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.     

THE KINETICS OF ORGANIC ACID UPTAKE BY THREE CHLOROPHYTA IN AXENIC CULTURE1

The uptake of 8 ¹⁴C-labelled dissolved carboxylic acids by Ankistrodesmus falcatus (Corda) Ralfs, Chlamydomonas segnis Ettl and Chlorella sp. was measured. The acids were acetic, citric, fumaric, glycollic, lactic, malic, pyruvic and succinic acids. For some substrates uptake was active, but for others it was apparently passive. The respiratory loss of substrates ranged from 5.0 to 95.0% of the total amount of C assimilated. The potential ability of these organisms to compete with bacterial populations for such substrates in natural waters was examined. In terms of the kinetics of assimilation it appeared that A. falcatus might compete successfully for glycollate, lactate, pyruvate and succinate; C. segnis for glycollate and lactate and Chlorella sp. for lactate, malate and possibly glycollate. Calculated replacement times of the algae on the 8 substrates did, however, indicate that although they might compete successfully for some substrates they were generally unable to grow upon them at the concentrations provided.     

On the acceptor specificity of glycollate oxidase of Nicotiana tabacum

Summary The effect of compounds on the activity of ammonium sulphate preparations of glycollate oxidase from Nicotiana tabacum cv. John Williams' Broadleaf and the aurea mutant Su/su is reported. Coupling to DCPIP as terminal oxidant under anaerobic conditions gave greater rates of glycollate oxidation than when measured as O₂ uptake in the presence of cyanide. The enzyme also linked to DCPIP in the presence of O₂, showing that it is a facultative aerobic dehydrogenase. Catalytic amounts of PMS stimulated enzyme-dependent oxygen uptake and DCPIP reduction under aerobic and anaerobic conditions. This further suggests that an intermediate carrier, or alternate acceptor, depending on concentration, exists before O₂ in vivo. Naturally occurring quinoid compounds may fulfill such a role, as evidenced by the enhancement of aerobic DCPIP reduction upon addition of catalytic amounts of caffeic and chlorogenic acid. The observation that PMS, caffeic and chlorogenic acid, biopterin, 6-hydroxy-2-amino-4-hydroxypteridine and a quinone extract of N. tabacum quenched the inhibitory effect of blue light on tobacco glycollate oxidase, is in accordance with the possible function of such compounds in glycollate oxidation.Abbreviation DCPIP 2,6-dichlorophenolindophenol - FMN flavin mononucleotide - PMS phenazine methosulphate     

Untersuchung der Beziehung zwischen extracellulärer Glykolsäure-Ausscheidung und der photosynthetischen CO2-Aufnahme bei der Blaualge Anacystis nidulans

Summary The formations of transients in CO₂ exchange in the blue-green alga Anacystic nidulans is dependent on the temperature used during the measurements. The algae were grown in a low light intensity (4000 lux) under normal air conditions and measured in the same low CO₂ concentration (0.03 vol. %) but under a higher light intensity (10 000 lux). At a temperature of +20°C the stationary rate of CO₂ uptake was reached directly. At a temperature of +35°C, on the other hand, a maximum of CO₂ uptake could be observed at the beginning of the light period followed by a steady rate of photosynthesis, which was higher than at +20°C. In the beginning of the dark period a CO₂ outburst appeared at 35°C.Only at a low temperature (+20°C) did we find a light induced glycollate excretion; after a maximum at 7 1/2 minutes illumination the release of glycollate ceases and the level decreases to a lower value. A similar time course exists during illumination in red light (621 nm, 1.5·10^-8 einsteins) and a temperature of +20°C. In blue light (432 nm, 1,5·10^-8 einsteins, +20°C) and in white light at a high temperature (+35°C) we could not find any light induced glycollate excretion. Our results are discussed in reference to the photorespiration. We explain the formation of transients in CO₂ uptake of Anacystis at a high temperature (+35°C) and in blue light (+20°C) on the basis of the influence of photorespiration.     

Uptake and respiration of organic compounds and heterotrophic growth in Pediastrum duplex (Meyen)*

SUMMARY. Axenic cultures of Pediastrum duplex, a green alga prominent in Lake Kinneret, Israel, assimilated and respired amino acids, acetate, glucose, glycollate and glycerol under conditions of light or darkness. Increased rates of uptake and respiration were observed in nutrient (P and N) depleted medium. Although ineffective in moderate light (30–100 μ Einstein m^?2 s^?1) glycerol, glucose and leucine, but not glycollate, stimulated growth and yields under faint light (～ 2 μ Einstein m^?2 s^?1). In the dark, glycerol (and sometimes leucine) permitted growth. Kinetic studies with leucine indicated an active uptake mechanism effective at substrate concentrations from 0.5 to 47 μg 1^?1.     

Glycollate formation during the photoassimilation of acetate by Chlorella

Summary When ³H-¹⁴C-acetate was supplied to Chlorella pyrenoidosa in the light, glycollic acid became rapidly labelled with tritium and ¹⁴C. The ratio of glycollate was 10, whilst the ratio was 4 in the acetate added. Both ³H and ¹⁴C from acetate were present in glycollate before they were present in Calvin cycle intermediates, so that glycollate was not formed as a C₂-fragment from the Calvin cycle.     

Triterpeneglycosides as Inhibitors of Fungal Growth and Metabolism 3. Effect on Uptake of Potassium, Magnesium and Inorganic Phosphate

A venacin, the resistance factor in oat roots against Ophiobolus graminis var. graminis, and a related triterpeneglycoside, aescin, inhibited the uptake of K⁺ and Mg²⁺ in the fungal mycelium both in phosphate and succinate buffers. The uptake of the cations in Neurospora crassa was similarly inhibited when the inhibitors were dissolved in phosphate or acetatebuffer, while no decrease in the uptake of K⁺ and Mg²⁺ was observed when the inhibitors were dissolved in succinate buffer. The uptake of cations in Aspergillus niger and Pythium irregulare was more or less unaffected by aescin. The uptake of inorganic phosphate was in no case inhibited, but some decrease of the accumulation of inorganic phosphate in Ophiobolus graminis and Ncurospora crassa due to inhibitor treatment in phosphate buffer was observed. No accumulation of Ca²⁺ was observed in any of the tested fungi.     

The glycollate pathway during the photoassimilation of acetate by Chlorella

Summary When Chlorella pyrenoidosa photoassimilates ³H–¹⁴C-acetate glycollic acid rapidly becomes labelled with both tritium and ¹⁴C. The ³H/¹⁴C ratio was 10 in glycollate, (compared with 4 in the acetate added) and the only other intermediates showing similar ³H/¹⁴C ratios to glycollate were glycerate and serine. This suggests a glycollate pathway for the formation of serine was operating in Chlorella pyrenoidosa during the photoassimilation of acetate. When Chlorella pyrenoidosa assimilated ³H–¹⁴C-acetate in the dark glycollate was not labelled with either ¹⁴C or tritium. Although glycerate and serine both became labelled with ¹⁴C and tritium in the dark they did not show the high ³H/¹⁴C ratios recorded in the light. When cells were aerated with unlabelled 5% CO₂ during the photoassimilation of ³H–¹⁴C-acetate, the ³H/¹⁴C ratios of glycollate, glycerate and serine were slightly decreased. Similarly, under anaerobic conditions in the light the ³H/¹⁴C ratio was decreased compared with aerobic conditions.     

Glycollate metabolism of wheat and rice leaves during senescence and under the influence of growth regulators

The glycollate metabolism of wheat (Triticum vulgare Vill. cv. Sonalika) and rice (Oryza sativa L. ev. Jaya) leaves was studied during senescence by estimating the endogenous levels of glycollate and hydrogen peroxide (H₂O₂) and the activities of glycollate oxidase and catalase. In comparison with light incubation the incubation of excised leaves in the dark caused a decline in the glycollate content and in the activities of glycollate oxidase and catalase, and an increase in the H₂O₂ content, more marked in the leaves of rice than in the leaves of wheat. Glycollate oxidase activity gradually decreased with incubation time, and glycollate metabolism decreased during senescence. The glycollate oxidase in particular and glycollate metabolism of rice were more sensitive to incubation time than those of wheat. Kinetin increased the glycollate oxidase activity and glycollate metabolism during senescence, while ethrel (2-chloroethylpho-sphonic acid) and ABA (abscisic acid) reduced these activities in both plant species.     

Effects of Abscisic Acid, Salicylic Acid and Trans-cinnamic Acid on Phosphate Uptake, ATP-level and Oxygen Evolution in Scenedesmus

The effects of abscisic acid, salicylic acid and trans-cinnamic acid were tested on the light-induced phosphorylating reactions and oxygen evolution of the unicellular green alga Scenedesmus. It was found that abscisic acid and cinnamic acid had practically no influence on the total inorganic phosphate uptake, while salicylic acid in the concentration range of 10^-6 to 10^-3M gave a small decrease in the total inorganic phosphate uptake. The ATP level in the cells is in most cases increased when these three acids are given to the algae. The oxygen output is not significantly changed by abscisic acid or salicylic acid. Trans- cinnamic acid inhibits the oxygen evolution at concentrations of 10^-4–10^-3M None of the substances investigated caused such effects on photophosphorylation and oxygen evolution in Scenedesmus as those caused by the inhibitor β-complex from potatoes according to earlier reports. It is suggested that these effects are due to other components in the inhibitor β-complex.     

Mechanisms of phosphate uptake into brush-border membrane vesicles from goat jejunum

This study concerns the uptake of inorganic phosphate into brush-border membrane vesicles prepared from jejunal tissues of either control or Ca-and/or P-depleted goats. The brush-border membrane vesicles showed a time-dependent accumulation of inorganic phosphate with a typical overshoot phenomenon in the presence of an inwardly directed Na⁺ gradient. The Na⁺-dependent inorganic phosphate uptake was completely inhibited by application of 5 mmol·l^-1 sodium arsenate. Half-maximal stimulation of inorganic phosphate uptake into brush-border membrane vesicles was found with Na⁺ concentrations in the order of 5 mmol·l^-1. Inorganic phosphate accumulation was not affected by a K⁺ diffusion potential (inside negative), suggesting an electroneutral transport process. Stoichiometry suggested an interaction of two or more Na ions with one inorganic phosphate ion at pH 7.4. Na⁺-dependent inorganic phosphate uptake into jejunal brush-border membrane vesicles from normal goats as a function of inorganic phosphate concentration showed typical Michaelis-Menten kinetic with V _max=0.42±0.08 nmol·mg^-1 protein per 15 s^-1 and K _m=0.03±0.01 mmol·l^-1 (n=4, x ±SEM). Long-term P depletion had no effect on these kinetic parameters. Increased plasma calcitriol concentrations in Ca-depleted goats, however, were associated with significant increases of V _max by 35–80%, irrespective of the level of P intake. In the presence of an inwardly directed Na⁺ gradient inorganic phosphate uptake was significantly stimulated by almost 60% when the external pH was decreased to 5.4 (pH_out/pH_in=5.4/7.4). The proton gradient had no effect on inorganic phosphate uptake in absence of Na⁺. In summary, in goats Na⁺ and calcitriol-dependent mechanisms are involved in inorganic phosphate transport into jejunal brush-border membrane vesicles which can be stimulated by protons.Abbreviations AP activity of alkaline phosphatase - BBMV brush-border membrane vesicles - EGTA ethyleneglycol-triacetic acid - n _app apparent Hill coefficient - P _i inorganic phosphate - PTH parathyroid hormone     

Relationship between Carbon and Nitrogen Metabolisms in Photosynthesis. The Role of Photooxidation Processes

¹⁴CO₂ uptake in leaves of wheat plants (Triticum aestivum L.) fertilized by urea or Ca(NO₃)₂ (25 mol m^-3) was investigated. The Warburg effect (inhibition of ¹⁴CO₂ uptake by oxygen) under 0.03 vol. % CO₂ concentration was observed only in non-fertilized plants. Under 0.03 vol. % CO₂, the Warburg antieffect (stimulation of ¹⁴CO₂ uptake by oxygen) was detected only in plants fertilized by Ca(NO₃)₂. Under saturating CO₂ concentration (0.30 vol. %), the Warburg antieffect was observed in all variants. Under limitation of ribulose-1,5-bisphosphate carboxylase/oxygenase activity (0.30 vol. % CO₂ + 1 vol. % O₂), the rate of synthesis of glycollate metabolism products decreased in control and urea-fertilized plants but was enhanced in nitrate-fed plants. Hence, there was an activation of glycollate formation via transketolase reaction in fertilized plants, and the products of nitrate reduction function were oxidants in nitrate-fertilized plants whereas the superoxide radical played this role in urea-fertilized plants.     

Partial purification and characterization of mRNAs encoding glycollate oxidase and catalase

Polyadenylated mRNA was prepared from etiolated and greening leaves of Lens culinaris and cotyledons of Cucumis sativus during the transition from etiolated to photoautotrophic stage. These mRNA preparations were used to identify, by translation in vitro, the precursor forms of glycollate oxidase and catalase, both enzymes being markers of microbodies. The level (per fresh weight) of translatable RNA coding for glycollate oxidase was found to increase ten fold during the first 3 d of illumination of etiolated leaves. For catalase mRNA activity, this increase was less pronounced. Characterizing the products of in-vitro translation directed by the mRNA prepared, we observed a 43-kDa species of glycollate oxidase and a 56-kDa species of apo-catalase. Limited proteolysis of the in-vitro-formed proteins and comparison with the respective mature enzymes present in vivo revealed differences between the cucumber and the lens protein but not between the monomeric precursor and the subunit of mature glycollate oxidase from Lens culinaris. Messenger RNA coding for glycollate oxidase was highly purified by electrophoresis on low-melting-point agarose in the presence of methylmercuric hydroxide. The size of the mRNA was determined to be 1.47 kb. By this procedure, the mRNA for glycollate oxidase in the subfraction could be enriched in such a way that the activity, assayed by translation in a reticulocyte lysate, amounted to 30% of the total translation activity.Abbreviations PAGE polyacrylamide gel electrophoresis - poly(A)⁺ RNA polyadenylated RNA - SDS sodium dodecyl sulfate     

Effets du GA3 et de la BAP sur la respiration,l’absorption et le métabolisme du phosphate chez des disques foliaires dePelargonium zonale après survie

Respiration rate of foliarPelargonium discs was insensitive to ageing. The addition of BAP or GA₃ to the ageing medium did not produce any effect. The presence of GA₃ or BAP in the ageing medium induced an increase (27 %) or a decrease (45 %) of the phosphate uptake. The analysis of phosphorylated compound labelling showed that these two hormones decreased³²P incorporation in the non-acid soluble fraction and increased³²P incorporation in the acid soluble organic fraction. GA₃ and BAP had little effect on the distribution of radioactivity between the different acid soluble compounds, but they increased the ATP level. These results suggest that both GA₃ and BAP increase the basal metabolism, but they seem to act differently on the development of the uptake mechanism during ageing.     

Biochemical studies of morpholine catabolism by an environmental mycobacterium

Summary When Mycobacterium strain MorG was grown with morpholine as sole source of carbon and nitrogen, enzymes for ethanolamine catabolism (via the ethanolamine-O-phosphate pathway) and glycollate catabolism (via the glycerate pathway) were strongly induced. Almost all morpholine-negative (Mor^–) mutants of MorG failed to utilize glycollate as a carbon source and were shown to be effective in one or more enzymes for its metabolism via the glycerate pathway. Growth of MorG with morpholine also induced the jacoby and Fredericks pathway for pyrrolidine catabolism, Mor^– mutants had invariably lost the ability to grow on pyrrolidine and 2(2-aminoethoxy)acetate was shown to be an intermediate in morpholine catabolism. This indicates that morpholine is initially catabolised by an analogous route to pyrrolidine, producing 2(2-aminoethoxy)acetate which can be oxidatively cleaved to give rise directly to glycollate and indirectly to ethanolamine. Offprint requests to: W. A. Venables     

Hydrogen peroxide production and the release of carbon dioxide during glycollate oxidation in leaf peroxisomes

Summary The rate at which H₂O₂ becomes available during glycollate oxidation for further oxidation reactions, especially that of glyoxylate to formate and CO₂, in peroxisomes from spinach-beet (Beta vulgaris L., var. vulgaris) leaves has been determined by measuring O₂ uptake in the presence and absence of added catalase. The rates observed under air and pure O₂ were sufficient to account for the ¹⁴CO₂ released from [l-¹⁴C]glycollate under these conditions; the two reactions showed similar characteristics. In the course of the reaction, a fall in catalase activity was observed concomitant with an increase in ¹⁴CO₂ release. There is no evidence that catalase was disproportionately lost from the peroxisomes during isolation, and it is argued that the CO₂ release observed contributes to the photorespiratory CO₂ loss in intact leaves.Abbreviations DCPIP 2,6-dichlorophenolindophenol - FMN Flavin mononucleotide     

Effects of Hydroxyurea and Deoxyadenosine on the Synthesis of Deoxycytidine Phosphate and on the Uptake of Nucleosides in Excised Root Tips of Vicia faba

The effects of hydroxyurea and deoxyadenosine on the synthesis of deoxycytidine phosphate was studied by measuring the incorporation of [¹⁴C]-cytidine into acid soluble deoxycytidine phosphate in root tips of Vicia faba. Hydroxyurea and deoxyadenosine both markedly depressed the incorporation of [¹⁴C]-cytidine. Deoxyadenosine had the additional effect of inhibiting the uptake of [¹⁴C]-cytidine. Furthermore, millimolar concentrations of deoxyadenosine inhibited the uptake of micromolar concentrations of adenosine, thymidine, and deoxycytidine. The incorporation of [¹⁴C]-cytidine into RNA was only slightly affected by hydroxyurea. Deoxyadenosine inhibited the incorporation into RNA to about the same extent as the uptake of [¹⁴C]-cytidine. It is suggested that hydroxyurea reduced the incorporation of radioactive cytidine into deoxycytidine phosphate mainly by interfering with ribonucleotide reduction. The depression of [¹⁴C]-cytidine incorporation into deoxycytidine phosphate in the presence of deoxyadenosine is believed to be the result of an inhibition of both ribonucleotide reduction and [¹⁴C]-cytidine uptake.     

The influence of radioactive phosphate level on the absorption of phosphate by plants and on the determination of labile soil phosphate

Summary Previous work has suggested that the presence of P³² in fertilizers inhibits the uptake of the applied phosphate from the soil by plants, and also that if the applied phosphate is not incorporated uniformly in the soil there will be preferential uptake from regions of low specific activity. This made it desirable to determine the effect of P³²-level on phosphate uptake and the determination ofL-values in pot experiments in which the labelled phosphate source is added as discrete particles of the phosphate form of an anion-exchange resin.Increasing the level of P³² from 0.05 to 1.25 mo per gram of phosphorus in the added phosphate did not have a significant effect on the fresh weight, dry weight or total phosphorus uptake of the ryegrass crop. The measuredL-value showed a significant increase, about 15 per cent for a five-fold increase in P³² level, on each of the four soil types used, as would be expected if P³² depressed the uptake of labelled fertilizer phosphate.Although a significant effect of P³² was observed this does not invalidate a comparison of soils with respect toL-value.     

Uptake by Yeast: Interaction of Rb+, Na+ and Phosphate

It has been shown that addition of phosphate to phosphate deficient yeast gives rise to an immediate increase in the rate of Na⁺ uptake and an immediate decrease in the rate of Rb⁺ uptake. In addition, phosphate uptake is enhanced specifically by Na ions presumably by a process with a very high affinity for phosphate with a K_m of about 2 × 10⁻⁶M at pH 7.2, whereas the K_m for phosphate uptake of the Na⁺ independent process amounts to 1.3 × 10⁻⁴M.     

Use of carboxylic acids by Thiobacillus A2

Thiobacillus A2 can grow on acetate, glycollate, succinate and citrate as sole carbon and energy sources. Results of growth and transport experiments indicated that separate transport systems existed for the four acids although acetate uptake by bacteria grown on glycollate was very rapid. Citrate was a potentially toxic substrate in that low concentrations had to be supplied to adapt organisms to growth on citrate following autotrophic culture on thiosulphate. Apparent Ks values for transport by whole organisms were around 10(-4) M. The effects of uncoupling agents, phosphate and arsenate, on acid uptake did not allow identification of the mechanisms of transport, but indicated energy-requiring processes possibly involving anion participation. The ratio of carbon assimilated from the -CH2- and the -COOH carbons of succinate was about 5:1, reflecting very rapid decarboxylation of succinate following uptake into the cell.