Chemical inhibition of the glycolate pathway in soybean leaf cells

Isolated soybean (Glycine max [L.] Merr.) leaf cells were treated with three inhibitors of the glycolate pathway in order to evaluate the potential of such inhibitors for increasing photosynthetic efficiency. Preincubation of cells under acid conditions in α-hydroxypyridinemethanesulfonic acid increased ¹⁴CO₂ incorporation into glycolate, but severely inhibited photosynthesis. Isonicotinic acid hydrazide (INH) increased the incorporation of ¹⁴CO₂ into glycine and reduced label in serine, glycerate, and starch. Butyl 2-hydroxy-3-butynoate (BHB) completely and irreversibly inhibited glycolate oxidase and increased the accumulation of ¹⁴C into glycolate. Concomitant with glycolate accumulation was the reduction of label in serine, glycerate, and starch, and the elimination of label in glycine. The inhibitors INH and BHB did not eliminate serine synthesis, suggesting that some serine is synthesized by an alternate pathway. The per cent incorporation of ¹⁴CO₂ into glycolate by BHB-treated cells or glycine by INH-treated cells was determined by the O₂/CO₂ ratio present during assay. Photosynthesis rate was not affected by INH or BHB in the absence of O₂, but these compounds increased the O₂ inhibition of photosynthesis. This finding suggests that the function of the photorespiratory pathway is to recycle glycolate carbon back into the Calvin cycle, so if glycolate metabolism is inhibited, Calvin cycle intermediates become depleted and photosynthesis is decreased. Thus, chemicals which inhibit glycolate metabolism do not reduce photorespiration and increase photosynthetic efficiency, but rather exacerbate the problem of photorespiration.     

Induction of glycolate oxidase by SO2 in Nicotiana tabacum

In contrast to the inhibitory action of sulfite on glycolate oxidase, the specific activity of the enzyme in tobacco leaves exposed to SO₂ for 18 hr increases in proportion to the SO₂ concentration. This increase is strongly reduced by pretreatment with cycloheximide. As a consequence of induced de novo synthesis of glycolate oxidase the glycolate content of the leaves is markedly reduced after 18 hr exposure to SO₂.     

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.     

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.     

The occurrence of glycolate dehydrogenase and glycolate oxidase in green plants: an evolutionary survey

Homogenates of various lower land plants, aquatic angiosperms, and green algae were assayed for glycolate oxidase, a peroxisomal enzyme present in green leaves of higher plants, and for glycolate dehydrogenase, a functionally analogous enzyme characteristic of certain green algae. Green tissues of all lower land plants examined (including mosses, liverworts, ferns, and fern allies), as well as three freshwater aquatic angiosperms, contained an enzyme resembling glycolate oxidase, in that it oxidized l- but not d-lactate in addition to glycolate, and was insensitive to 2 mm cyanide. Many of the green algae (including Chlorella vulgaris, previously claimed to have glycolate oxidase) contained an enzyme resembling glycolate dehydrogenase, in that it oxidized d- but not l-lactate, and was inhibited by 2 mm cyanide. Other green algae had activity characteristic of glycolate oxidase and, accordingly, showed a substantial glycolate-dependent O₂ uptake. It is pointed out that this distribution pattern of glycolate oxidase and glycolate dehydrogenase among the green plants may have phylogenetic significance.     

Evidence for the presence in tobacco leaves of multiple enzymes for the oxidation of glycolate and glyoxylate

The enzymic oxidation of glycolate to glyoxylate and glyoxylate to oxalate by preparations purified from tobacco (Nicotiana tabacum var Havana Seed) leaves was studied. The K_m values for glycolate and glyoxylate were 0.26 and 1.0 millimolar, respectively. The ratio of glycolate to glyoxylate oxidation was 3 to 4 in crude extracts but decreased to 1.2 to 1.5 on purification by (NH₄)₂SO₄ fractionation and chromatography on agarose A-15 and hydroxylapatite. This level of glyoxylate oxidation activity was higher than that previously found for glycolate oxidase (EC 1.1.3.1). The ratio of the two activities was changed by reaction with the substrate analog 2-hydroxy-3-butynoate (HBA) which at all concentrations inhibited glyoxylate oxidation to a greater extent than glycolate oxidation. The ratio of the two activities could also be altered by changing the O₂ concentration. Glycolate oxidation increased 3.6-fold when the O₂ atmosphere was increased from 21 to 100%, whereas glyoxylate oxidation increased only 1.6-fold under the same conditions. These changes in ratio during purification, on inhibition by HBA, and under varying O₂ concentrations imply that tobacco leaves contain at least two enzymes capable of oxidizing glycolate and glyoxylate.     

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.     

Glycolate synthesis in a C3 C4 and intermediate photosynthetic plant type

Excised leaves of a C₃-photosynthetic type, Hordeum vulgare,a C₄-type, Panicum miliaceum, and an intermediate-type, Panicummilioides, were allowed to take up through their cut ends a1 mM solution of butyl hydroxybutynoate (BHB), an irreversibleinactivator of glycolate oxidase. After 30 to 60 min in BHB,extractable glycolate oxidase activity could not be detectedin the distal quarter of the leaf blades. Following this pretreatment,recovery of ¹⁴C-glycolate from ¹⁴CO₂ incorporated in a 10 minperiod was nearly maximal for each of the three plant types.Labeled glycolate was 51% of the total ¹⁴CO₂ incorporated forthe C₃-species, 36% for the intermediate-species, and 27% forthe C₄-species Increased labeling of glycolate was compensatedfor primarily by decreased labeling of the neutral and basicfractions for the C₃ and intermediate-type species. In the C₄-type,label decreased primarily in the neutral and insoluble fractions,but increased in the basic fraction. A lower rate of glycolatesynthesis is indicative of a lower rate of photorespirationand consistent with a lower O₂/CO₂ ratio present in the bundle-sheathcells of C₄-plants. We conclude that both decreased glycolatesynthesis and the refixation of photorespiratory-released CO₂are important in maintaining a lower rate of photorespirationin C₄-plants compared to C₃ plants. Intermediate glycolate synthesisin Panicum milioldes is consistent with its intermediate levelof O₂ inhibition of photosynthesis and intermediate rate ofphotorespiration. (Received May 6, 1978; )     

Synthesis of oxalic Acid by enzymes from lettuce leaves

A rapid purification of lactate dehydrogenase and glycolate oxidase from lettuce (Lactuca sativa) leaves is described. The kinetics of both enzymes are reported in relation to their possible roles in the production of oxalate. Lettuce lactate dehydrogenase behaves like mammalian dehydrogenase, catalyzing the dismutation of glyoxylate to glycolate and oxalate. A model is proposed in which glycolate oxidase in the peroxisomes and lactate dehydrogenase in the cytosol are involved in the production of oxalate. The effect of pH on the balance between oxalate and glycolate produced from glyoxylate suggests that in leaves lactate dehydrogenase may function as part of an oxalate-based biochemical, pH-stat.     

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.     

Identity of aliphatic L- -hydroxyacid oxidase and glycolate oxidase from rat livers

l-α-Hydroxyacid oxidase and glycolate oxidase have been partially purified from rat livers and found to be identical, judging by substrate specificities, K_m values for certain substrates and coenzyme (FMN), activation energy, inhibition rates by various reagents and pH optimum. K_m values are as follows; glycolate, 2.4 × 10^?4m; l-α-hydroxyisocaproate, 1.26 × 10^?3; glyoxylate, 1.41 × 10^?4m; and FMN, 1.13 × 10^?6m. K_m values for glycolate and FMN are one-tenth and one-twentieth the literature values for hepatic glycolate oxidase. Sucrose density gradient centrifugation establishes that this enzyme is located in hepatic peroxisomes.     

Source of glycolate and cyclic changes in photosynthetic and photorespiratory activity during the development of barley leaves

¹⁴CO₂ assimilation, RuBP earboxylase and PEP carboxylase activities show cyclic changes during the development of barley leaves. Cyclic changes, but in phase opposition with respect to carboxylating enzymes, are shown by RuBP oxygenase, phosphoglycolate phosphatase, glycolate oxidase and nitrate reductase activities. The oxygenase function of RuBP carboxylase appears to be the primary source of glycolate in young leaves, whereas in old ones glycolate could be supplied from some source in addition to RuBP oxygenase activity.     

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.     

Travels in a world of small science*

As a boy, I read Sinclair Lewis's Arrowsmithand dreamed of doing research of potential benefit to society. I describe the paths of my scientific career that followed. Several distinguished scientists served as my mentors and I present their profiles. Much of my career was in a small department at a small institution where independent researchers collaborated informally. I describe the unique method of carrying on research there. My curiosity about glycolate metabolism led to unraveling the enzymatic mechanism of the glycolate oxidase reaction and showing the importance of H₂O₂ as a byproduct. I discovered enzymes catalyzing the reduction of glyoxylate and hydroxypyruvate. I found α-hydroxysulfonates were useful competitive inhibitors of glycolate oxidase. In a moment of revelation, I realized that glycolate metabolism was an essential part of photorespiration, a process that lowers net photosynthesis in C₃ plants. I added inhibitors of glycolate oxidase to leaves and showed: (1) glycolate was synthesized only in light as an early product of photosynthetic CO₂ assimilation, (2) the rate of glycolate oxidation consumed a sizable fraction of net photosynthesis in C₃ but not in C₄ plants, and (3) that glycolate metabolism increased greatly at higher temperatures. For a while I studied the control of stomatal opening in leaves, and this led to the finding that potassium ions are a key solute in guard cells. I describe experiments that show that when photorespiration rates are high, as occurs at higher temperatures, genetically increasing leaf catalase activity reduces photorespiration and increases net photosythetic CO₂ assimilation. This revised version was published online in June 2006 with corrections to the Cover Date.     

Peroxisomal enzyme activity in Todea barbara gametophytes and sporophytes

Catalase and glycolate oxidase activity were observed in cultured gametophytes and sporophytes of the fern Todea barbara (L.) Moore. The biochemical characteristics of the glycolate-oxidizing enzyme in both plants indicates it is a glycolate oxidase. The results suggest that these plants are capable of photorespiration by a process similar to that occurring in leaves of higher plants.     

A Study of Formate Production and Oxidation in Leaf Peroxisomes during Photorespiration

When glycolate was metabolized in peroxisomes isolated from leaves of spinach beet (Beta vulgaris L., var. vulgaris) formate was produced. Although the reaction mixture contained glutamate to facilitate conversion of glycolate to glycine, the rate at which H₂O₂ became “available” during the oxidation of [1-¹⁴C]glycolate was sufficient to account for the breakdown of the intermediate [1-¹⁴C]glyoxylate to formate (C₁ unit) and ¹⁴CO₂. Under aerobic conditions formate production closely paralleled ¹⁴CO₂ release from [1-¹⁴C]glycolate which was optimal between pH 8.0 and pH 9.0 and was increased 3-fold when the temperature was raised from 25 to 35 C, or when the rate of H₂O₂ production was increased artificially by addition of an active preparation of fungal glucose oxidase.     

The Effect of Various Alkaline Salts on the Glycolate Oxidase of Salicornia europaea and Pisum sativum in vitro

The response of glycolate oxidase from shoots of Salicornia europaea L. and from leaves of Pisum sativum L. to salt treatment during assay was studied by DCPIP reduction and O₂ uptake. In Pisum there was found up to five times more glycolate oxidase activity per gram fresh weight than in Salicornia. However, the calculation of the specific activity pointed out that this result was caused only by the high level of enzyme protein in Pisum, and that specific activity from both species was of equal size. By the DCPIP method it was shown that in test media containing up to 1.0 M NaCl or KCl glycolate oxidase of Salicornia was of equal size compared with the control (medium without additional salts). With 2.0 M NaCl or KCl the activity decreased by about 80 and 30% respectively. Glycolate oxidase of Pisum was somewhat more salt sensitive. 1.0 M NaCl or KCl reduced the activity by about 35%. In the presence of 2.0 M NaCl or KCl the enzyme activity from Pisum was inhibited to about 80 and 60% respectively. By substituting sulfates for chlorides the activity of glycolate oxidase from both Salicornia and Pisum was stimulated strongly. 1.5 M Na₂SO₄ and 0.5 M K₂SO₄ (both are saturated solutions) caused an increase of glycolate activity from Salicornia of about 225 and 185% respectively, and from Pisum of about 50 and 30% respectively. Studying the response of glycolate oxidase to salt treatment by O₂ uptake one must establish that with this method the degree of inhibition of enzyme activity at higher salt concentrations was always more severe than with dye reduction. Addition of 1.0 M NaCl or KCl to the assay medium caused an inhibition of glycolate oxidase activity from Salicornia of about 50% and from Pisum of about 60%. 2.0 M NaCl or KCl reduced the enzyme activity of both Salicornia and Pisum to nearly 10% of control activity. Furthermore, in contrast to DCPIP reduction no stimulating effect of sulfates on glycolate oxidase activity was detectable. Indeed, the inhibitory effect of sulfates was very slight. 1.0 M Na₂SO₄ caused a mean inhibition of glycolate oxidase activity of only 15% with both species, and in the presence of 1.5 M Na₂SO₄ 50% of control activity was measured. At maximal K₂SO₄ concentrations (0.5 M) glycolate oxidase from both Salicornia and Pisum was also unaffected. It is supposed that the described salt tolerance of glycolate oxidase in vitro, possibly is due to an adaptation of the enzyme to high salt levels within peroxisomes in vivo.     

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.     

Glycolate oxidase content of microbodies as affected by nitrate

Glycolate oxidase is loosely held by microbodies obtained from etiolated barley (Hordeum vulgare L.) leaves depleted of nitrate. Defined centrifugation conditions cause the complete detachment of the enzyme from the microbodies. Addition of nitrate to these plants brings about a greater retention of glycolate oxidase by the microbodies. Synthesis of a nitrate-induced protein seems to be responsible for the enhanced retention of glycolate oxidase. Catalase, on the contrary, is strongly attached to the microbodies under all nutritional and experimental conditions considered.     

Cytochemical localization of glycolate oxidase in microbodies (glyoxysomes and peroxisomes) of higher plant tissues with the CeCl3 technique

Summary Alpha hydroxy acid oxidase activity (using glycolate as substrate) was demonstrated cytochemically in leaf-type peroxisomes, glyoxysomes, and unspecialized peroxisomes of higher plant tissues with the CeCl₃ technique in which cerous ions react with enzyme-generated H₂O₂ to form insoluble, electron-dense cerium perhydroxide. In all peroxisomes examined, reaction product was deposited throughout the matrices. None of the three types of microbody inclusions (crystals, amorphous nucleoids, or fibrillar, threadlike structures) observed in leaftype peroxisomes showed cytochemical reactivity. However, results with crystal-containing peroxisomes of guayule and tobacco leaves indicate an intimate association of glycolate oxidase with the crystals; reaction product was deposited in the spaces between the structural units of the crystal.Prolonged (18- versus 3-hour) incubation with glycolate and CeCl₃ were required for reliable cytochemical reactivity in glyoxysomes of castor bean endosperm and unspecialized peroxisomes of barley coleoptile, both of which contain relatively low enzyme activity. The CeCl₃ procedure may prove useful for helping identify microbodies observed with the electron microscope as peroxisomes. The lack of significant background deposits, and resolution of reaction product within crystals, illustrate qualities of the CeCl₃ procedure superior to those of the ferricyanide-reduction method, which was previously used to localize glycolate oxidase in higher plant microbodies.