Glyoxylate Cycle Enzymes Research Articles

Involvement of cyanide-resistant and rotenone-insensitive pathways of mitochondrial electron transport during oxidation of glycine in higher plants

Metabolism of glycine in isolated mitochondria and protoplasts was investigated in photosynthetic, etiolated (barley and pea leaves) and fat-storing (maize scutellum) tissues using methods of [1- 14C]glycine incorporation and counting of 14CO 2 evolved, oxymetric measurement of glycine oxidation and rapid fractionation of protoplasts incubated in photorespiratory conditions with consequent determination of ATP/ADP ratios in different cell compartments. The involvement of different paths of electron transport in mitochondria during operation of glycine decarboxylase complex (GDC) was tested in different conditions, using aminoacetonitrile (AAN), the inhibitor of glycine oxidation in mitochondria, rotenone, the inhibitor of Complex I of mitochondrial electron transport, and inhibitors of cytochrome oxidase and alternative oxidase. It was shown that glycine has a preference to other substrates oxidized in mitochondria only in photosynthetic tissue where succinate and malate even stimulated its oxidation. Rotenone had no or small effect on glycine oxidation, whereas the role of cyanide-resistant path increased in the presence of ATP. Glycine oxidation increased ATP/ADP ratio in cytosol of barley protoplasts incubated in the presence of CO 2, but not in the CO 2-free medium indicating that in conditions of high photorespiratory flux oxidation of NADH formed in the GDC reaction passes via the non-coupled paths. Activity of GDC in fat-storing tissue correlated with the activity of glyoxylate-cycle enzymes, glycine oxidation did not reveal preference to other substrates and the involvement of paths non-connected with proton translocation was not pronounced. It is suggested that the preference of glycine to other substrates oxidized in mitochondria is achieved in photosynthetic tissue by switching to rotenone-insensitive intramitochrondrial NADH oxidation and by increasing of alternative oxidase involvement in the presence of glycine.

Isocitrate lyase from Aspergillus nidulans: crystallization and X-ray analysis of a glyoxylate cycle enzyme.

Isocitrate lyase (ICL) from the filamentous fungus Aspergillus nidulans catalyzes the first committed step of the carbon-conserving glyoxylate bypass. This enzyme has been crystallized by the hanging-drop method of vapour diffusion using polyethylene glycol 2000 as the precipitant. Diffraction patterns show that the crystals diffract to beyond 2.5 A and are probably in space group P4(2)2(1)2 with unit-cell dimensions of a = b = 91.9 and c = 152.7 A, with one molecule in the asymmetric unit. The elucidation of the structure of this enzyme to high resolution will advance the understanding of how the metabolic branch point between the tricarboxylic acid cycle and the glyoxylate bypass is controlled by the affinity of ICL for its substrate isocitrate and contribute to a programme of rational drug design.

Natural Senescence of Pea Leaves (An Activated Oxygen-Mediated Function for Peroxisomes).

We studied the activated oxygen metabolism of peroxisomes in naturally and dark-induced senescent leaves of pea (Pisum sativum L.). Peroxisomes were purified from three different types of senescent leaves and the activities of different peroxisomal and glyoxysomal enzymes were measured. The activities of the O2-- and H2O2-producing enzymes were enhanced by natural senescence. Senescence also produced an increase in the generation of active oxygen species (O2- and H2O2) in leaf peroxisomes and in the activities of two glyoxylate-cycle marker enzymes. A new fraction of peroxisomes was detected at an advanced stage of dark-induced senescence. Electron microscopy revealed that this new peroxisomal fraction varied in size and electron density. During senescence, the constitutive Mn-superoxide dismutase (SOD) activity of peroxisomes increased and two new CuZn-SODs were induced, one of which cross-reacted with an antibody against glyoxysomal CuZn- SOD. This fact and the presence of glyoxylate-cycle enzymes support the idea that foliar senescence is associated with the transition of peroxisomes into glyoxysomes. Our results indicate that natural senescence causes the same changes in peroxisome-activated oxygen metabolism as dark-induced senescence, and reinforce the hypothesis of an effective role of peroxisomes and their activated oxygen metabolism in this stage of the life cycle.

Induction of Glyoxylate Cycle Expression in Caenorhabditis elegans: A Fasting Response throughout Larval Development

The mRNA and the bifunctional protein for the two glyoxylate cycle (GC) enzymes, isocitrate lyase and malate synthase, are expressed in a tissue- and stage-specific pattern in Caenorhabditis elegans. Since expression of the two enzymes for the carbon-conserving glyoxylate cycle is regulated by the availability of carbon sources in microorganisms, we have studied the bifunctional GCP gene expression under fasting conditions and in certain mutants of C. elegans in order to understand possible mechanisms regulating its expression during nematode development. The GCP mRNA and protein levels were elevated in early larvae which were never fed, a result consistent with previous enzyme activity measurements (Khan, F.R., & McFadden, B.A. (1982) Exp. Parasitol. 54, 48-54]. However, larvae of later stages also expressed higher levels of GCP mRNA and protein when they were shifted from normal to fasting growing conditions. The GCP expression appeared to be regulated primarily at the transcriptional level throughout development. Although the expression of both the GCP gene and lin-14 peaks at about the same time during development and are induced by fasting, the regulation of the GCP gene is independent of the heterochronic lin-14 control mechanism of postembryonic lineages, as demonstrated by the fact that there was no significant change of the GCP at both mRNA and protein levels in the heterochronic lin-4 (lf) and lin-14 (gf) mutants compared to the wild type.

Transcriptional regulation of peroxisomal glyoxylate cycle enzymes of an n-alkane-assimilating yeast, Candida tropicalis.

Transcriptional regulation of peroxisomal glyoxylate cycle enzymes of an n-alkane-assimilating yeast, Candida tropicalis.

Induction of glyoxylate cycle enzymes in rat liver upon food starvation

The key enzymes of the glyoxylate cycle, isocitrate lyase and malate synthase, have been detected in liver of foodstarved rats. Activities became measurable 3 days and peaked 5 days after the beginning of starvation. Both enzymes were found in the peroxisomal cell fraction after organelle fractionation by isopycnic centrifugation. Isocitrate lyase was purified 112-fold by ammonium sulfate precipitation, and chromotography on DEAE-cellulose and Toyopearl HW-65. The specific activity of the purified enzyme was 9.0 units per mg protein. The K m(isocitrate) was 68 μM and the pH optimum was at pH 7.4. Malate synthase was enriched 4-fold by ammonium sulfate precipitation. The enzyme had a K m(acetyl-CoA) of 0.2 μM, a K m(glyoxylate) of 3 mM and a pH optimum of 7.6.

CHANGES IN CELL COMPOSITION AND LIPID METABOLISM MEDIATED BY SODIUM AND NITROGEN AVAILABILITY IN THE MARINE DIATOM PHAEODACTYLUM TRICORNUTUM (BACILLARIOPHYCEAE)1

ABSTRACTThe effects of nitrogen starvation in the presence or absence of sodium in the culture medium were monitored in batch cultures of the marine diatom Phaeodactylum tricornutum Bohlin. During nitrogen starvation in the presence of sodium, cell nitrogen and chlorophyll a decreased, mainly as a consequence of continued cell division. These decreases were accompanied by decreases in the rates of photosynthesis and respiration. There was no change in either cell volume or carbohydrate, but both carbon and lipid increased. During nitrogen starvation in the absence of sodium, cell division ceased. Cell nitrogen and chlorophyll a remained constant, and respiration did not decrease, but the changes in the photosynthetic rate and the lipid content per cell were similar to cultures that were nitrogen‐starved in the presence of sodium. The carbon‐to‐nitrogen ratio increased in both cultures. Nitrogen, in the form of nitrate, and sodium were resupplied to cultures that had been preconditioned in nitrogen‐ and sodium‐deficient medium for 5 d. Control cultures to which neither nitrate or sodium were added remained in a static state with respect to cell number, volume, and carbohydrate but showed slight increases in lipid. Cells in cultures to which 10 mM nitrate alone was added showed a similar response to cultures where no additions were made. Cells in cultures to which 50 mM sodium alone was added divided for 2 d, with concomitant small decreases in all measured constituents. Cell division resumed in cultures to which both sodium and nitrate were added. The lipid content fell dramatically in these cells and was correlated to metabolic oxidation via measured increases in the activity of the glyoxylate cycle enzyme, isocitrate lyase. We conclude that lipids are stored as a function of decreased growth rate and are metabolized to a small extent when cell division resumes. However, much higher rates of metabolism occur if cell division resumes in the presence of a nitrogen source.

Diauxic Growth in Rice Suspension Cells Grown on Mixed Carbon Sources of Acetate and Glucose.

Diauxic growth was observed in rice (Oryza sativa L.) suspension cells growing on acetate (10 mM) and glucose (10 mM). Cells used acetate during the first growth phase and the acetate level in the medium was rapidly decreased, whereas the level of glucose remained essentially unchanged. After acetate was depleted from the medium, cells started to use glucose, forming the second growth phase. It appears that uptake of [14C]glucose was repressed during the first growth phase and became active during the second growth phase. In contrast, uptake of [14C]acetate occurred actively throughout the diauxic growth. By further demonstrating the specific induction of isocitrate lyase (EC 4.1.3.1), a glyoxylate cycle enzyme, and hexokinase (EC 2.7.1.1), a glycolysis enzyme, during the first and second growth phases, respectively, it was clearly shown that rice cells use acetate first and do not use both carbon sources simultaneously. This kind of diauxic growth pattern has been observed in bacteria. To our knowledege, this study is the first report demonstrating the presence of diauxic growth in plant cells.

RTG Genes in Yeast That Function in Communication between Mitochondria and the Nucleus Are Also Required for Expression of Genes Encoding Peroxisomal Proteins

In Saccharomyces cerevisiae cells with dysfunctional mitochondria, such as in petites, the CIT2 gene encoding the peroxisomal glyoxylate cycle enzyme, citrate synthase 2 (CS2), is transcriptionally activated by as much as 30-fold, a phenomenon we call retrograde regulation. Two genes, RTG1 and RTG2, are required for both basal and elevated expression of CIT2 (Liao, X., and Butow, R. A. (1993) Cell 72, 61-71). Different blocks in the tricarboxylic acid cycle also elicit an increase in CIT2 expression, but not to the extent observed in petites. We have examined whether other genes of the glyoxylate cycle exhibit retrograde regulation and the role of RTG1 and RTG2 in their expression. Of the glyoxylate cycle genes tested, CIT2 is the only one that shows retrograde regulation, suggesting that CS2 may be an important control point for metabolic cross-feeding from the glyoxylate cycle to mitochondria. Surprisingly, RTG1 and RTG2 are required for efficient growth of cells on medium containing oleic acid, a condition which induces peroxisome biogenesis; these genes are also required together for oleic acid induction of three peroxisomal protein genes tested, POX1 and CTA1 involved beta-oxidation of long chain fatty acids and PMP27, which encodes the most abundant protein of peroxisomal membranes. These data indicate that, in addition to their role in retrograde regulation of CIT2, the RTG genes are important for expression of genes encoding peroxisomal proteins and are thus key components in a novel, three-way path of communication between mitochondria, the nucleus, and peroxisomes.

Evidences of glyoxylate cycle in peroxisomes of senescent cotyledons

The metabolic pathway of the glyoxylate cycle has been investigated in peroxisomes isolated from senescent pumpkin ( Cucurbita sp.) cotyledons. β-oxidation activity, as well as activities of glyoxylate cycle enzymes isocitrate lyase (EC 4.1.3.1), malate synthase (EC 4.1.3.2), malate dehydrogenase (EC 1.1.1.37) and citrate synthase (EC 4.1.3.7) were detected. In order to establish if there is a channelling of acetyl CoA into the glyoxylate cycle, peroxisomes have been incubated with various substrates. The incubations show acetyl CoA utilization by the glyoxylate cycle. When the incubation medium for citrate formation is used, all the label from [1- 14C]acetyl CoA is recovered in citrate, whereas only 18% of the added radioactivity is recovered in malic acid (by isocitrate lyase and malate synthase) after a long incubation time (3 h). Only by feeding peroxisomes with [1,5- 14C]citric acid and exogenous aconitase (EC 4.2.1.3) can a weak formation of other organic acids (glyoxylate and succinate) be note. The requirement for exogenous aconitase to carry out the peroxisomal glyoxylate cycle points towards the isocitric acid step as a crucial factor for the operation of the global cycle.

Derepression of the glyoxylate cycle in mutants of Neurospora crassa accelerated for growth on acetate.

Two spontaneous allelic mutations have been isolated with the unusual semi-dominant phenotype of faster-than-wild-type growth on acetate as sole carbon source. The mutants were designated Aag-1 (accelerated acetate growth) and mapped on linkage group II. Upon re-isolation of both the Aag-1 alleles from repeated back-crosses to wild-type, between 1 and 6% of the progeny were found to be acu (acetate non-utilizing) mutants. Ten of these were selected for heterokaryon complementation analysis with known acu mutants; nine proved to be new alleles of acu-5 (deficient in acetyl-CoA synthetase), and one was a new acetate non-utilizing class, designated acu-14. Although the Aag-1 mutants clearly have no acetate-growth-related enzyme deficiencies, they did exhibit significant constitutive enzyme levels for acetyl-CoA synthetase and the glyoxylate cycle enzymes (isocitrate lyase and malate synthase) on the non-inducing carbon source, sucrose. The derepression was restricted to these enzymes, as representative enzymes from other carbon-assimilatory pathways remained repressed and subject to carbon catabolite repression. Northern blot analysis of the mRNA levels of acetyl-CoA synthetase and the glyoxylate cycle enzymes from the mutants demonstrated the derepression to occur at the level of transcription. These data suggest that the physiological explanation for the accelerated acetate growth phenotype lies in the standing levels of the acetate-assimilatory enzymes, which enable the mutants to forgo some of the normal time required for adaption to growth on acetate.

Mitochondrial Alcohol Dehydrogenase from Ethanol-Grown Euglena gracilis

The inducing effects of ethanol on alcohol dehydrogenase and the key enzymes of the glyoxylate cycle, isocitrate lyase and malate synthase, in Euglena cells were investigated. Ethanol as the sole carbon source resulted in increases in alcohol dehydrogenase and the two glyoxylate cycle enzymes. The experimental results indicated that ethanol is assimilated by alcohol dehydrogenase and the glyoxylate cycle in Euglena. Mitochondria from aerobically grown Euglena contain a unique type of alcohol dehydrogenase that accounts for their ability to respire with ethanol as a substrate. This alcohol dehydrogenase was purified to homogeneity from ethanol-grown Euglena gracilis. The mitochondrial alcohol dehydrogenase was NAD(+)-specific but not NADP(+)-specific. Ethanol was the most active substrate, but the enzyme was also active towards 1-butanol, 1-heptanol, cinnamyl alcohol, and myristyl alcohol. These results indicated that mitochondrial alcohol dehydrogenase participated in alcohol metabolism in Euglena gracilis.

NADP+‐isocitrate dehydrogenase in germinating and senescing pumpkin cotyledons

NADP+‐isocitrate dehydrogenase (IDH, EC 1.1.1.42) was studied during the post‐germinative growth of pumpkin (Cucurbita pepo L. cv. Alberello di Sarzana) seedlings. In cotyledons. IDH activity increased in the dark and declined after illumination. Native PAGE showed that at least two isozymes of low electrophoretic mobility are present in cotyledons and absent in other pumpkin tissues. Anion exchange chromatography performed on extracts both from 4‐day‐old etiolated cotyledons and from illuminated cotyledons confirmed the trend of the IDH isoforms. In senescing cotyledons an additional IDH isoform with higher electrophoretic mobility appears. Overall the data indicate the presence of specific NADP+‐IDH isoforms in etiolated cotyledons and senescing cotyledons, when glyoxylate cycle enzymes are active. A possible role for these IDH isoforms is proposed.

Bifunctional Glyoxylate Cycle Protein of Caenorhabditis elegans: A Developmentally Regulated Protein of Intestine and Muscle

The reaction of an abundant 106-kDa polypeptide with a specific monoclonal antibody has been localized in intestinal and muscle cells of the nematode Caenorhabditis elegans . This protein was first detected in 4-6 cells of the clonal E lineage of 100-cell embryos. This lineage is committed to the intestinal cell fate. The antigen continued to be expressed in the differentiating gut and then appeared in early differentiating body wall muscle cells of 400- to 500-cell embryos. Molecular cloning and sequencing showed that the largest cDNA clone contained 3274 bp and encoded a sequence of 1005 amino acids. The predicted polypeptide of 112,799 MW contains separate domains for the glyoxylate cycle enzymes isocitrate lyase and malate synthase. Their enzymatic activities had been shown previously to be highest in embryos and L1 larvae (Khan, F. R., and McFadden, B. A. (1980). FEBS Lett. 115, 312-314; Khan, F. R., and McFadden, B. A. (1982). Exp. Parasitol. 54, 48-54; Wadsworth, W. G., and Riddle, D. L. (1989). Dev. Biol. 132, 167-173). The domain-specific sequences were shown to be contiguous in genomic DNA and are separated by an intron of 68 bp. A single polypeptide and both enzymatic activities are precipitated by the antibody, and peptide fragments resulting from limited proteolytic digestion contained amino acid sequences which overlap the predicted junctional region. The physical localization of the gene correlates with a small region of the left arm of Linkage Group V to which multiple embryonic lethal mutations have been mapped.

Glyoxylate Cycle Enzyme Synthesis During the Irreversible Phase of Senescence of Cucumber Cotyledons

Summary Glyoxylate cycle enzymes isocitrate lyase (ICL; EC 4.1.3.1) and malate synthase (MS; EC 4.1.3.2) are synthesised during the terminal irreversible phase of cucumber cotyledon senescence when the endomembrane system (endoplasmic reticulum, Golgi and tonoplast) is disassembled. This follows the period of reversible senescence when most chloroplast thylakoid membrane disassembly occurs. During the terminal stage of senescence, nuclear integrity is maintained for gene transcription, and mitochondria and peroxisomes are thought to participate in reactions of the glyoxylate cycle and gluconeogenesis.

The isocitrate lyase gene of cucumber: Isolation, characterisation and expression in cotyledons following seed germination

The cucumber (Cucumis sativus L.) genome contains only a single gene encoding the glyoxylate cycle enzyme isocitrate lyase (ICL). The cucumber icl gene has been isolated and sequenced, revealing only two small introns. The predicted amino acid sequence is more than 85% identical to ICL from other higher plants, and contains the C-terminal tripeptide Ser-Arg-Met which resembles a peroxisomal targeting sequence. The icl gene is coordinately expressed with the malate synthase (ms) gene after seed germination in both the light and the dark, suggesting that these genes may contain similar DNA elements regulating transcription. The start of transcription of the icl gene was determined and the DNA sequences upstream compared with the region of the ms gene promoter known to regulate transcription. This comparison revealed a highly conserved DNA sequence at similar positions in each gene.

Metabolic regulation of glyoxylate-cycle enzyme synthesis in detached cucumber cotyledons and protoplasts

When cotyledons are detached from cucumber (Cucumis sativus L. cv. Masterpiece) plants 14 d after seed imbibition, accumulation of the glyoxylate-cycle enzymes malate synthase (MS) and isocitrate lyase (ICL) occurs rapidly in the dark but not in the light. This is primarily due to an increase in transcript abundance. It has been proposed that glyoxylate-cycle enzymes are synthesised in response to lipid degradation. However, MS and ICL synthesis in detached cucumber cotyledons begins before a decline in lipid can be detected. Furthermore, the activation of MS and ICL gene expression does not correlate with loss of chlorophyll, carotenoid, protein or RNA. These results are discussed in terms of the activation of genes encoding glyoxylate-cycle enzymes and their possible function in detached and senescing organs. Previous work (Graham et al, 1992, Plant Cell 3, 349–357) has suggested that sucrose or other carbohydrates can repress the synthesis of MS and ICL. Consistent with this proposal, incubation of detached cotyledons in the light leads to a fivefold increase in sucrose content. However, when MS and ICL synthesis occurs in the dark, no change in the amount of sucrose is detected. Incubation of cotyledons in the dark in the presence of 25 mM sucrose leads to a complete inhibition of the accumulation of MS and ICL. Similarly, cotyledon protoplasts incubated in darkness accumulate MS and ICL, but not if 25 mM sucrose is included in the medium. These results collectively support the view that MS and ICL synthesis responds to the metabolic activities of plant cells.

The function and specificity of the C-terminal tripeptide glyoxysomal targeting signal in Neurospora crassa.

The function of the C-terminal tripeptide targeting signal responsible for microbody targeting in many eukaryotes has been investigated in the filamentous fungus Neurospora crassa. Using an in-vivo targeting assay that employs transformants carrying C-terminally-modified versions of the bacterial enzyme chloramphenicol acetyltransferase (CAT), it has been demonstrated that C-terminal tripeptide-dependent import occurs most efficiently in response to nutritional acetate-induction. Under these conditions Neurospora generates a specialized organelle, the glyoxysome, which carries the enzymes responsible for the glyoxylate cycle and can be distinguished from peroxisome-like microbodies that contain catalase. Moreover, several C-terminal peptides have been tested in this system to extend the tripeptide targeting consensus to A/C/G/S-H/K/Q/R-I/L/V. However, the tripeptide analogue, ARM, found at the C-terminus of the glyoxylate cycle enzyme isocitrate lyase in higher plants, does not apparently function here.

Postharvest lipid loss, malate accumulation, and appearance of malate synthase activity in asparagus spear tips

Lipid levels, glyoxylate-cycle enzymes and respiratory quotients were measured in 3 cm tips of asparagus spears (Asparagus officinalis L.) during 5 days of storage at 20°C, to determine whether lipids are mobilised as in some other senescing plant tissues. Within 12 h, the respiratory quotient dropped from near unity to 0.66 before stabilising around 0.75 after 2 days. Over 5 days of storage, almost half of the total lipid in spear tips was lost. Malate synthase (EC 4.1.3.2) activity, which was not detectable at harvest, appeared after 1–2 days and continued to increase throughout the remaining 3 days. Malate concentration increased 3- to 4-fold in 4–5 days. Isocitrate lyase (EC 4.1.3.1) activity was not detected at any time during storage, and it is unlikely that the glyoxylate cycle functions in tips of harvested asparagus spears. These results are discussed in relation to other postharvest metabolic events in asparagus.

β-oxidation of fatty acids is linked to the glyoxylate cycle in the aleurone but not in the embryo of germinating barley

An assay for the measurement of overall β-oxidation was optimized for use with extracts from germinating barley. The most important adaptation is the addition of commercially available acyl-CoA synthetase to the incubation medium in order to reactivate the palmitate formed by the high thioesterase activity of the extracts. The β-oxidation activity determined by the overall assay originated exclusively from microbodies, since inhibitors of the electron-transport chain and alternative respiration in mitochondria did not affect the total β-oxidation activity. Activity patterns of β-oxidation upon germination were determined both for barley aleurone and embryo. In the aleurone, β-oxidation activity was already present in the quiescent grain and increased during germination, reaching an optimum 4 days after germination. In addition, in the embryo β-oxidation activity could be demonstrated in the quiescent grain, and it increased upon germination. Within the growing embryo, β-oxidation activity was present in leaves, roots and scutellum. The maximal activity levels reached in the various embryo parts were significantly lower than those in the aleurone. Organelle separation by density gradient centrifugation of homogenates from scutellum suggests that, in this tissue, microbodies constitute the only subcellular compartment carrying out β-oxidation. In the aleurone, the developmental activity profiles of glyoxylate cycle enzymes closely resemble those of β-oxidation indicating that, in this tissue, β-oxidation is localized in so-called glyoxysomes. Inability to detect glyoxylate cycle enzyme activities in the embryo indicates different metabolic functions of the β-oxidation route in aleurone and embryo.