Oxidative phosphorylation during glycollate metabolism in mitochondria from phototrophic Euglena gracilis.

Mitochondria were isolated by gradient centrifugation on linear sucrose gradients from broken cell suspensions of phototrophically grown Euglena gracilis. An antimycin A-sensitive but rotenone-insensitive glycollate-dependent oxygen uptake was demonstrated in isolated mitochondria. The partial reactions of glycollate-cytochrome c oxidoreductase and cytochrome c oxidase were demonstrated by using Euglena cytochrome c as exogenous electron acceptor/donor. Isolated mitochondria contain glycollate dehydrogenase and glyoxylate-glutamate aminotransferase and oxidize exogenous glycine. A P:O ratio of 1.7 was obtained for glycollate oxidation, consistent with glycollate electrons entering the Euglena respiratory chain at the flavoprotein level. The significance of these results is discussed in relation to photorespiration in algae.     

The localization of glycollate-pathway enzymes in Euglena.

Isolation of organelles from broken-cell suspensions of phototrophically grown Euglena gracilis Klebs was achieved by isopycnic centrifugation on sucrose gradients. 2. Equilibrium densities of 1.23g/cm3 for peroxisome-like particles, 1.22g/cm3 for mitochondria and 1.17g/cm3 for chloroplasts were recorded. 3. The enzymes glycollate dehydrogenase, glutamate-glyoxylate aminotransferase, serineglyoxylate aminotransferase, aspartate-alpha-oxoglutarate aminotransferase, hydroxy pyruvate reductase and malate dehydrogenase were present in peroxisome-like particles. 4. Unlike higher plants glycollate dehydrogenase and glutamate-glyoxylate aminotransferase were present in the mitochondria of Euglena. 5. Rates of glycollate and D-lactate oxidation were additive in the mitochondria, and, although glycollate dehydrogenase was inhibited by cyanide, D-lactate dehydrogenase activity was unaffected. 6. Glycollate oxidation was linked to O2 uptake in mitochondria but not in peroxisome-like particles. This glycollate-dependent O2 uptake was inhibited by antimycin A or cyanide. 7. The physiological significance of glycollate metabolism in Euglena mitochondria is discussed, with special reference to its role in photorespiration in algae.     

Enzymes of the glycollate pathway in relation to greening in Euglena gracilis

Summary The kinetics of chlorophyll formation during the greening of dark-grown Euglena gracilis was accompanied by marked increases in activity of the enzymes of glycollate metabolism; glycollate: DCPIP¹ oxidoreductase and phosphoglycollate phosphatase (E.C. 3.1.3.18.). Inhibitors of protein synthesis indicated a de novo synthesis of these enzymes during the development of the photosynthetic system. The inhibitory effects of chloramphenicol and cycloheximide, together with a non-aqueous localisation of glycollate: DCPIP oxidoreductase in photoautotrophically-grown cells, indicated that this enzyme is synthesized and located in the cytoplasm, while phosphoglycollate phosphatase was synthesized in the chloroplast. Glycollate: DCPIP oxidoreductase did not increase above the low level in heterotrophic cells when exogenous glycollate, in the presence or absence of glucose, was supplied in the dark.     

Enzymic evidence for peroxisomes in a mutant of Chlorella vulgaris

Summary Isopycnic sucrose density gradient centrifugation of cell-free extracts of a yellow mutant of Chlorella vulgaris and its green parent strain showed a distribution of catalase and glycollate oxidoreductase activity consistent with their association with a particle/organelle fraction. Gradient centrifugation starting from a pellet of cell-free material resulted in a concentration of enzyme activity in the 1.5 M to 2.0 M sucrose fractions which coincided with a microbody-containing fraction as determined by electron microscopy. The algal glycollate-oxidizing enzyme coupled to oxygen, oxidized both d- and l-lactate and was insensitive to cyanide in vitro, showing it to be similar to that of higher plants. The association of glycollate oxidase together with catalase, with the microbody fraction, may be taken as evidence for the presence of algal peroxisomes in these organisms.Abbreviations DCPIP 2,6-dichlorophenolindophenol     

Relationships between glycollate and folate metabolism in Euglena gracilis

When division synchronized cultures of Euglena gracilis Klebs (strain Z) were aerated with 5% CO₂ in air the specific activity of glycollate dehydrogenase was only 13% of that in cultures receiving unsupplemented air. The concentrations of 10-formyltetrahydrofolate synthetase (EC 6.3.4.3) and formylfolate derivatives were also lowered by this treatment. In contrast, the specific activity of serine hydroxymethyltransferase (EC 2.1.2.1) and the concentration of methylfolates were raised by supplying CO₂-supplemented air. These effects on enzyme levels were reversed when air was supplied following a period of CO₂ treatment. The levels of glycollate dehydrogenase, 10-formyl-tetrahydrofolate synthetase and formylfolate derivatives were decreased when cells were aerated in media containing 5 mM α-hydroxy-2-pyridinemethane sulphonate. Cell free extracts had the ability to decarboxylate glyoxylate, producing ca equal amounts of CO₂ and formate from C-1 and C-2 respectively. Cells receiving 5% CO₂ in air had a decreased ability to incorporate formate-[¹⁴C] into serine and methionine. It is concluded that during growth at low CO₂ concentrations glycollate metabolism will provide substrate for the formyltetrahydrofolate synthetase reaction.     

Glycollate oxidation by thylakoids of the cyanobacteria Anabaena cylindrica,Nostoc muscorum and Chlorogloea fritschii

The intracellular distribution of glycollate dehydrogenase EC 1.2.1.17 has been investigated in extracts of the cyanobacteria (blue-green algae) Anabaena cylindrica, Nostoc muscorum and Chlorogloea fritschii. Most of the enzyme activity was associated with a chlorophyll-containing cell-free pellet, which also exhibited Photosystem I and II activities. Sucrose density gradient centrifugation of this washed pellet resulted in the formation of a green band within which maximal chlorophyll concentration and enzymic glycollate oxidation coincided. Antiserum raised to this fraction obtained from A. cylindrica inhibited glycollate dehydrogenase and Photosystem II activity. The data indicate that most of the cyanobacterial glycollate dehydrogenase is associated with the thylakoids and thus provide evidence for the dual role of these membranes in photosynthetic and respiratory processes.Abbreviations DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - DCPIP 2,6-dichlorophenolindophenol - DPC diphenylcarbazide     

The presence of glycollate oxidase and dehydrogenase in Dunaliella primolecta

Homogenates of Dunaliella primolecta, D. salina and D. tertiolecta were assayed for glycollate oxidase and glycollate dehydrogenase. Both D. primolecta and D. salina but not D. tertiolecta showed substantial glycollate-dependent O₂-uptake which is characteristic of glycollate oxidase. L-Lactate was an alternative substrate and both glycollate- and L-lactate-dependent O₂ uptake were insensitive to 2 mM cyanide. Glycollate dehydrogenase, measured by following the glycollate-dependent reduction of 2,6-dichlorophenolindophenol under aerobic conditions, was present in D. primolecta, D. salina and D. tertiolecta. In the presence of glycollate and D-lactate, rates were additive so both glycollate and D-lactate dehydrogenases are present in the homogenates. Glycollate and D-lactate oxidation were both inhibited by 2 mM cyanide. Organelles released from phototrophically grown cells of D. primolecta were separated by isopycnic centrifugation on sucrose gradients. Glycollate oxidase was present in the peroxisome fraction at an equilibrium density of 1.25 g/cm³, while the major peak of glycollate dehydrogenase activity was in the mitochondrial fraction at an equilibirium density of 1.22 g/cm³.     

Glycollate production and excretion by Alcaligenes eutrophus.

Autotrophic cultures of the facultative chemolithotroph Alcaligenes eutrophus have been found to excrete glycollate. This excretion was greatly stimulated by the incorporation of up to 20% (v/v) oxygen in the hydrogen used for gassing. The stimulatory effect of oxygen was prevented by the addition of 10% (v/v) CO2 to the gassing mixture. Glycollate excretion only in the presence of oxygen was increased by the addition of 2-pyridyl-hydroxymethane sulphonic acid (HPMS), an inhibitor of glycollate oxidation, indicating that glycollate formation itself was stimulated by oxygen. No glycollate excretion by cultures grown heterotrophically on pyruvate was detected, either in the absence or presence of HPMS, under heterotrophic or autotrophic cells showed phosphoglycollate phosphatase and glycollate oxidoreductase activities, which were considerably lower in extracts prepared from pyruvate- or fructose-grown (heterotrophic) cells. The increase in activity of both enzymes upon cell transfer from heterotrophic to autotrophic growth was prevented by chloramphenicol and resembled the induction of D-ribulose 1,5-diphosphate carboxylase under the same conditions.     

Biosynthesis of streptomycin enzymic oxidation of dihydrostreptomycin (6-phosphate) to streptomycin (6-phosphate) with a particulate fraction of Streptomyces griseus

Resting cells and to a greater extent permeabilized cells of Streptomyces griseus can oxidize dihydrostreptomycin to streptomycin. The dihydrostreptomycin oxidoreductase activity was localized in the 100 000 × g particulate fraction. Sucrose density gradient centrifugation of the particulate suspension gave a band at a density of 1.09 which consisted mainly of membrane vesicles. This fraction had high dihydrostreptomycin oxidoreductase activity. S. griseus protoplasts also contain high oxidoreductase activity. These data are consistent with localization of the enzyme in the cell membrane. Dihydrostreptomycin and dihydrostreptomycin 6-phosphate can both serve as substrates for the oxidoreductase, but the phosphate was the better substrate in the cell free system. Addition of cofactors was not required for the bound dihydrostreptomycin oxidoreductase. The electron acceptor for the oxidation is unknown. Oxidation of dihydrostreptomycin 6-phosphate to streptomycin 6-phosphate very probably represents the penultimate step in the biosynthesis of streptomycin.     

Deoxyribonuclease II purified from Euglena gracilis SM-ZK, a chloroplast-lacking mutant: comparison with porcine spleen deoxyribonuclease II

An acid deoxyribonuclease was extracted from Euglena gracilis SM-ZK, a chloroplast-lacking strain, by homogenizing the cells in 50 mM sodium acetate (pH 4.6). The enzyme was then purified by heat treatment and a series of chromatographic separations. The molecular mass of the Euglena acid DNase was estimated to be 45 kDa by sensitive activity staining in an SDS-polyacrylamide gel using SYBR Green. Treatment of the Euglena enzyme with a reducing agent prior to electrophoresis destroyed its DNase activity in the gel, indicating that disulfide bridging is essential for its enzyme activity. Nucleolytic properties of this enzyme are essentially the same as to those of porcine DNase II. The Euglena enzyme acts on both double-stranded (ds) and single-stranded DNA, but acts preferentially on dsDNA with an optimum pH at approximately 5.3. EDTA did not inhibit its enzyme activity. Euglena DNase makes double-strand breaks in circular DNA substrate and generates a terminus with 3'-phosphate and 5'-OH. These results indicate that the Euglena acid DNase is in fact a member of the DNase II family.     

Glycollate production and excretion byAlcaligenes eutrophus

Autotrophic cultures of the facultative chemolithotrophAlcaligenes eutrophus have been found to excrete glycollate. This excretion was greatly stimulated by the incorporation of up to 20% (v/v) oxygen in the hydrogen used for gassing. The stimulatory effect of oxygen was prevented by the addition of 10% (v/v) CO₂ to the gassing mixture. Glycollate excretion only in the presence of oxygen was increased by the addition of 2-pyridyl-hydroxymethane sulphonic acid (HPMS), an inhibitor of glycollate oxidation, indicating that glycollate formation itself was stimulated by oxygen. No glycollate excretion by cultures grown heterotrophically on pyruvate was detected, either in the absence or presence of HPMS, under heterotrophic or autotrophic conditions.Extracts from autotrophic cells showed phosphoglycollate phosphatase and glycollate oxidoreductase activities, which were considerably lower in extracts prepared from pyruvate- or fructose-grown (heterotrophic) cells. The increase in activity of both enzymes upon cell transfer from heterotrophic to autotrophic growth was prevented by chloramphenicol and resembled the induction ofd0ribulose 1,5-diphosphate carboxylase under the same conditions.Abbreviations DTE dithioerythritol - EDTA ethylenediamine tetraacetate - HPMS 2-pyridyl-hydroxymethane sulphonie acid - RuDP d-ribulose 1,5-diphosphate     

Mevalonic Acid Kinase in Euglena gracilis

The isolation and partial purification of mevalonic acid kinase from Euglena gracilis is described. The product of the reaction MVA-5-P has been characterized by paper chromatography. The apparent Km values for l-mevalonic acid, ATP, and Mg(2+) are 3 x 10(-5)m, 6 x 10(-3)m, and 9 x 10(-3)m, respectively. A concentration of 1 x 10(-3)mp-hydroxymercuribenzoate completely inactivates the enzyme. A distribution study has shown that mevalonic acid kinase is present in most higher plants and the algae Euglena gracilis and Chlamydomonas. No enzymatic activity could be detected in several species of photosynthetic bacteria or blue-green algae.     

Intracellular localization of aldehyde dehydrogenase in rat liver

1. Distribution of aldehyde dehydrogenase activity in rat liver was studied by measuring the rate of disappearance of acetaldehyde in the presence of each of the subcellular fractions. These were obtained by rough separation of particulate fractions from the soluble portion of the cell, by differential centrifugation, and by isopycnic gradient centrifugation. 2. The maximal rate of acetaldehyde oxidation was 3.7 mumol/min per g, with an apparent K(m) value below 10(-5)m. The highest rate of activity was observed in phosphate buffers of high P(i) concentration (above 60mm). 3. The activity measured was completely dependent on NAD(+). 4. The microsomal fraction and the nuclei were inactive in the assay. Of the total activity 80% was found in the mitochondrial fraction and the remaining 20% in the cytoplasm. 5. The distribution pattern is important from the point of view of acetaldehyde oxidation during ethanol metabolism. The apparent discrepancy of the results obtained by different workers and the localization of acetaldehyde oxidation in vivo is discussed.     

Intracellular Localization of GDP-Fucose: Polysaccharide Fucosyl Transferase in Corn Roots (Zea mays L.)

The intracellular site of synthesis of the fucose-rich polysaccharide slime secreted by corn roots was localized by monitoring the distribution of GDP-fucose:polysaccharide fucosyl transferase activity in subcellular fractions of corn roots. Root tip sections were chopped in the presence of 0.56 molar sucrose and 100 millimolar Tris (pH 7.0). After a brief centrifugation, the homogenate was applied to a Sepharose 4B column (1.5 × 30 cm). The turbid, particulate portion of the supernatant fraction eluted at the void volume. Ninety per cent of the enzyme activity was found in the pooled particulate fractions. The particulate fraction was purified on linear sucrose gradients. Gradient fractions were characterized by buoyant density, 280 nanometer absorbance, electron microscope observation, and distributions of NADH-cytochrome c oxidoreductase and fucosyl transferase activities.     

Intracellular localization of glycolate dehydrogenase in a blue-green alga

Glycolate dehydrogenase activity was detected in cell-free extracts of Oscillatoria sp. prepared by osmotic lysis of spheroplasts in 0.05 m potassium phosphate buffer, pH 7.5, containing 0.3 m mannitol. Most of the enzyme activity was found in a particulate fraction and localized in the photosynthetic lamellae after centrifugation in a discontinuous sucrose density gradient. Enzyme activity was detected in this fraction both in the presence and absence of the artificial electron acceptor 2,6-dichlorophenolindophenol (DPIP) and a low rate of O(2) uptake was detected in this lamellar fraction. Activity was lost from the lamellar fraction by repeated washing or by treatment with 0.005% Triton X-100 and the solubilized enzyme activity was DPIP-dependent. The data indicate that both glycolate dehydrogenase and its natural electron acceptor are bound to the photosynthetic lamellae in vivo. In contrast, catalase activity was found in the soluble cytoplasmic fraction.     

Enzymes of glycerol metabolism in the storage tissues of Fatty seedlings

Various enzymes of glycerol metabolism in the extracts of 5-day-old eastor bean (Ricinus communis L. var. Hale) endosperm and 4-day-old peanut (Archis hypogaea L.) cotyledon were studied. NAD-glycerol dehydrogenase and NAD-α-glycerolphosphate dehydrogenase were not detected. Glycerol kinase was detected in the soluble fractions and an α-glycerolphosphate oxidoreductase was found in the particulate fractions. The particulate fractions were separated into various organelle fractions by sucrose gradient centrifugation and the α-glycerolphosphate oxidoreductase was shown to be present in the mitochondria. The properties of the castor bean mitochondrial α-glycerolphosphate oxidoreductase resembled those of a similar enzyme present in the mitochondria of many animal tissues. A survey showed that the α-glycerolphosphate oxidoreductase was present in great amount only in the storage tissues of fatty seedlings but not in other nonfatty plant tissues. It is concluded that in the storage tissues of fatty seedlings, the soluble glycerol kinase and the mitochondrial cytochrome-linked α-glycerolphosphate oxidoreductase are the two enzymes responsible for the initial conversion of glycerol to hexose.     

Plant leaf alanine: 2-oxoglutarate aminotransferase. Peroxisomal localization and identity with glutamate:glyoxylate aminotransferase.

The distribution of alanine:2-oxoglutarate aminotransferase (EC 2.6.1.2) in spinach (Spinacia oleracea) leaf homogenates was examined by centrifugation in a sucrose density gradient. About 55% of the total homogenate activity was localized in the peroxisomes and the remainder in the soluble fraction. The peroxisomes contained a single form of alanine:2-oxoglutarate aminotransferase, and the soluble fraction contained two forms of the enzyme. Both the peroxisomal enzyme and the soluble predominant form (about 90% of the total soluble activity) were co-purified with glutamate:glyoxylate aminotransferase to homogeneity; it had been reported to be present exclusively in the peroxisomes of plant leaves and to participate in the glycollate pathway in leaf photorespiration [Tolbert (1971) Annu. Rev. Plant Physiol. 22, 45-74]. The evidence indicates that alanine:2-oxoglutarate aminotransferase and glutamate:glyoxylate aminotransferase activities are associated with the same protein. The peroxisomal and soluble enzyme preparations had nearly identical properties, suggesting that the soluble predominant alanine aminotransferase activity is from broken peroxisomes and about 96% of the total homogenate activity is located in peroxisomes.     

Euglena gracilis rhodoquinone:ubiquinone ratio and mitochondrial proteome differ under aerobic and anaerobic conditions

Euglena gracilis cells grown under aerobic and anaerobic conditions were compared for their whole cell rhodoquinone and ubiquinone content and for major protein spots contained in isolated mitochondria as assayed by two-dimensional gel electrophoresis and mass spectrometry sequencing. Anaerobically grown cells had higher rhodoquinone levels than aerobically grown cells in agreement with earlier findings indicating the need for fumarate reductase activity in anaerobic wax ester fermentation in Euglena. Microsequencing revealed components of complex III and complex IV of the respiratory chain and the E1beta subunit of pyruvate dehydrogenase to be present in mitochondria of aerobically grown cells but lacking in mitochondria from anaerobically grown cells. No proteins were identified as specific to mitochondria from anaerobically grown cells. cDNAs for the E1alpha, E2, and E3 subunits of mitochondrial pyruvate dehydrogenase were cloned and shown to be differentially expressed under aerobic and anaerobic conditions. Their expression patterns differed from that of mitochondrial pyruvate:NADP(+) oxidoreductase, the N-terminal domain of which is pyruvate:ferredoxin oxidoreductase, an enzyme otherwise typical of hydrogenosomes, hydrogen-producing forms of mitochondria found among anaerobic protists. The Euglena mitochondrion is thus a long sought intermediate that unites biochemical properties of aerobic and anaerobic mitochondria and hydrogenosomes because it contains both pyruvate:ferredoxin oxidoreductase and rhodoquinone typical of hydrogenosomes and anaerobic mitochondria as well as pyruvate dehydrogenase and ubiquinone typical of aerobic mitochondria. Our data show that under aerobic conditions Euglena mitochondria are prepared for anaerobic function and furthermore suggest that the ancestor of mitochondria was a facultative anaerobe, segments of whose physiology have been preserved in the Euglena lineage.     

Particulate guanylate cyclase of skeletal muscle: effects of Ca2+ and other divalent cations on enzyme activity.

The properties of particulate guanylate cyclase (GTP pyrophosphate-lyase (cyclizing), EC 4.6.1.2) from purified rabbit skeletal muscle membrane fragments were studied. Four membrane fractions were prepared by sucrose gradient centrifugation and the fractions characterized by analysis of marker enzymes. Guanylate cyclase activity was highest in the fraction possessing enzymatic properties typical of sarcolemma, while fractions enriched with sarcoplasmic reticulum had lower activities. In the presence of suboptimal Mn2+ concentrations, Mg2+ stimulated particulate guanylate cyclase activity both before and after solubilization in 1% Triton X-100. Guanylate cyclase activity was biphasic in the presence of Ca2+. Increasing the Ca2+ concentration from 10(-8) to 10(-5) M decreased the specific activity. As the Ca2+ concentration was further increased to 5 . 10(-4) M enzyme activity again increased. After solubilization of the membranes in 1% Triton X-100, Ca2+ suppressed enzyme activity. Studies utilizing ionophore X537A indicated that the altered effect of Ca2+ upon the solubilized membranes was independent of asymmetric distribution of Ca2+ and Mg2+.     

A new type of a multifunctional beta-oxidation enzyme in euglena

The biochemical and molecular properties of the beta-oxidation enzymes from algae have not been investigated yet. The present study provides such data for the phylogenetically old alga Euglena (Euglena gracilis). A novel multifunctional beta-oxidation complex was purified to homogeneity by ammonium sulfate precipitation, density gradient centrifugation, and ion-exchange chromatography. Monospecific antibodies used in immunocytochemical experiments revealed that the enzyme is located in mitochondria. The enzyme complex is composed of 3-hydroxyacyl-coenzyme A (-CoA) dehydrogenase, 2-enoyl-CoA hydratase, thiolase, and epimerase activities. The purified enzyme exhibits a native molecular mass of about 460 kD, consisting of 45.5-, 44.5-, 34-, and 32-kD subunits. Subunits dissociated from the complete complex revealed that the hydratase and the thiolase functions are located on the large subunits, whereas two dehydrogenase functions are located on the two smaller subunits. Epimerase activity was only measurable in the complete enzyme complex. From the use of stereoisomers and sequence data, it was concluded that the 2-enoyl-CoA hydratase catalyzes the formation of L-hydroxyacyl CoA isomers and that both of the different 3-hydroxyacyl-CoA dehydrogenase functions on the 32- and 34-kD subunits are specific to L-isomers as substrates, respectively. All of these data suggest that the Euglena enzyme belongs to the family of beta-oxidation enzymes that degrade acyl-CoAs via L-isomers and that it is composed of subunits comparable with subunits of monofunctional beta-oxidation enzymes. It is concluded that the Euglena enzyme phylogenetically developed from monospecific enzymes in archeons by non-covalent combination of subunits and presents an additional line for the evolutionary development of multifunctional beta-oxidation enzymes.