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.     

Untersuchungen über die Aufnahme von Glykolat bei Chlorogonium

Summary The alga Chlorogonium was cultured either heterotrophically or autotrophically under different partial pressures of CO₂ by aerating with pure air of air enriched with 2% CO₂. Cells were harvested in the logarithmic phase, transferred to phosphate buffer containing 0.01 M 1C¹⁴-glycolate and incubated with shaking in the dark. Under these conditions the rate of glycolate uptake was higher when the cells had been grown in the light. Cells grown in the light at the lower CO₂-concentration took up more glycolate than those grown with 2% CO₂. Approximately 90% of the radioactivity taken up with the glycolate was released as CO₂. The radioactivity remaining in the algae was somewhat higher in those cells which had been cultured heterotrophically or autotrophically under air than in cells grown autotrophically under air enriched with 2% CO₂.Addition of glycolate increased the uptake of oxygen by the cells. The consumption of the oxygen was quantitatively correlated to the uptake of glycolate.     

The synthesis of oxalate from hydroxypyruvate by isolated perfused rat liver the mechanism of hyperoxaluria in L-lyceric aciduria

Hydroxypyruvate and glycolate inhibited the oxidation of [U-¹⁴C]glyoxylate to [¹⁴C]oxalate in isolated perfused rat liver, but stimulated total oxalate and glycolate synthesis. [¹⁴C]Oxalate synthesis from [¹⁴C]glycine similarly inhibited by hydroxypyruvate, but conversion of [¹⁴C₁]glycolate to [⁴C]oxalate was increased three-fold. Pyruvate had no effect on the synthesis of [¹⁴C]oxalate or total oxalate. The inhibition studies suggest that hydroxypyruvate is a precursor of glycolate and oxalate and that the conversion of glycolate to oxalate does not involve free glyoxylate as an intermediate. [¹⁴C₃]Hydroxypyruvate, but not [¹⁴C₁]hydroxypyruvate, was oxidized to [¹⁴C]oxalate in isolated perfused rat liver. Isotope dilution studies indicate the major pathway involves the decarboxylation of hydroxypyruvate forming glycolaldehyde which is subsequently oxidized to oxalate via glycolate. The oxidation of serine to oxalate appears to proceed predominantly via hydroxypyruvate rather than glycine or ethanolamine. The hyperoxaluria of L-glyceric aciduria, primary hyperoxaluria type II, is induced by the oxidation of the hydroxypyruvate, which accumulates because of the deficiency of D-glyceric dehydrogenase, to oxalate.     

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.     

Interactions of glycolate-, HCO 3 - -, Cl--, and H+-balance of Scenedesmus obliquus

Excretion and absorption of glycolate by young cells of Scenedesmus obliquus (Turp.) Krüger strain D₃ grown synchronously with 2% CO₂ was compared after no pretreatment with air (CO₂-adapted) or after a 2 h adaptation to normal air (0.03% CO₂) (air-adapted). At 21% O₂, excretion occurred only from CO₂-adapted cells at high pH (pH 8.0). Under conditions where no excretion occurred, external glycolate (0.2 mM) was taken up by both air-and CO₂-adapted cells at a much faster rate at pH 5 than at pH 8. The uptake was accompanied by an apparent stoichiometric uptake of H⁺. CO₂-adapted algae exhibited high uptake rates that were even higher in the dark than in the light. Air-adapted algae showed high uptake rates in the light but only minimal uptake in the dark. The uptake rate was decreased to about 1/3 with 5% CO₂, except with CO₂-adapted cells in the light, in which a slight stimulation occurred. Cl^- ions inhibited glycolate uptake by air-adapted cells in the light; conversely, light-stimulated Cl^- uptake of these cells was inhibited by glycolate. A hypothesis is discussed according to which the internal pH regulates the uptake and release of Cl^-, HCO ₃ ^- , and glycolate.Abbreviations DCMU 3-(3,4 dichlorophenyl)-1, 1-dimethyl urea - FCCP carbonyl cyanide p-trifluoro-methoxyphenylhydrazone - HEPES 2-(4-(2-hydroxyethyl)-piperazinyl) ethanesulfonic acid - HPMS -hydroxypyridinemethanesulfonate - MES 2-morpholinoethanesulfonic acid - PCV packed cell volume     

An investigation of glycolate excretion in two species of blue-green algae

Summary The amount of ¹⁴C-glycolate excreted by Oscillatoria sp. and Anabaena flos-aquae is less than 1% of the ¹⁴C fixed by the algae during photosynthesis. Transfer of cells grown on 5% CO₂ in air to a medium of low bicarbonate concentration or treatment of the cells with isonicotinyl hydrazide (INH) during photosynthesis, caused little increase in glycolate excretion. -Hydroxysulfonates failed to stimulate massive excretion of glycolate. Although these blue-green algae excreted little glycolate, a significant proportion of the photosynthetically fixed carbon was excreted in the form of basic, neutral and acidic compounds, and such excretion was greater in 5% CO₂-grown cells than in air-grown cells.     

Isolation and characterization of a desulforubidin-containing sulfate-reducing bacterium growing with glycolate

Sulfate-dependent degradation of glycolate was studied with a new sulfate-reducing bacterium, strain PerGlyS, enriched and isolated from marine anoxic sediment. Cells were gram-negative, motile rods with a DNA G+C content of 56.2±0.2 mol%. Cytochromes of theb- andc-type and menaquinone-5 were detected. A sulfite reductase of the desulforubidin-type was identified by characteristic absorption maxima at 279, 396, 545, and 580 nm. The purified desulforubidin is a heteropolymer consisting of three subunits with molecular masses of 42.5 (α), 38.5 (β), and 13 kDa (γ). Strain PerGlyS oxidized glycolate completely to CO₂. Lactate, malate, and fumarate were oxidized incompletely, yielding more sulfide and less acetate than expected for typical incomplete oxidation of these substrates. Part of the acetate residues formed was oxidized through the CO-dehydrogenase pathway. The biochemistry of glycolate degradation was investigated in cell-free extracts. A membrane-bound glycolate dehydrogenase, but no glyoxylate-metabolizing enzyme activity was detected; the further degradation pathway is unclear. Dedicated to Prof. Norbert Pfennig on the occasion of his 70th birthday     

Sequence of Formation of Phosphoglycolate and Glycolate in Photosynthesizing Chlorella pyrenoidosa

In Chlorella pyrenoidosa which have been photosynthesizing in either 1.5% ¹⁴CO₂ or 0.05% ¹⁴CO₂ in air, gassing with 100% O₂ results in rapid formation of phosphoglycolate which is apparently converted to glycolate. However, only about one-third to one-half of the rate of glycolate formation can be accounted for by this route. The remaining glycolate formation may be the result of the oxidation of sugar monophosphates. The rates of formation of both glycolate and phosphoglycolate are about four times greater with algae that have been photosynthesizing in 1.5% ¹⁴CO₂ than with algae which have been photosynthesizing with air, when the algae are then gassed with 100% O₂.     

EXCRETION OF GLYCOLATE BY A SPECIES OF CHLORELLA (CHLOROPHYCEAE)1

The claim that Chlorella sp. (CCAP 211/8p), sometimes referred to as C. fusca, Shihira and Krauss, does not excrete glycolate has been reexamined. Chlorella sp. grown on 5% CO₂in air, excreted glycolate when incubated in light in 10 mM bicarbonate. Excretion ceased 30–60 min after transfer of the cells to air and no excretion could be detected with air-grown cells or with cells grown on 5% CO₂in media buffered at pH 8.0. Incubation with 10 mM isonicotinyl hydrazide, a glycolate pathway inhibitor, caused excretion in air-grown cells and stimulated excretion in CO₂-grown cells indicating that both the rate of glycolate synthesis and metabolism is higher in CO₂grown cells than in air-grown cells. Enhanced glycolate synthesis and excretion in CO₂-grown cells is correlated with law photosynthetic rate in 10 mM bicarbonate, and the photosynthetic rate of these cells doubles over a period of 2–2.5 h after initial transfer from high CO₂to bicarbonate. This correlation of photosynthetic induction with cessation of glycolate excretion is similar to that reported in a bluegreen alga and thought to occur in other green algae. These results indicate that glycolate excretion and its regulation in this species of Chlorella is not different from that in other algae.     

Oxidation of Ethylene Glycol by a Salt-Requiring Bacterium

Bacterium T-52, cultured on ethylene glycol, readily oxidized glycolate and glyoxylate and exhibited elevated activities of ethylene glycol dehydrogenase and glycolate oxidase. Labeled glyoxylate was identified in reaction mixtures containing [¹⁴C]-ethylene glycol, but no glycolate was detected. The most likely pathway of ethylene glycol catabolism by bacterium T-52 is sequential oxidation to glycolate and glyoxylate.     

Light-Dependent Oxygen Uptake, Glycolate, and Ammonia Release in l-Methionine Sulfoximine-Treated Chlamydomonas

Glycolate and ammonia excretion plus oxygen exchanges were measured in the light in l-methionine-dl-sulfoximine treated air-grown Chlamydomonas reinhardii. At saturating CO₂ (between 600 and 700 microliters per liter CO₂) neither glycolate nor ammonia were excreted, whereas at the CO₂ compensation concentration (<10 microliters per liter CO₂) treated algae excreted both glycolate and ammonia at rates of 37 and 59 nanomoles per minute per milligram chlorophyll, respectively. From the excretion values we calculate the amount of O₂ consumed through the glycolate pathway. The calculated value was not significantly different from the component of O₂ uptake sensitive to CO₂ obtained from the difference between O₂ uptake of the CO₂ compensation point and at saturating CO₂. This component was about 40% of stationary O₂ uptake measured at the CO₂ compensation point. From these data we conclude that glyoxylate decarboxylation in air-grown Chlamydomonas represents a minor pathway of metabolism even in conditions where amino donors are deficient and that processes other than glycolate pathway are responsible for the O₂ uptake insensitive to CO₂.     

OCCURRENCE OF GLYCOLATE DEHYDROGENASE AND GLYCOLATE OXIDASE IN SOME COCCOID,ZOOSPORE-PRODUCING GREEN ALGAE1

Twenty-seven species of coccoid, zoospore-producing green algae representing 16 genera in the Chlorococcales and Chlorosarcinales were assayed for glycolate oxidase or glycolate dehydrogenase. Only Planophila terrestris Groover & Bold and Fasciculochloris boldii Trainor, contained glycolate oxidase whereas the others contained glycolate dehydrogenase. Representative algae were grown under varying conditions and assayed to determine any effects on these glycolate enzymes. Although specific rates of enzyme activity often varied widely, the form of glycolate enzyme present was not affected.     

Formation and metabolism of glycolate in the cyanobacterium Coccochloris peniocystis

The formation and metabolism of glycolate in the cyanobacterium Coccochloris peniocystis was investigated and the activities of enzymes of glycolate metabolism assayed. Photosynthetic ¹⁴CO₂ incorporation was O₂ insensitive and no labelled glycolate could be detected in cells incubated at 2 and 21% O₂. Under conditions of 100% O₂ glycolate comprised less than 1% of the acid-stable products indicating ribulose 1,5 bisphosphate (RuBP) oxidation only occurs under conditions of extreme O₂ stress. Metabolism of [1-¹⁴C] glycolate indicated that as much as 62% of ¹⁴C metabolized was released as ¹⁴CO₂ in the dark. Metabolism of labelled glycolate, particularly incorporation of ¹⁴C into glycine, was inhibited by the amino-transferase inhibitor amino-oxyacetate. Metabolism of [2-¹⁴C] glycine was not inhibited by the serine hydroxymethyltransferase inhibitor isonicotinic acid hydrazide and little or no labelled serine was detected as a result of ¹⁴C-glycolate metabolism. These findings indicate that a significant amount of metabolized glycolate is totally oxidized to CO₂ via formate. The remainder is converted to glycine or metabolized via a glyoxylate cycle. The conversion of glycine to serine contributes little to glycolate metabolism and the absence of hydroxypyruvate reductase confirms that the glycolate pathway is incomplete in this cyanobacterium.Abbreviations AAN aminoacetonitrile - AOA aminooxyacetate - DIC dissolved inorganic carbon - INH isonicotinic acid hydrazide - PEP phosphoenolpyruvate - PEPcase phosphoenolpyruvate carboxylase - PG phosphoglycolate - PGA phosphoglyceric acid - PGPase phosphoglycolate phosphatase - PR photorespiration - Rubisco ribulose-1,5-bisphosphate carboxylase oxygenase - TCA trichloroacetic acid - RuBP ribulose-1,5-bisphosphate     

Studies on regulation of tetradecane oxidation inPseudomonas aeruginosa

Summary Using mainly the Warburg technique, the effect of intermediates of tetradecane metabolism on tetradecane oxidation inPseudomonas aeruginosa was investigated. Precultivation and simultaneous incubation of bacteria with acetate, succinate, fumarate, glycolate and malonate inhibit the oxidation of tetradecane as well as of all primary oxidation products including myristic acid in a different manner. C₂-units have a key function in the regulation of tetradecane oxidation. The inhibitory effect of tetradecane and its primary oxidation products on substrate oxidations may be unspecific.During the oxidation of tetradecane or its primary oxidation products O₂-consumption increases exponentionally but CO₂-production becomes stationary. The RQ-values obtained with myristic acid growing cells decrease to O.1, indicating that myristic acid is only partially oxidized via citric acid cycle.     

PURIFICATION AND CHARACTERIZATION OF GLYCOLATE OXIDASE FROM THE BROWN ALGA SPATOGLOSSUM PACIFICISM (PHAEOPHYTA)1

Glycolate oxidase was purified to apparent homogeneity from the brown alga Spatoglossum pacificum Yendo. The 1326-fold purified glycolate oxidase enzyme exhibited a specific activity of 22. 4 micromoles glyoxylate formed ·min^?1·mg protein^?1. The molecular weight of the native enzyme was estimated to be 230,000 by gel filtration. The subunit molecular weight of the enzyme was determined to be 49,000 by sodium dodecyl sulfate–polyacrylamide gel electrophoresis, suggesting that the native enzyme is a tetramer. There were two absorption peaks at 345 and 445 nm, indicating that glycolate oxidase is a flavoprotein. This enzyme had a high isoelectric point (pI 9.6) and a high pH optimum (pH 8.3). The K_m values for glycolate and l -lactate were 0.49 and 5.5 mM, respectively. This enzyme also had a broad specificity for other straight-chain α-hydroxy acids but not for β-hydroxyacids. Cyanide, azide, N-ethylmaleimide, and p-chloromercuribenzoic acid did not affect the enzyme, whereas 2-pyridylhydroxymethanesulfonic acid strongly inhibited it. These properties of glycolate oxidase from the brown alga S. pacificum are similar to the properties of the glycolate oxidasesfrom higher plants. Polyclonal antibodies raised against the polypeptide fragment of Spatoglossum glycolate oxidase could recognize glycolate oxidase from Spinacia oleracea L., although the cross-reactivity was weak. The N-terminal sequence of two internal polypeptide fragments of the enzyme from S. pacificum showed a high degree of similarity to that of glycolate oxidase from higher plants. These results suggest that glycolate oxidase from higher plants and brown algae share the same ancestral protein.     

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.     

Localization of glycolate dehydrogenase in two species of Dunaliella

The occurrence of glycolate oxidase in addition to glycolate dehydrogenase in Dunaliella salina and D. primolecta, as reported in the literature, could not be confirmed. Both species were demonstrated to possess only glycolate dehydrogenase. After separation of organelles by gradient centrifugation, glycolate dehydrogenase along with hydroxypyruvate reductase was found exclusively in the mitochondria. Thus the peroxisomes from Dunaliella are not of the leaf-type: because of their content of catalase, uricase and hydroxyacyl-CoA dehydrogenase they appear to be of the same type as in Eremosphaera and other chlorophycean algae. No activity of glycolate dehydrogenase was found in the chloroplast fraction when the 2,6-dichlorophenol-indophenol test was used.This work was supported by the Deutsche Forschungsgemeinschaft.     

A comparative study of ethylene oxidation inVicia faba andMycobacterium paraffinicum

Vicia faba L. Herz Freya (fababean) cotyledons andMycobacterium paraffinicum Bardane strain (MPB) cells were studied to describe and compare physiological and biochemical factors regulating ethylene oxidation. Both organisms demonstrated a linear rate of ethylene uptake as a function of concentration from 1 ppm to 1,000 ppm. CO₂ did not influence ethylene oxidation by either organism. Zero degree temperatures and CO inhibited ethylene oxidation by fababeans but not by MPB.An N₂ gas phase blocked ethylene consumption by fababeans. In contrast, MPB continued to consume ethylene at a reduced rate under anaerobic conditions. Hydrocarbon oxidation was limited to alkenes. Alkanes were not oxidized by either organism. Both organisms were sensitive to diethyldithiocarbamic acid, o-phenanthroline, carbonyl cyanidem-chlorophenyl hydrazone, and CS₂. The possibility that CS₂ acted as a suicide substrate is discussed. Evidence is presented that hydrocarbon gas oxidation by fababeans is not a part of, or reflection of, the way ethylene acts as a hormone.     

海水中藻菌共培养体系对碳氮磷的吸收转化

海洋环境中,细菌和微藻之间的物质交换是生源要素在自然界中迁移转化的重要方式。为进一步了解生源要素的生物地球化学循环,在实验室模拟条件下,研究了共培养体系中营养盐和有机物在细菌和微藻之间的转换。通过纯培养中肋骨条藻(Skeletonema costatum)、东海原甲藻(Prorocentrum donghaiense)、天然海水中的细菌以及藻菌混合培养,分析了营养盐和有机物随藻菌生物量的变化情况,并计算了溶解有机碳(DOC)和溶解有机氮(DON)的浓度比值[(DOC/DON)a]。结果发现,在共培养体系中,细菌对中肋骨条藻的生长有抑制作用,对东海原甲藻影响不明显;中肋骨条藻有利于细菌生长,东海原甲藻抑制细菌生长,这种不同可能与微藻的粒径有关。海洋细菌在2种藻的指数生长均期均会促进微藻吸收氨氮(NH_4-N),但在生长末期NH_4-N以释放为主。硝氮(NO_3-N)的浓度与藻的生长呈负相关,但在衰亡期NO_3-N略有增加,表明NO_3-N再生所需时间较长。细菌对硝氮的吸收量较少,但对其再生有贡献。细菌和中肋骨条藻对磷酸盐(PO_4-P)的吸收存在竞争,但与东海原甲藻的竞争关系不明显。不同培养体系中DOC浓度变化不同,在藻菌共培养体系中增加较快,纯藻培养体系中增加缓慢,在纯菌培养体系中缓慢减少。通过对DOC与DON浓度比值的分析,发现用判断颗粒有机碳(POC)来源的方法可以分析DOC的来源。     

Changes in Chlorophyll a/b Ratio and Products of CO(2) Fixation by Algae Grown in Blue or Red Light

Chlamydomonas and Chlorella were grown for 10 days in white light. 955 μw/cm² blue light (400-500 mμ) or 685 μw/cm² red light (above 600 mμ). Rates of growth in blue or red light were initially slow, but increased over a period of 5 days until normal growth rates were reestablished. During this adaptation period in blue light, total chlorophyll per volume of algae increased 20% while the chlorophyll a/b ratio decreased. In red light no change was observed in the total amount of chlorophyll or in the chlorophyll a/b ratio. After adaptation to growth in blue light and upon exposure to ¹⁴CO₂ with either blue or white light for 3 to 10 minutes, 30 to 36% of the total soluble fixed ¹⁴C accumulated in glycolate-¹⁴C which was the major product. However, with 1 minute experiments, it was shown that phosphate esters of the photosynthetic carbon cycle were labeled before the glycolate. Glycolate accumulation by algae grown in blue light occurred even at low light intensity. After growth of the algae in red light, ¹⁴C accumulated in malate, aspartate, glutamate and alanine, whereas glycolate contained less than 3% of the soluble ¹⁴C fraction.