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Early reports have demonstrated the occurrence of glyoxylate cycle enzymes in several Leishmania species. However, these results have been underestimated because genes for the two key enzymes of the cycle, isocitrate lyase (ICL) and malate synthase (MS), are not annotated in Leishmania genomes. We have re-examined this issue in promastigotes of Leishmania amazonensis. Enzyme activities were assayed spectrophotometrically in cellular extracts and characterized partially. A 40 kDa band displaying ICL activity was visualized on zymograms of the extracts. By immunoblotting with mouse antibodies against ICL from Bacillus stearothermophilus, a band of approximately 40 kDa was identified, coincident with the relative molecular mass of the activity band revealed on zymograms. Indirect immunofluorescence of intact promastigotes showed that the recognized antigen is distributed as a punctuated pattern, mainly distributed beneath the subpellicular microtubules, over a diffused cytoplasmic stain. These results clearly demonstrate the existence of an apparent ICL activity in L. amazonensis promastigotes, which is associated to a 40 kDa polypeptide and distributed both diffused and as punctuate aggregates in the cytoplasm. The relevance of this activity is discussed. [ABSTRACT FROM AUTHOR]

We cultured Chlamydomonas reinhardtii cells in a minimal culture medium supplemented with various concentrations of acetate, fatty acids, ethanol, fatty alcohols, or sucrose. The presence of acetate (0.5 or 1.0 %, w/v) was advantageous for cell growth. To determine whether peroxisomes are involved in fatty acid and fatty alcohol metabolism, we investigated the dynamics of peroxisomes, including changes in their number and size, in the presence of acetate, ethanol, and sucrose. The total volume of peroxisomes increased when cells were grown with acetate, but did not change when cells were grown with ethanol or sucrose. We analyzed cell growth on minimal culture medium supplemented with various fatty acids (carbon chain length ranging from one to ten) to investigate which fatty acids are metabolized by C. reinhardtii. Among them, acetate caused the greatest increase in growth when added to minimal culture media. We analyzed the transcript levels of genes encoding putative glyoxysomal enzymes. The transcript levels of genes encoding malate synthase, malate dehydrogenase, isocitrate lyase, and citrate synthase increased when Chlamydomonas cells were grown on minimal culture medium supplemented with acetate. Our results suggest that Chlamydomonas peroxisomes are involved in acetate metabolism via the glyoxylate cycle. [ABSTRACT FROM AUTHOR]

Oilseed crops are an important natural resource because they can be used for food and renewable energy production. However, oilseed seedling establishment and vigor depend upon the capacity to overcome functional transition, a developmental stage characterized by the consumption of the remaining oil reserves, through β-oxidation and glyoxylate cycle, and the onset of autotrophic metabolism. The increased growth and the acclimation to full photosynthetic activity lead to production of reactive oxygen species and a reorganization of the cell antioxidant systems to achieve a new redox homeostasis. In the present study, catalase (CAT) was inhibited by 3-amino-1,2,4-triazole application during functional transition in sunflower and safflower seedlings to understand the effect of this antioxidant enzyme impairment on the mRNA expression of the glyoxylate cycle enzymes isocitrate lyase (ICL) and malate synthase (MLS), as well as the superoxide dismutase (SOD) activity and ascorbate peroxidase (APX) activity and expression. CAT inhibition led to significant seedling growth reduction and increases in HO content, SOD activity, and mRNA expression of CAT and APX in both species. However, APX activity was induced only in safflower plants. Additionally, ICL and MLS mRNA expressions were upregulated after 6 h of treatment when compared to the control values. These results indicate that under CAT impairment conditions, redox homeostasis at the functional transition phase was partially supported by the SOD and APX antioxidant systems to maintain the seedling photosynthetic establishment. [ABSTRACT FROM AUTHOR]

Malate synthase (Mls), a key enzyme in the glyoxylate cycle, is required for virulence in microbial pathogens. In this study, we identified the AoMls gene from the nematode-trapping fungus Arthobotrys oligospora. The gene contains 4 introns and encodes a polypeptide of 540 amino acids. To characterize the function of AoMls in A. oligospora, we disrupted it by homologous recombination, and the Δ AoMls mutants were confirmed by PCR and Southern blot analyses. The growth rate and colony morphology of the Δ AoMls mutants showed no obvious difference from the wild-type strains on potato dextrose agar (PDA) plate. However, the disruption of gene AoMls led to a significant reduction in conidiation, failure to utilize fatty acids and sodium acetate for growth, and its conidia were unable to germinate on minimal medium supplemented with sodium oleate. In addition, the trap formation was retarded in the Δ AoMls mutants, which only produced immature traps containing one or two rings. Moreover, the nematicidal activity of the Δ AoMls mutants was significantly decreased. Our results suggest that the gene AoMls plays an important role in conidiation, trap formation and pathogenicity of A. oligospora. [ABSTRACT FROM AUTHOR]

2-Ketoisocaproate (KIC) is used as a therapeutic agent, and a KIC-producing organism may serve as a platform for products deriving from this 2-keto acid. We engineered Corynebacterium glutamicum for the production of KIC from glucose by deletion of ltbR and ilvE, encoding the transcriptional repressor LtbR and transaminase B, respectively, and additional overexpression of ilvBNCD, encoding acetohydroxyacid synthase, acetohydroxyacid isomeroreductase, and dihydroxyacid dehydratase. The KIC-producing strain was improved by deletion of the methylcitrate synthase genes and by decreasing citrate synthase activity by exchange of the native promoter of the citrate synthase gene. In shake-flask fermentations under l-leucine limitation, the newly constructed strain C. glutamicum VB (pJC4 ilvBNCD) produced 31 ± 2 mM (4.0 ± 0.3 g l) KIC and showed a product yield of about 0.26 ± 0.02 mol per mole (0.19 ± 0.01 g per gram) of glucose. As by-product, the strain formed about 33 mM 2-ketoisovalerate, which is a precursor of KIC. KIC production was further improved by additional expression of an isopropylmalate synthase allele ( leuA), encoding an enzyme resistant towards l-leucine inhibition, and by addition of acetate as additional substrate. With glucose and acetate, the newly constructed strain produced 71 ± 3.2 mM (9.2 ± 0.4 g l) KIC with a yield of 0.24 ± 0.01 mol C (KIC) per mole C (in both substrates) and with nearly no 2-ketoisovalerate by-product formation (<2 mM). Investigating the activities and regulation of the native isopropylmalate synthase and dehydratase of C. glutamicum, we observed competitive and noncompetitive inhibition, respectively, by KIC. [ABSTRACT FROM AUTHOR]

The glyoxylate cycle is an anabolic pathway that is necessary for growth on nonfermentable carbon sources such as vegetable oils and is important for riboflavin production by the filamentous fungus Ashbya gossypii. The aim of this study was to identify malate synthase in the glyoxylate cycle of A. gossypii and to investigate its importance in riboflavin production from rapeseed oil. The ACR268C gene was identified as the malate synthase gene that encoded functional malate synthase in the glyoxylate cycle. The ACR268C gene knockout mutant lost malate synthase activity, and its riboflavin production and oil consumption were 10- and 2-fold lower, respectively, than the values of the wild-type strain. In contrast, the ACR268C gene-overexpressing strain showed a 1.6-fold increase in the malate synthase activity and 1.7-fold higher riboflavin production than the control strain. These results demonstrate that the malate synthase in the glyoxylate cycle has an important role not only in riboflavin production but also in oil consumption. [ABSTRACT FROM AUTHOR]

Malate synthase (EC 2.3.3.9, formerly EC 4.1.2.2) has been investigated in the unicellular green algae Chlamydomonas reinhardtii. The molecular characteristics and the regulation of gene expression have been investigated for the enzyme. A full-length malate synthase cDNA has been isolated, containing an open reading frame of 1,641 bp encoding a polypeptide of 546 amino acids. This protein shares the conserved signature of the malate synthase family, along with the catalytic residues essential for enzymatic activity and a C-terminal motif that matches the consensus for glyoxysome import. Functionality studies have been facilitated by heterologous expression of the malate synthase cDNA in Escherichia coli. The remarkable metabolic versatility of the alga has been used to analyse the metabolic control of malate synthase gene expression. The data strongly support the role of acetate and light as the main regulatory effectors, and the existence of cross-talk between the two signalling pathways. [ABSTRACT FROM AUTHOR]

Key enzymes of the glyoxylate cycle, isocitrate lyase and malate synthase, were identified in pupas of the butterfly Papilio machaon L. The activities of these enzymes in pupas were 0.056 and 0.108 unit per mg protein, respectively. Isocitrate lyase was purified by a combination of various chromatographic steps including ammonium sulfate fractionation, ion- exchange chromatography on DEAE-Toyopearl, and gel filtration. The specific activity of the purified enzyme was 5.5 units per mg protein, which corresponded to 98-fold purification and 6% yield. The enzyme followed Michaelis-Menten kinetics (Km for isocitrate, 1.4 mM) and was competitively inhibited by succinate (Ki = 1.8 mM) and malate (R = 1 mM). The study of physicochemical properties of the enzyme showed that it is a homodimer with a subunit molecular weight of 68 ± 2 kD and a pH optimum of 7.5 (in Tris-HCl buffer). [ABSTRACT FROM AUTHOR]

A phylogenetic study of the largest tribe of palms, the Areceae, was conducted using sequences of two low-copy nuclear genes. Previous morphological and plastid DNA studies have not supported the monophyly of the tribe, but have placed its members in a large clade that includes the monophyletic tribes Geonomeae, Cocoeae, Podococceae, and Hyophorbeae. We analyzed this large clade to test the monophyly of tribe Areceae with nuclear data, to explore relationships among its subtribes, and to identify other monophyletic groups. For 54 palm species, including members of all 17 subtribes of tribe Areceae, we sequenced regions of the malate synthase (MS) and phosphoribulokinase (PRK) genes. Simultaneous analysis of these regions revealed 52 shortest trees, all of which resolved tribe Areceae as polyphyletic. Subtribes Iguanurinae, Dypsidinae, Oncospermatinae, and Arecinae were also resolved as polyphyletic. A clade of Indo-Pacific taxa was resolved with strong support, and would be a suitable target for more focused study. [ABSTRACT FROM AUTHOR]

Malate synthase is an essential metabolic enzyme of the glyoxylate bypass that makes possible the replenishment of carbon intermediates to cells grown on acetate. A polymerase chain reaction (PCR)-based molecular screening investigation of full-length malate synthase genes from Streptomyces spp. was initiated by our group. To this end, consensus primers were designed based on known streptomycete malate synthase sequences and successful amplification was obtained for Streptomyces griseus, S. fimbriatus and S. lipmanii. The putative full-length malate synthase gene from S. griseus was subsequently cloned, sequenced and expressed. Sequence analysis of this gene showed very high identity with other streptomycete malate synthase genes. Furthermore, high malate synthase activity was detected after heterologous expression in Escherichia coli, thus demonstrating successfully the rapid cloning and functional verification of a streptomycete malate synthase gene. Growth studies of S. griseus revealed that malate synthase activity was induced by the presence of acetate, which is a two-carbon source. Interestingly, the activity peaked during late growth phase when the biomass was declining, suggesting that the enzyme may have a late role in metabolism. [ABSTRACT FROM AUTHOR]

The glyoxylate cycle, catalysed by two unique enzymes: isocitrate lyase (ICL; EC 4.1.3.1) and malate synthase (MS; EC 4.1.3.2), is necessary for the net conversion of acetate into glucose. This metabolic pathway operates in microorganisms, higher plants and nematodes. Two bacterial genes, encoding ICL and MS, were modified in order to introduce them into the mouse germ line. The ovine metallothionein-Ia (MT-Ia) promoter- aceB gene-ovine growth hormone (GH) gene (3′ GH sequence) construct was fused to the ovine MT-Ia promoter- aceA gene-ovine GH gene (3′ GH sequence). Therefore, in this single DNA sequence, both aceA and aceB are under independent MT-Ia promoter control and can be induced by zinc. Transgenic mice were generated by pronuclear microinjection of the aceB-aceA gene construct. We now report the establishment of four mouse lines carying these two transgenes. Studies on the progeny of these lines indicate that one line (No. 91) is expressing both genes at the mRNA and enzyme levels in the liver and intestine, whereas another line (No. 66) has a much lower expression. Both enzyme activities were detected in the liver and intestine at levels up to 25% of those measured in fully derepressed Escherichia coli cells. [ABSTRACT FROM AUTHOR]

In previous work, we have demonstrated that oleate induces a massive proliferation of microbodies (peroxisomes) in Aspergillus nidulans. Although at a lower level, proliferation of peroxisomes also occurrs in cells growing under conditions that induce penicillin biosynthesis. Here, microbodies in oleate-grown A. nidulans cells were characterized by using several antibodies that recognize peroxisomal enzymes and peroxins in a broad spectrum of eukaryotic organisms such as yeast, and plant, and mammalian cells. Peroxisomes were immunolabeled by anti-SKL and anti-thiolase antibodies, which suggests that A. nidulans conserves both PTS1 and PTS2 import mechanisms. Isocitrate lyase and malate synthase, the two key enzymes of the glyoxylate cycle, were also localized in these organelles. In contrast to reports of Neurospora crassa, our results demonstrate that A. nidulans contains only one type of microbody (peroxisomes) that carry out the glyoxylate cycle and contain 3-ketoacyl-CoA thiolase and proteins with the C-terminal SKL tripeptide. [ABSTRACT FROM AUTHOR]

Bradyrhizobium japonicum, the nitrogen-fixing symbiotic partner of soybean, was grown on various carbon substrates and assayed for the presence of the glyoxylate cycle enzymes, isocitrate lyase and malate synthase. The highest levels of isocitrate lyase [165–170 nmol min–1 (mg protein)–1] were found in cells grown on acetate or β-hydroxybutyrate, intermediate activity was found after growth on pyruvate or galactose, and very little activity was found in cells grown on arabinose, malate, or glycerol. Malate synthase activity was present in arabinose- and malate-grown cultures and increased by only 50–80% when cells were grown on acetate. B. japonicum bacteroids, harvested at four different nodule ages, showed very little isocitrate lyase activity, implying that a complete glyoxylate cycle is not functional during symbiosis. The apparent K m of isocitrate lyase for d, l-isocitrate was fourfold higher than that of isocitrate dehydrogenase (61.5 and 15.5 μM, respectively) in desalted crude extracts from acetate-grown B. japonicum. When isocitrate lyase was induced, neither the V max nor the d, l-isocitrate K m of isocitrate dehydrogenase changed, implying that isocitrate dehydrogenase is not inhibited by covalent modification to facilitate operation of the glyoxylate cycle in B. japonicum. [ABSTRACT FROM AUTHOR]

In the amino-acid-producing microorganism Corynebacterium glutamicum, the specific activities of the acetate-activating enzymes acetate kinase and phosphotransacetylase and those of the glyoxylate cycle enzymes isocitrate lyase and malate synthase were found to be high when the cells were grown on acetate (0.8, 2.9, 2.1, and 1.8 U/mg protein, respectively). When the cells were grown on glucose or on other carbon sources such as lactate, succinate, or glutamate, the specific activities were two- to fourfold (acetate kinase and phosphotransacetylase) and 45- to 100-fold (isocitrate lyase and malate synthase) lower, indicating that the synthesis of the four enzymes is regulated by acetate in the growth medium. A comparative Northern (RNA) analysis of the C. glutamicum isocitrate lyase and malate synthase genes ( aceA and aceB) and transcriptional cat fusion experiments revealed that aceA and aceB are transcribed as 1.6- and 2.7-kb monocistronic messages, respectively, and that the regulation of isocitrate lyase and malate synthase synthesis is exerted at the level of transcription from the respective promoters. Surprisingly, C. glutamicum mutants defective in either acetate kinase or phosphotransacetylase showed low specific activities of the other three enzymes (phosphotransacetylase, isocitrate lyase, and malate synthase or acetate kinase, isocitrate lyase, and malate synthase, respectively) irrespective of the presence or absence of acetate in the medium. This result and a correlation of a high intracellular acetyl coenzyme A concentration with high specific activities of isocitrate lyase, malate synthase, acetate kinase, and phosphotransacetylase suggest that acetyl coenzyme A or a derivative thereof may be a physiological trigger for the genetic regulation of enzymes involved in acetate metabolism of C. glutamicum. [ABSTRACT FROM AUTHOR]

The malate synthase gene, MLS1, of the yeast Saccharomyces cerevisiae is transcriptionally regulated by the carbon source in the growth medium. A MLS1-lacZ fusion gene, expressed at a basal level in the presence of 2% glucose, is derepressed more than 100-fold under conditions of sugar limitation. No evidence for MLS1 induction by oleic acid was found. By deletion analysis of the MLS1 control region, we identified two sites, UAS1 and UAS2, as important for efficient derepression of the gene. Both sites contain sequences that resemble the previously characterized carbon source-responsive element (CSRE) found in the promoter of the isocitrate lyase gene ICL1. Indeed, UAS1 and UAS2 in the MLS1 upstream region turn out to be functional CSRE sequence variants. This finding allowed us to define a modified version of the CSRE consensus sequence (CCRTYSRNCCG). Protein binding to UAS1MLS1 was observed with extracts from derepressed but not from repressed cells, and could be competed for by an excess of the unlabelled CSRE( ICL1) sequence. No competition was observed with a mutated CSRE variant. Site-directed mutagenesis of both CSREs in the MLS1 promoter reduced gene activation under derepressing conditions to 20% of the wild-type level. The same decrease was observed with the wild-type MLS1 promoter in a cat8 mutant, lacking an activator of CSRE-dependent transcription. The CSRE/Cat8p-independent activation of MLS1 is mediated by constitutive UAS elements. The pleiotropic transcription factor Abf1p, which binds to the MLS1 upstream region, may contribute to constitutive activation. Thus, in order to ensure the severe glucose repression of MLS1 observed, repressor elements that respond to the carbon source must counteract constitutive activation. In summary, ICL1 and MLS1 share common cis-acting elements, although a distinct mechanism of carbon source control also contributes to MLS1 regulation. [ABSTRACT FROM AUTHOR]

Copyright of Chemical Monthly / Monatshefte für Chemie is the property of Springer Nature and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Copyright of Chemical Monthly / Monatshefte für Chemie is the property of Springer Nature and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Copyright of Chemical Monthly / Monatshefte für Chemie is the property of Springer Nature and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Analytical ultracentrifugation is a powerful tool for investigating the size of proteins in solution, especially by measuring sedimentation and diffusion coefficients and molar masses. Several further molecular parameters such as frictional ratios, axial ratios of hydrodynamic models, and Stokes radii allow a rough estimate of the protein overall structure. Sedimentation analysis may also be applied efficaciously for monitoring conformational changes of proteins occurring upon ligand binding or denaturation. For the determination of very small changes in shape, however, great care and a series of precautions are required. We investigated the enzymes citrate synthase and malate synthase in the absence and in the presence of ligands, in order to study the structural properties of the proteins and their ligand complexes. We also compared the results of the ultracentrifugal analysis with the results of other solution techniques such as UV absorption, fluorescence spectroscopy, circular dichroism, and small-angle x-ray scattering on the one hand, and the crystallographic 3D structure of citrate synthase on the other. The spectroscopic methods may be used as efficient and rapid tools for screening the occurrence of conformational changes caused by alterations of chromophores and fluorophores. The structural information provided by small-angle scattering (e.g., radii of gyration, maximum particle diameters, vclumes and surface areas) can be used to establish quantitative correlations between solution scattering and hydrodynamic data. In this context, however, knowledge or qualified assumptions of partial specific volumes and hydration are additionally required. Good agreement was reached between small-angle scattering and ultracentrifugal data, and also with crystallographic data if protein hydration was considered properly. The given approaches may be used to predict hydrodynamic properties if x-ray data are available, and for many verifications of other structural data, e.g., Stokes radii, diffusion coefficients, axial and frictional ratios determined by independent methods. [ABSTRACT FROM AUTHOR]

The cucumber malate synthase (MS) gene, including 1856 bp of 5′ non-trnascribed sequence, has been transferred into Petunia (Mitchell) and Nicotiana plumbaginifolia plants using an Agrobacterium binary vector. The transferred gene is found in variable copy number in different transformants, and is stably transmitted in each case as a single Mendelian character. Transgene mRNA accumulates in the seedling during the first three days of germination, then declines in amount as the cotyledons emerge from the seed. The decline is more pronounced in light-grown seedlings than in dark-grown seedlings. Expression of the MS transgene is also detected at a low level in petals of transformed Petunia plants. In these respects the pattern of MS gene expression is similar in cucumber and in trnasformed plants, showing that the transferred DNA fragment contains a functional MS gene. A 1076 bp fragment of 5′ sequence was linked to the β-glucuronidase reporter gene and transferred into Nicotiana, where it was shown to direct temporal and spatial patterns of expression similar to that of the complete MS gene. However, histochemical localisation of β-glucuronidase activity demonstrated that the chimaeric gene is expressed not only in cotyledons of transgenic plants, but also in endosperm and some hypocotyl cells during early germination. The relevance of these findings to the control of malate synthase gene expression is discussed. [ABSTRACT FROM AUTHOR]

The temporal, nonconcerted development of activities of malate synthase (MS), isocitrate lyase (ICL), and catalase (Cat) was explored in more detail in maturing and germinated cotton ( Gossypium hirsutum L.) seeds. RNA was extracted at six intervals beginning at 17 days post anthesis (DPA) through 72 hours post imbibition (HPI). In vitro translations revealed that mRNAs for each enzyme were translatable at all intervals. Enzyme activities and immunoselected proteins also were found at all intervals. Similar specific activities throughout maturation indicated that embryo cells were not accumulating inactive protein. The steady-state level of mRNAs encoding each enzyme exhibited different patterns of change during seed maturation, and each peaked at least 24 h before peak enzyme activities in germinated seeds. All three enzymes occur together as early as 17 DPA in a coordinate manner; however, the subsequent, nonconcerted increases in protein, activity, and mRNA for each enzyme indicate that developmental expression in cotton seed embryos is regulated in a noncoordinate fashion by as yet unidentified specific control mechanism(s). [ABSTRACT FROM AUTHOR]

The complete sequences of a full-length cDNA clone and a genomic clone encoding the Cucumis sativus glyoxysomal enzyme malate synthase, have been determined. The sequences have enabled us to identify putative control regions at the 5′ end of the gene, three introns, and possible alternative polyadenylation sites at the 3′ end. The deduced amino acid sequence predicts a polypeptide of 64961 molecular weight, which has 48% identity with that of Escherichia coli. Comparison of the sequence of malate synthase from cucumber with that from E. coli and with other glyoxysomal and peroxisomal enzymes, shows that a conserved C-terminal tripeptide is a common feature of those enzymes imported into microbodies. [ABSTRACT FROM AUTHOR]

Cotton ( Gossypium hirsutum L.) embryos excised from bolls 38-43 d after anthesis and cultured in vitro for 4 d on a nutrient agar medium containing 3.8 μM abscisic acid (ABA) developed enzyme activity and accumulated insoluble protein, neutral lipid, and dry weight similar to embryos maturing on the plant. Inclusion of ABA in the medium prevented precosious germination and allowed continued increases in catalase, malate dehydrogenase, citrate synthase, aspartate aminotransferase, and β-oxidation enzyme activities as well as de-novo synthesis of malate synthase. Isocitrate lyase activity was not detectable in ABA-cultured embryos nor normally-developed embryos. Omission of sucrose from the medium resulted in near-doubling of the development of malate synthase activity, with minimal effects on the other enzyme activities. Addition of Actinomycin D, cordycepin, or cycloheximide to ABA-containing cultures did not overcome the observed inhibition of germination, but severely reduced both the appearance of new malate synthase activity and further production of other related enzyme activities. Thus, development of these enzyme activities in the presence of ABA appears dependent on transcription and translation, while inhibition of germination by ABA at this stage of development is not sensitive to the RNA- and protein-synthesis inhibitors. The results indicate that ABA does not prevent vivipary by suppressing translation of m-RNAs coding for isocitrate lyase and its companion enzymes, as previously proposed. [ABSTRACT FROM AUTHOR]

The degradation of storage fat in the cotyledons of mustard seedlings is unaffected by phytochrome and photosynthesis (irradiation with continuous red or far-red light from sowing of the seeds) although light imposes a strong constraint on the translocation of organic matter from the cotyledons into the seedling axis. Likewise, the development and disappearance of glyoxysomal enzyme activities (isocitrate lyase, malate synthase, citrate synthase) takes place independently of light. It is concluded that the mobilization of storage fat (fat→carbohydrate transformation) is independent of photomorphogenesis. The surplus of carbohydrate produced from fat in the light seems to be converted to starch grains in the plastids, which function as a secondary storage pool in the cotyledons. [ABSTRACT FROM AUTHOR]

This study addresses the still open question of whether or not in oily storage tissues, e.g. cotyledons of germinating rape ( Brassica napus L.) seedlings' lipase (triacylglycerol acylhydrolase, EC 3.1.1.3) and the β-oxi-dation system of fatty acids are located in one or more membrane-bounded organelles. The organelles were isolated carefully and identified by marker-enzyme activities. Activities of neither lipase nor acylester acylhydrolase (EC 3.1.1) could be detected either in glyoxysomes or in mitochondria, even when various substrate emulsions were employed. Only after long-term incubations could the presence of a low lipolytic activity be demonstrated for different organellar fractions. This alkaline carboxylic ester hydrolase, whose activity is below the detection limit of various standard tests, cannot play a role in the lipolytic function of glyoxysomes. In addition, a complete set of enzyme activities necessary for the conversion of saturated fatty acids to acetyl CoA was found only in the glyoxysomal cell fraction. The low β-oxidation activity discovered in the mitochondrial cell fraction is evidently due to glyoxysomal contamination. Enzyme activities unique to the mitochondrial β-oxidation system such as carnitine palmitoyltransferase (EC 2.3.1.21), carnitine acetyltransferase (EC 2.3.1.7), and acyl-CoA dehydrogenase (EC 1.3.99.3) were absent, indicating that mitochondria are not involved in fatty acid metabolism. In addition, on Western blots, antibodies raised against malate synthase (EC 4.1.3.2) and acyl-CoA oxidase (EC 1.1.3) recognized three polypeptides with molecular masses of 45, 63, and 70 kDa only in glyoxysomal fractions. Obviously, in the fatty rape seed neither glyoxysomes nor mitochondria are involved in triacylglycerol hydrolysis, and β-oxidation of fatty acids occurs exclusively in glyoxysomes. [ABSTRACT FROM AUTHOR]

In order to investigate a possible association between soybean malate synthase (MS; l-malate glyoxylate-lyase, CoA-acetylating, EC 4.1.3.2) and glyoxysomal malate dehydrogenase (gMDH; ( S)-malate: NAD oxidoreductase, EC 1.1.1.37), two consecutive enzymes in the glyoxylate cycle, their elution profiles were analyzed on Superdex 200 HR fast protein liquid chromatography columns equilibrated in low- and high-ionicstrength buffers. Starting with soluble proteins extracted from the cotyledons of 5-d-old soybean seedlings and a 45% ammonium sulfate precipitation, MS and gMDH coeluted on Superdex 200 HR (low-ionic-strength buffer) as a complex with an approximate relative molecular mass (M) of 670000. Dissociation was achieved in the presence of 50 mM KCl and 5 mM MgCl, with the elution of MS as an octamer of M 510000 and of gMDH as a dimer of M 73 000. Polyclonal antibodies raised to the native copurified enzymes recognized both denatured MS and gMDH on immunoblots, and their native forms after gel filtration. When these antibodies were used to screen a λ ZAP II expression library containing cDNA from 3-d-old soybean cotyledons, they identified seven clones encoding gMDH, whereas ten clones encoding MS were identified using an antibody to SDS-PAGE-purified MS. Of these cDNA clones a 1.8 kb clone for MS and a 1.3-kb clone for gMDH were fully sequenced. While 88% identity was found between mature soybean gMDH and watermelon gMDH, the N-terminal transit peptides showed only 37% identity. Despite this low identity, the soybean gMDH transit peptide conserves the consensus R(X)HL motif also found in plant and mammalian thiolases. [ABSTRACT FROM AUTHOR]

Crude particulate homogenates from leaves of barley ( Hordeum vulgare L.), rice ( Oryza sativa L.), leaf-beet ( Beta vulgaris var. cicla L.) and pumpkin ( Cucurbita pepo L.) cotyledons were separated on sucrose density gradients. The peroxisomal fractions appeared at a buoyant density of 1.25 g·cm and contained most of the activities of catalase (EC 1.11.1.6), and hydroxypyruvate reductase (EC 1.1.1.81) on the gradients. In peroxisomal fractions from detached leaves and green cotyledons incubated in permanent darkness we detected the presence of isocitrate lyase (EC 4.1.3.1) and malate synthase (EC 4.1.3.2), key enzymes of the glyoxylate cycle, and β-oxidation activity (except in pumpkin). As proposed by H. Gut and P. Matile (1988, Planta 176, 548-550) the glyoxylate cycle may be functional during leaf senescence, and the presence of two key enzymes indicates a transition from leaf peroxisome to glyoxysome; for pumpkin cotyledons in particular a double transition occurs (glyoxysome to leaf peroxisome during greening, and leaf peroxisome to glyoxysome during senescence). [ABSTRACT FROM AUTHOR]

The presence of 30 μM abscisic acid inhibited development of malate-synthase activity in the endosperm of germinating castor-bean seeds. Malate synthase was purified from castor-bean endosperms and an antibody to it was prepared from rabbit serum. This antibody was used to measure the amounts of malate-synthase mRNA using an in-vitro translation system. The effect of abscisic acid appeared to be greater on malate-synthase mRNA than on the bulk of mRNA, indicating some specificity of abscisic-acid action. The extent of the inhibition of malate-synthase activity and of malate-synthase mRNA accumulation were similar. This indicates that abscisic acid inhibits malate-synthase activity by lowering levels of translatable malate-synthase mRNA rather than by affecting the translation rate of this mRNA. [ABSTRACT FROM AUTHOR]

Malate synthase, one of the key enzymes in the glyoxylate cycle, was purified from peroxisomes of alkane-grown yeast, Candida tropicalis. The enzyme was mainly localized in the matrix of peroxisomes, judging from subcellular fractionation followed by exposure of the organelles to hypotonic conditions. The molecular mass of this peroxisomal malate synthase was determined to be 250,000 daltons by gel filtration on a Sepharose 6B column as well as by ultracentrifugation. On sodium dodecylsulfate/polyacrylamide slab-gel electrophoresis, the molecular mass of the subunit of the enzyme was demonstrated to be 61,000 daltons. These results revealed that the native form of this enzyme was homo-tetrameric. Peroxisomal malate synthase showed the optimal activity pH at 8.0 and absolutely required Mg for enzymatic activity. The K values for Mg, acetyl-CoA and glyoxylate were 4.7 mM, 80 μM and 1.0 mM, respectively. [ABSTRACT FROM AUTHOR]

The key enzymes of the glyoxylate cycle, isocitrate lyase and malate synthase, were present in cell-free extracts of the phototrophic, green, thermophilic bacterium Chloroflexus aurantiacus grown with acetate as the sole organic carbon source. The optimum temperature of these enzymes was 40° C, and their specific activities were high enough to account for the observed growth rate. Lower levels of the enzymes were found in extracts from cells grown on a complete medium. Itaconate was shown to inhibit isocitrate lyase from C. aurantiacus 96% at a concentration of 0.25 mM and also had a profound effect on the growth of the organism on acetate, 0.25 mM inhibiting completely. Itaconate also inhibited the growth when added to the complex medium, but in this case much higher concentrations were required. [ABSTRACT FROM AUTHOR]

The production of poly (A) RNA was determined over the cell-cycle in Euglena cultures division-synchronized by 14 h light periods alternating with 10 h dark periods. The amount of poly (A) RNA per culture aliquot was constant from the beginning until near the end of the light phase of the cell-cycle when it increased in a sharp burst after the time of DNA replication. Poly (A) RNA from all stages of the cell-cycle promoted protein synthesis with equal efficiency in a mRNA-dependent reticulocyte lysate translation system. Malate synthase mRNA was detected using malate synthase antibody to precipitate nascent malate synthase protein in the translation system, binding the IgG-antigen complex to Staphylococcus aureus, concentrating, eluting the IgG-antigen complex and analysing the products by sodium dodecyl sulfatepolyacrylamide gel electrophoresis. Malate synthase mRNA was absent from phototrophic cells and was only produced in darkened cultures following the addition of acetate. [ABSTRACT FROM AUTHOR]

An Arthrobacter sp. (strain 9006), isolated from lake water, accumulated nitrite up to about 15 mg N/l, but no nitrate. In a mineral medium supplemented with tryptone, yeast extract, acetate and ammonium, the cells released nitrite into the medium parallel to growth or when growth had virtually ceased. The nitrite formed was proportional to the initial acetate concentration, indicating an involvement of acetate metabolism with nitrification. The organism grew with a wide variety of organic carbon sources, but washed cells formed nitrite from ammonium only in the presence of citrate, malate, acetate or ethanol. Magnesium ions were required for nitrification of ammonium and could not be replaced by other divalent metal ions. Analysis of the glyoxylate cycle key enzymes in washed suspensions incubated in a minimal medium revealed that isocitrate lyase and malate synthase were most active during the nitrification phase. Nitrite accumulation but not growth was inhibited by glucose, tryptone and yeast extract. A possible explanation for the different nitrification patterns during growth is based on the regulatory properties of glyoxylate cycle enzymes. [ABSTRACT FROM AUTHOR]

When Rhodopseudomonas gelatinosa was grown on acetate aerobically in the dark both enzymes of the glyoxylate bypass, isocitrate lyase and malate synthase, could be detected. However, under anaerobic conditions in the light only isocitrate lyase, but not malate synthase, could be found. The reactions, which bypass the malate synthase reaction are those catalyzed by alanine glyoxylate aminotransferase and the enzymes of the serine pathway. Other Rhodospirillaceae were tested for isocitrate lyase and malate synthase activity after growth with acetate; they could be divided into three groups: I. organisms possessing both enzymes; 2. organisms containing malate synthase only; 3. R. gelatinosa containing only isocitrate lyase when grown anaerobically in the light. [ABSTRACT FROM AUTHOR]

The potential of tobacco BY-2 suspension-cultured cells for examining in vivo targeting and import of proteins into plant peroxisomes was shown recently in our laboratory. In the current study, the necessity and sufficiency of putative C-terminal targeting signals on cottonseed malate synthase and bacterial chloramphenicol acetyl-transferase (CAT) were examined in BY-2 cells. Cotton suspension cells also were evaluated as another in vivo peroxisome targeting system. Ultrastructural views of BY-2 cells showed that the peroxisomes were relatively small (0.1-0.3 μm diameter), a characteristic of so-called 'unspecialized' peroxisomes, Peroxisomes in cotton and tobacco cells were identified with anti-cottonseed catalase IgGs as distinct immunofluorescent particles clearly distinguishable from abundant immunofluorescent mitochondria and plastids, marked with antibodies to β-ATPase and stearoyl-ACP Δ 9 desaturase, respectively. The C-terminal ser-lys-leu (SKL) motif is a well-established peroxisome targeting signal (PTS 1) for mammals and yeasts, but not for plants. Antiserum raised against SKL peptides recognized proteins only in peroxisomes in cotton and tobacco cells. The necessity of SKL-COOH for targeting of proteins to plant peroxisomes had not been demonstrated; we showed that SKL-COOH was necessary for directing cottonseed malate synthase to BY-2 peroxisomes. KSRM-COOH, a conservative modification of SKL-COOH, was shown by others to be sufficient for redirecting CAT in stably-transformed Arabidopsis plants to the leaf peroxisomes. Here we show with the same CAT constructs (e.g., pMON316CAT-KSRM) that KSRM is sufficient for targeting transiently-expressed passenger proteins to unspecialized BY-2 peroxisomes. These results provide new direct evidence for the necessity of SKL-COOH (a type 1 PTS) and sufficiency of a conservative modification of the PTS 1 (KSRM-COOH) for in vivo, heterologous targeting of proteins to plant peroxisomes. [ABSTRACT FROM AUTHOR]

Enzymatic levels and subcellular localization of malate synthase in maturing seeds of castor bean ( Ricinus communis cv. Hale) are reported. Extracts of maturing seeds exhibited moderately high specific activity (9.68 nmoles/min/mg protein) at 15-20 DAP and lower specific activity (0.49) in mature, dry seeds. Subcellular localization of the enzyme during seed maturation was primarily cytosolic (85%). The remainder of the activity in sucrose gradients was located at high density (1.21 g/cm). Dry seeds did not contain organelle-bound malate synthase activity. In extracts of 4-day germinated seeds the enzyme was present at high specific activity (12.8 nmoles/min/mg protein) with better than 85% of the total activity in glyoxysomes (1.24 g/cm). Two polypeptides, 62kDa and 66kDa, reactive with anti-malate synthase were detected at high density in sucrose gradients of homogenates of late-maturing seeds (60 DAP); dry seeds; and seeds imbibed for 6 h. One polypeptide, 62 kDa, in 4-day germinated seeds, reacted with anti-malate synthase. Immunoreactive polypeptides in late-maturing and dry seeds were present at approximately 1/760 of the level found in 4-day germinated seeds. We conclude that malate synthase activity is prominent during early seed maturation but is very low and minimally compartmentalized during late maturation. The rapidly sedimenting immunoreactive polypeptides from dry seeds are enzymatically inactive and are presumed to be of no physiological significance. [ABSTRACT FROM AUTHOR]

Recently it has been demonstrated that detached leaves show glyoxysomal enzyme activities when incubated in darkness for several days. In this report glyoxylate-cycle enzymes have been detected in leaves of rice ( Oryza sativa L.) and wheat ( Triticum durum L.) from either naturally senescing or dark-treated plants. Isolated peroxisomes of rice and wheat show isocitrate lyase (EC 4.1.3.1), malate synthase (EC 4.1.3.2) and β-oxidation activities. Leaf peroxisomes from dark-induced senescing leaves show glyoxylic-acid-cycle enzyme activities two to four times higher than naturally senescing leaves. The glyoxysomal activities detected in leaf peroxisomes during natural foliar senescence may represent a reverse transition of the peroxisomes into glyoxysomes. [ABSTRACT FROM AUTHOR]

The activities of two key enzymes of the glyoxylic-acid cycle, isocitrate lyase and malate synthase, can barely be detected in mature, presenescent primary leaves of barley ( Hordeum vulgare L.) but are apparently induced in senescent leaf tissue. Upon incubation of leaf segments in permanent darkness, the activities appear and increase dramatically up to the sixth day and thereafter decline. The glyoxylic-acid cycle may thus be functional during foliar senescence. The main period of galactolipid loss is characterized by RQ values as low as 0.63, indicating that long-chain fatty acids produced from thylakoidal acyl-lipids may be utilized for gluconeogenesis involving corresponding glyoxisomal metabolic pathways. Foliar senescence may be characterized by a peroxisomeglyoxysome transition analogous to the glyoxisome-peroxisome transition in greening cotyledons of fat-storing seeds. [ABSTRACT FROM AUTHOR]