic of the oxidase class of flavoproteins: it stabilizes the anionic flavin semiquinone and forms a reversible covalent flavin-sulfite complex. The approximately 300 mV separation between the two FAD redox potentials is in accordance with the high amount of the anionic semiquinone formed on photoreduction. GO can be distinguished from d-amino acid oxidase by its low catalytic efficiency and high apparent K(m) value for d-alanine. A number of active site ligands have been identified; the tightest binding is observed with glycolate, which acts as a competitive inhibitor with respect to sarcosine. The presence of a carboxylic group and an amino group on the substrate molecule is not mandatory for binding and catalysis.     

Purification and properties of glycolate oxidase from plants with different photosynthetic pathways: Distinctness of C4 enzyme from that of a C3 species and a C3–C4 intermediate

Glycolate oxidase (GO; EC 1.1.3.1) was purified from the leaves of three plant species:Amaranthus hypochondriacus L.(NAD-ME type C₄ dicot),Pisum sativum L. (C₃ species) andParthenium hysterophorus L. (C₃–C₄. intermediate). A flavin moiety was present in the enzyme from all the three species. The enzyme from the C₄ plant had a low specific activity, exhibited lower K_M for glycolate, and required a lower pH for maximal activity, compared to the C₃ enzyme. The enzyme from the C₄ species oxidized glyoxylate at <10% of the rate with glycolate, while the GO from the C₃ plant oxidized glyoxylate at a rate of about 35 to 40% of that with glycolate. The sensitivity of GO from C₄ plant to -hydroxypyridinemethane sulfonate, 2-hydroxy-3-butynoate and other inhibitors was less than that of the enzyme from C₃ source. The properties of GO fromParthenium hysterophorus, were similar to those of the enzyme fromPisum sativum. The characteristics of glycolate oxidase from leaves of a C₄ plant,Amaranthus hypochondriacus are different from those of the C₃ species or the C₃–C₄ intermediate.     

Glycolate metabolism and subcellular distribution of glycolate oxidase in Spatoglossum pacificum (Phaeophyceae, Chromophyta)

Seven enzymes participating in glycolate metabolism were demonstrated to be present in crude extract of the brown alga Spatoglossum pacificum Yendo. These were phosphoglycolate phosphatase, glycolate oxidase, glutamate-glyoxylate aminotransferase, serine hydroxymethyltransferase, amino acid-hydroxy-pyruvate aminotransferase, hydroxypyruvate reductase and catalase. Malate synthase, which is involved in glycolate metabolism in the xanthophycean alga, could not be detected. On demonstration of subcellular distribution of glycolate oxidizing enzymes by linear sucrose density gradient centrifugation, glycolate oxidase was detected in the same fraction at a density of 1.23 g cm^?3 with catalase: that is, the marker enzyme of peroxisome and serine hydroxymethyltransferase was found in the same fraction at a density of 1.21 g cm^?3 with isocitrate dehydrogenase, the marker of mitochondria. From the present data, it is proposed that the brown alga Spatoglossum possesses the ability to metabolize glycolate to glycerate via the pathway which may be similar to that of higher plants.