Enzymes of the Glyoxylate Cycle in Chlorella vulgaris

The detection and assay of the enzymes of the glyoxylate cyclein Chlorella are described. The activity of the enzymes in cellsgrown on acetate is about adequate to account for the rate ofgrowth with acetate as sole carbon source. Isocitratase activity increases markedly when cells are incubatedwith acetate in darkness; malate synthetase activity also increasesbut the activity of the other enzymes is unaffected. Isocitrataseactivity does not increase when glucose is added as well asacetate or the cells are illuminated and supplied with carbondioxide. When cells are given acetate as sole carbon sourcethere is a lag of 24 hrs. before cell division begins; duringthis period, isocitratase activity increases greatly.     

Carbon Catabolite Repression Regulates Glyoxylate Cycle Gene Expression in Cucumber

We have previously proposed that metabolic status is important in the regulation of cucumber malate synthase (MS) and isocitrate lyase (ICL) gene expression during plant development. In this article, we used a cell culture system to demonstrate that intracellular metabolic status does influence expression of both of these genes. Starvation of cucumber cell cultures resulted in the coordinate induction of the expression of MS and ICL genes, and this effect was reversed when sucrose was returned to the culture media. The induction of gene expression was closely correlated with a drop in intracellular sucrose, glucose, and fructose below threshold concentrations, but it was not correlated with a decrease in respiration rate. Glucose, fructose, or raffinose in the culture media also resulted in repression of MS and ICL. Both 2-deoxyglucose and mannose, which are phosphorylated by hexokinase but not further metabolized, specifically repressed MS and ICL gene expression relative to a third glyoxylate cycle gene, malate dehydrogenase. However, the addition of 3-methylglucose, an analog of glucose that is not phosphorylated, did not result in repression of either MS or ICL. It is proposed that the signal giving rise to a change in gene expression originates from the intracellular concentration of hexose sugars or the flux of hexose sugars into glycolysis.     

The Induction of Glyoxylate Cycle Enzymes in Tissues of Starving Rats

The induction of glyoxylate cycle enzyme activities was revealed in the liver and other organs of starving rats. A five day deprivation of food was followed by the appearance of isocitrate lyase (ICL) and malate synthase activities and the increase of malate dehydrogenase (MDH) and citrate synthase activities. The induction of MDH was associated with the appearance of its new isoform with Rf 0.52. ICL activity was revealed in the liver, blood, pancreas, kidney, lungs, heart, and skeletal muscles of starving rats, reaching a peak on day 5 of food deprivation. No significant changes of blood glucose level in starving rats were revealed until day 9. A homogeneous ICL preparation with a specific activity of 12.4 IU per mg protein was obtained as the result of a five-stage purification procedure.     

Activities of Tricarboxylic Acid Cycle Enzymes, Glyoxylate Cycle Enzymes, and Fructose Diphosphatase in Bakers' Yeast During Adaptation to Acetate Oxidation

Bakers' yeast oxidizes acetate at a high rate only after an adaptation period during which the capacity of the glyoxylate cycle is found to increase. There was apparently no necessity for the activity of acetyl-coenzyme A synthetase, the capacity of the tricarboxylic acid cycle, or the concentrations of the cytochromes to increase for this adaptation to occur. Elevation of fructose 1,6 diphosphatase occurred only when acetate oxidation was nearly maximal. Cycloheximide almost completely inhibited adaptation as well as increases in the activities of isocitrate lyase and aconitate hydratase, the only enzymes assayed. p-Fluorophenylalanine was partially effective and chloramphenicol did not inhibit at all. The presence of ammonium, which considerably delayed adaptation of the yeast to acetate oxidation, inhibited the increases in the activities of the glyoxylate cycle enzymes to different degrees, demonstrating noncoordinate control of these enzymes. Under the various conditions, the only enzyme activity increase consistently related to the rising oxygen uptake rate was that of isocitrate lyase which apparently limited the activity of the cycle.     

Development of Enzymes of the Glyoxylate Cycle during Senescence of Pumpkin Cotyledons

The presence and activities of isocitrate lyase (EC 4.1.3.1 [EC] )and malate synthase (EC 4.1.3.2 [EC] ) were studied during senescenceof pumpkin cotyledons (Cucurbita sp. Amakuri Nankin). Afterincubation of detached cotyledons in permanent darkness, theactivities appeared and increased up to the eighth day and thendeclined, while the activities of catalase (EC 1.11.1.6 [EC] ), glycolateox-idase (EC 1.1.3.1 [EC] ), and hydroxypyruvate reductase (EC 1.1.1.81 [EC] )decreased dramatically. After fractionation of cell organellesby sucrose density gradient, we detected isocitrate lyase andmalate synthase activities in peroxisomal fractions. The activityof the two key enzymes of the glyoxylate cycle also increasedduring senescence in vivo and we confirmed the presence of thetwo enzymes in the peroxisomal fractions after sucrose gradientcentrifugation. At every point examined, the level of malatesynthase was demonstrated by immunoblotting. It is concludedthat the development of isocitrate lyase and malate synthaseactivities represents the transition from leaf peroxisomes toglyoxysomes and that such a phenomenon is associated with senescence. (Received January 25, 1991; Accepted March 22, 1991)     

Glyoxylate Cycle Enzymes in Peroxisomes Isolated from Petals of Pumpkin (Cucurbita sp.) during Senescence

The activities of the two unique enzymes of the glyoxylate cycle,isocitrate lyase (EC 4.1.3.1 [EC] ) and malate synthase (EC 4.1.3.2 [EC] ),were undetectable in petals of pumpkin (Cucurbita sp. AmakuriNankin) until the end of blooming, but they appeared duringsenescence. The activity of catalase (EC 1.11.1.6 [EC] ) increased,glycolate oxidase (EC 1.1.3.1 [EC] ) activity did not change, whilehydroxypyruvate reductase (EC 1.1.1.81 [EC] ) activity peaked at fullblooming stage and declined thereafter. After fractionationof cellular organelles on a sucrose density gradient, we detectedisocitrate lyase and malate synthase activities in peroxisomalfractions only from petals at the senescing stage. Northernblot analysis revealed that malate synthase mRNA increased duringpetal senescence. Citrate synthase (EC 4.1.3.7 [EC] ) and malate dehydrogenase(EC 1.1.1.37 [EC] ) activities were also present, while aconitase(EC 4.2.1.3 [EC] ) was not detectable in peroxisomal fractions. Moreoverthe presence of 3-hydroxyacyl-CoA dehydrogenase (EC 1.1.1.35 [EC] )and urate oxidase (EC 1.7.3.3 [EC] ) in the peroxisomal fractionsfrom senescing petals indicates that peroxisomes could be involvedboth in the ß-oxidation pathway and in the purinecatabolism during petal senescence. (Received May 25, 1991; Accepted September 25, 1991)     

Autotrophic CO2 Fixation by Chloroflexus aurantiacus: Study of Glyoxylate Formation and Assimilation via the 3-Hydroxypropionate Cycle

In the facultative autotrophic organism Chloroflexus aurantiacus, a phototrophic green nonsulfur bacterium, the Calvin cycle does not appear to be operative in autotrophic carbon assimilation. An alternative cyclic pathway, the 3-hydroxypropionate cycle, has been proposed. In this pathway, acetyl coenzyme A (acetyl-CoA) is assumed to be converted to malate, and two CO(2) molecules are thereby fixed. Malyl-CoA is supposed to be cleaved to acetyl-CoA, the starting molecule, and glyoxylate, the carbon fixation product. Malyl-CoA cleavage is shown here to be catalyzed by malyl-CoA lyase; this enzyme activity is induced severalfold in autotrophically grown cells. Malate is converted to malyl-CoA via an inducible CoA transferase with succinyl-CoA as a CoA donor. Some enzyme activities involved in the conversion of malonyl-CoA via 3-hydroxypropionate to propionyl-CoA are also induced under autotrophic growth conditions. So far, no clue as to the first step in glyoxylate assimilation has been obtained. One possibility for the assimilation of glyoxylate involves the conversion of glyoxylate to glycine and the subsequent assimilation of glycine. However, such a pathway does not occur, as shown by labeling of whole cells with [1,2-(13)C(2)]glycine. Glycine carbon was incorporated only into glycine, serine, and compounds that contained C(1) units derived therefrom and not into other cell compounds.     

Role of Photosynthetic Electron Transfer in Light Activation of Calvin Cycle Enzymes

The effect of light in activating fructose-1,6 biphosphate phosphatase (E.C. 3.1.3.11), sedoheptulose-1,7, biphosphate phosphatase (E.C. 3.1.3.11), ribulose-5 phosphate kinase (E.C. 2.7.1.19), ribulose-1,5 biphosphate carboxylase (E.C. 4.1.1.39) and (NADPH) glyceraldehyde-3 phosphate dehydrogenase (E.C. 1.2.1.13) in intact spinach chloroplasts in the presence of antimycin A, tetramethylethylenediamine (TMEDA) or chlorophenyl-1,1-dimethylurea (CMU) was examined. Antimycin A and TMEDA were added as stimulating agents for photosynthetic electron transfer in intact chloroplasts while CMU was added for its inhibitory characteristics. Light exerted its control through the mediation of the photosynthetic electron transfer. Antimycin A and TMEDA promoted the light activation. CMU nullified the light activation as well as the stimulatory effect of antimycin A and TMEDA. Thus the control by light of the activities of the Calvin cycle enzymes involves a reduced agent formed by the photosynthetic electron transport chain. From the presently available evidence, it seems appropriate to hypothesize that the light activation of the enzymes is not a single mechanism. In fact three types of enzymes can be distinguished: Ru-5 P kinase and (NADPH) G-3 P dehydrogenase, maximal activation of which appears within the first minute of illumination and is promoted by antimycin A and by TMEDA; F-1,6 P2 phosphatase and S-1,7 P2 phosphatase, ferredoxin-dependent enzymes, activation of which is slightly slower but is also promoted by antimycin A and by TMEDA; finally Ru-1,5 P2 carboxylase, activation of which is still slower and characterized by the absence of any response to antimycin A as well as to TMEDA.     

Role of Glycine and Glyoxylate Decarboxylation in Photorespiratory CO(2) Release

Mechanically isolated soybean leaf cells metabolized added glycolate by two mechanisms, the direct oxidation of glyoxylate and the decarboxylation of glycine. The rate of glyoxylate oxidation was dependent on the cellular glyoxylate concentration and was linear between 0.58 and 2.66 micromoles glyoxylate per milligram chlorophyll. The rate extrapolated to zero at a concentration of zero. The concentration and, therefore, the rate of oxidation of glyoxylate could be decreased by adding glutamate or serine to the cells. These substrates were amino donors for the transamination of glyoxylate to glycine. In the presence of these amino acids more CO₂ was released from added glycolate via the glycine decarboxylation reaction and less by the direct oxidation of glyoxylate.     

Cytosolic Aconitase Participates in the Glyoxylate Cycle in Etiolated Pumpkin Cotyledons

Two different aconitases are known to be expressed after thegermination of oil-seed plants. One is a mitochondrial aconitasethat is involved in the tricarboxylic acid cycle. The otherparticipates in the glyoxylate cycle, playing a role in gluconeogenesisfrom stored oil. We isolated and characterized a cDNA for anaconitase from etiolated pumpkin cotyledons. The cDNA was 3,145bp long and capable of encoding a protein of 98 kDa. N-terminaland C-terminal amino acid sequences deduced from the cDNA didnot contain mitochondrial or glyoxysomal targeting signals.A search of protein databases suggested that the cDNA encodeda cytosolic aconitase. Immuno blotting analysis with a specificantibody against the aconitase expressed in Escherichia colirevealed that developmental changes in the amount of the aconitasewere correlated with changes in levels of other enzymes of theglyoxylate cycle during growth of seedlings. Further analysisby subcellular fractionation and immunofluorescence microscopyrevealed that aconitase was present only in the cytosol andmitochondria. No glyoxysomal aconitase was found in etiolatedcotyledons even though all the other enzymes of the glyoxylatecycle are known to be localized in glyoxysomes. Taken together,the data suggest that the cytosolic aconitase participates inthe glyoxylate cycle with four glyoxysomal enzymes. (Received December 1, 1994; Accepted March 17, 1995)     

Glyoxylate Inhibition of Ribulosebisphosphate Carboxylase/Oxygenase Activation State in vivo

Photosynthetic CO₂ exchange in photorespiration mutants of Arabidopsisthaliana showed a time-dependent inhibition at 350 µl/literCO₂ in 50% O₂ but not in 2% O₂. In a glycolate-P phos-phatasedeficient mutant, inhibition of photosynthesis was due to adepletion of ribulosebisphosphate. In the remaining mutants,which have defects in photorespiratory enzymes which metabolizeamino acids, reduced photosynthesis was accompanied by a declinein the activation level of ribulosebisphosphate carboxylase/oxygenase(Chastain and Ogren 1985), a decline in ribulosebisphosphateconcentration, and an accumulation of glyoxylate. Addition ofglyoxylate at submillimolar concentrations to intact spinach(Spinacea oleracea L.) chloroplasts inhibited light activationof ribulosebisphosphate carboxylase/oxygenase (rubisco) andCO₂ fixation. Similar concentrations of glyoxylate had no effecton A. thaliana rubisco activity in vitro. These results suggestthat glyoxylate accumulation indirectly inhibited rubisco activationstate in vivo. The inhibition of photosynthesis in mutants whichaccumulate glyoxylate may be attributed to a decline in ribulosebisphosphateconcentration, a reduction in rubisco activation state, or acombination of both phenomena. ³Present address: CSIRO, Division of Plant Industry, GPO Box1600, Canberra, ACT 2601, Australia. (Received May 12, 1989; Accepted July 8, 1989)     

Nuclear Localization of Mitochondrial TCA Cycle Enzymes as a Critical Step in Mammalian Zygotic Genome Activation

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Suppression of Ripening-Associated Gene Expression in Tomato Fruits Subjected to a High CO2 Concentration

High concentrations of CO2 block or delay the ripening of fruits. In this study we investigated the effects of high CO2 on ripening and on the expression of stress- and ripening-inducible genes in cherry tomato (Lycopersicon esculentum Mill.) fruit. Mature-green tomato fruits were submitted to a high CO2 concentration (20%) for 3 d and then transferred to air. These conditions effectively inhibited ripening-associated color changes and ethylene production, and reduced the protein content. No clear-cut effect was observed on the expression of two proteolysis-related genes, encoding polyubiquitin and ubiquitin-conjugating enzyme E2, respectively. Exposure of fruit to high CO2 also resulted in the strong induction of two genes encoding stress-related proteins: a ripening-regulated heat-shock protein and glutamate decarboxylase. Induction of these two genes indicated that high CO2 had a stress effect, most likely through cytosolic acidification. In addition, high CO2 blocked the accumulation of mRNAs for genes involved in the main ripening-related changes: ethylene synthesis (1-aminocyclopropane-1-carboxylic acid synthase and 1-aminocyclopropane-1-carboxylic acid oxidase), color (phytoene synthase), firmness (polygalacturonase), and sugar accumulation (acid invertase). The expression of ripening-specific genes was affected by CO2 regardless of whether their induction was ethylene- or development-dependent. It is proposed that the inhibition of tomato fruit ripening by high CO2 is due, in part, to the suppression of the expression of ripening-associated genes, which is probably related to the stress effect exerted by high CO2.     

An Aspect of Urea Cycle Enzymes in Goat

A large amount of ammonia is produced in the rumen and some portion of the ammonia are absorbed into the portal blood through the rumen mucosa. Accordingly, it seems that ammonia detoxication is more necessary for the ruminant than for the non-ruminant. Activities of the urea cycle enzymes as principal instrument for ammonia detoxication in goat were investigated in this experiment.

The activities of the urea cycle enzymes of goat were found to be very similar to those of rat reported by other authors. The activities of the urea cycle enzymes were affected by the protein level of diet. Administration of magnesium aspartate increased the activities of argininosuccinate synthetase and arginase, and had some effect on the concentrations of citrulline, aspartic acid, and urea in the liver.