Enzyme Profiles in Seedling Development and the Effect of Itaconate, an Isocitrate Lyase-directed Reagent

Changes in levels of isocitrate lyase, malate synthase, and catalase have been investigated during germination of flax (Linum usitatissimum L.) in the presence and absence of itaconate. Germination was accompanied by a rapid increase in these enzymes during the first 3 days. The presence of 38 millimolar itaconate inhibited the incidence of seed germination and the growth of embryo axes as well as the appearance of isocitrate lyase but did not alter the levels of malate synthase, catalase, or NADP⁺-isocitrate dehydrogenase. The specific activity for the latter enzyme was constant throughout germination. Oxalate or succinate, each at 38 millimolar, had no effect upon germination of flax seeds. Itaconate did not inhibit the activities of malate synthase, catalase, or NADP⁺-isocitrate dehydrogenase in vitro but was a potent noncompetitive inhibitor of isocitrate lyase (K_i:17 micromolar at 30 C, pH 7.6). Itaconate (at 38 millimolar) did not alter the appearance of malate synthase but reduced the incidence of germination, onset of germination, and growth of the embryo axis as well as the specific activity of isocitrate lyase in seedlings of Zea mays, Vigna glabra, Glycine hispida, Vigna sinensis, Trigonella foenumgraecum, Lens culinaris, and Medicago sativa. The incidence and onset of germination of wheat seeds were unaltered by the same concentration of itaconate but seedlings did not contain isocitrate lyase or malate synthase. The data suggest that itaconate may be isocitrate lyase-directed in inhibiting the germination of fatty seeds.     

Metabolic Role, Regulation of Synthesis, Cellular Localization, and Genetic Control of the Glyoxylate Cycle Enzymes in Neurospora crassa

The glyoxylate shunt enzymes, isocitrate lyase and malate synthase, were present at high levels in mycelium grown on acetate as sole source of carbon, compared with mycelium grown on sucrose medium. The glyoxylate shunt activities were also elevated in mycelium grown on glutamate or Casamino Acids as sole source of carbon, and in amino acid-requiring auxotrophic mutants grown in sucrose medium containing limiting amounts of their required amino acid. Under conditions of enhanced catabolite repression in mutants grown in sucrose medium but starved of Krebs cycle intermediates, isocitrate lyase and malate synthase levels were derepressed compared with the levels in wild type grown on sucrose medium. This derepression did not occur in related mutants in which Krebs cycle intermediates were limiting growth but catabolite repression was not enhanced. No Krebs cycle intermediate tested produced an efficient repression of isocitrate lyase activity in acetate medium. Of the two forms of isocitrate lyase in Neurospora, isocitrate lyase-1 constituted over 80% of the isocitrate lyase activity in acetate-grown wild type and also in each of the cases already outlined in which the glyoxylate shunt activities were elevated on sucrose medium. On the basis of these results, it is concluded that the synthesis of isocitrate lyase-1 and malate synthase in Neurospora is regulated by a glycolytic intermediate or derivative. Our data suggest that isocitrate lyase-1 and isocitrate lyase-2 are the products of different structural genes. The metabolic roles of the two forms of isocitrate lyase and of the glyoxylate cycle are discussed on the basis of their metabolic control and intracellular localization.     

Acetate metabolism in Rhodopseudomonas gelatinosa and several other Rhodospirillaceae

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.     

Microbody-marker Enzymes during Transition from Phototrophic to Organotrophic Growth in Euglena

Transfer of Euglena gracilis Klebs Z cells from phototrophic to organotrophic growth on acetate results in derepression of the key enzymes of the glyoxylate cycle, malate synthase and isocitrate lyase, which appear coordinately regulated. The derepression of malate synthase and isocitrate lyase was accompanied by increased specific activities of succinate dehydrogenase, fumarase, and malate dehydrogenase, but hydroxypyruvate reductase activity was unaltered.     

Activity of the yeast MAP kinase homologue Slt2 is critically required for cell integrity at 37° C

We have analyzed the structure of genes encoding the glyoxylate cycle enzyme isocitrate lyase from Brassica napus L. and their expression during embryogeny and postgermination. Restriction mapping, nucleotide sequence, and DNA gel blot hybridization analyses of cDNA and genomic clones indicated that there are approximately six isocitrate lyase genes in the B. napus genome that can be divided into at least two subfamilies based upon their divergence in 5′ and 3′ untranslated regions. We showed previously that isocitrate lyase mRNA accumulates during late embryogeny and postgermination. Here, we present results which indicate that several isocitrate lyase genes are expressed at both stages of development. First, gene-specific probes were used to show that mRNAs encoded by representatives of both gene subfamilies accumulated in both late maturation stage embryos and in seedlings of B. napus. Second, a single B. napus isocitrate lyase gene, together with 3.5 kb and 1.4 kb of 5′ and 3′ flanking regions, respectively, was expressed in both embryos and seedlings of transgenic tobacco plants. The results indicated that accumulation of isocitrate lyase in late embryogeny and postgermination does not result from the alternate expression of distinct members of the gene family.     

When is a peroxisome not a peroxisome?

It is time to drop the glyoxysome name. Recent functional genomics analysis together with cell biology studies emphasize the unifying features of peroxisomes rather than their differences. Plant peroxisomes contain 300 or more proteins, the functions of which are dominated by activities related to fatty acid oxidation (>70 enzymes). By comparison, relatively few proteins are committed to metabolism of reactive oxygen species ( approximately 20) and to photorespiration ( approximately 10). Analysis of triglyceride metabolism in Arabidopsis seedlings now indicates that only two enzymes (isocitrate lyase and malate synthase) potentially distinguish glyoxysomes from other peroxisomes. Future research is best served by focusing on the common features of peroxisomes to establish how these dynamic organelles contribute to energy metabolism, development and responses to environmental challenges.     

OXIDATIVE METABOLISM AND THE GLYOXYLATE CYCLE IN PSEUDOMONAS INDIGOFERA.

McFadden, Bruce A. (Washington State University, Pullman, Wash.) and William V. Howes. Oxidative metabolism and the glyoxylate cycle in Pseudomonas indigofera. J. Bacteriol. 84:72-76. 1962.-Oxidative patterns of Pseudomonas indigofera have been investigated. Intact cells oxidize acetate, ethanol, fumarate, glyoxylate, alpha-ketoglutarate, malate, oxaloacetate, pyruvate, and succinate to greater than 35% of completion. Isocitrate is oxidized to 21% of completion. Citrate is not oxidized by whole cells but is oxidized by cell-free preparations, as are fumarate, isocitrate, malate, and succinate. These patterns are suggestive of the operation of the tricarboxylic acid cycle. Investigations of levels of isocitrate lyase and malate synthase as functions of growth substrate have been conducted. Assays for these enzymes in "soluble" preparations were performed under ostensibly optimal conditions for catalysis. Growth substrates used at 0.3% were: (i) ethanol, (ii) glucose, (iii) succinic acid, and (iv) yeast extract. Specific activities of isocitrate lyase were: for (i) 3.80, (ii) 0.61, (iii) 1.47, and (iv) 1.33; activities of malate synthase were: for (i) 0.18, (ii) 0.032, (iii) 0.021, and (iv) 0.029. Additionally, the isocitrate lyase level from butyrate-grown cells was similar to that for ethanol-grown cells; the specific activity of malate synthase was about 60% as high. Specific activities of these enzymes were reproducible when conditions of sonic disruption were standardized. Longer durations of disruption decreased both activities.     

Regulation of glyoxysomal enzymes during germination of cucumber. Temporal changes in translatable mRNAs for isocitrate lyase and malate synthase

The relative levels of translatable messenger RNA for isocitrate lyase and malate synthase were determined in the dry seed and for the first seven days of development of cucumber cotyledons. After extraction and quantification of total and poly(A)-rich RNA each day, the RNA fractions were translated in an optimized wheat germ system and the specific polypeptides were immunoprecipitated quantitatively. The radiolabeled isocitrate lyase and malate synthase polypeptides were then fractionated on dodecylsulphate/polyacrylamide gels, visualized by exposure to X-ray film and quantified densitometrically. The relative levels of translatable messenger RNA for these enzymes rise and fall with a developmental program similar to the enzyme activities, but preceding the latter by about one day. This implies that the rise in enzyme activity is dependent upon a prior postgerminative increase in translatable messenger RNA for the enzymes. These studies also suggest that messenger RNA levels may be regulated, at least in part, by light.     

Activities and subcellular localization of enzymes responsible for lipolysis and gluconeogenesis during the germination of Brassica campestris cv. esculenta seeds

During the growth of turnip seedlings, two new lipases have been demonstrated, one with a maximum activity at pH 4.5 (acid lipase) and the other with a maxima at pH 8.6 (alkaline lipase). Many different enzymes are involved in gluconeogenesis: catalase, isocitrate lyase, malate synthetase, malate dehydrogenase, aconitase, citrate synthetase, fumarase, glycolate oxidase, phosphoenol-pyruvate carboxykinase. All of these show maximum activity coinciding with the stage in which lipid hydrolysis is maximal and when the accumulation of soluble carbohydrates has also reached its peak. The alkaline lipase as found to be located mainly in the spherosomes, whereas the glyoxysomes contained the following main activities: catalase, isocitrate lyase, malate synthetase, malate dehydrogenase and citrate synthetase. Aconitase, together with cytochrome oxidase and fumarase showed their highest activity in the mitochondria, and the presence of malate dehydrogenase, citrate synthetase and glycolate oxidase was also observed in these organelles. In the membrane-bound fraction, the activities of cytochrome reductase, glycolate oxidase and phosphoenol-pyruvate kinase were marked, although the latter enzyme was even more active in the soluble fraction.     

Control of Enzyme Activities in Cotton Cotyledons during Maturation and Germination: II. Glyoxysomal Enzyme Development in Embryos

The sequence of glyoxysomal enzyme development was investigated in cotyledons of cotton (Gossypium hirsutum L. cv. Deltapine 16) embryos from 16 to 70 days after anthesis (DAA). Catalase, malate dehydrogenase, and citrate condensing enzyme activities were barely detectable prior to 22 DAA, but showed dramatic increases from 22 to 50 DAA. Development of malate synthase activity, however, was delayed during this period, rising to peak activity from 45 to 50 DAA (just prior to desiccation) in the absence of any detectable isocitrate lyase activity. Substantial activities of all of these enzymes (except isocitrate lyase) persisted in the dry seeds. Isopycnic centrifugations on sucrose gradients demonstrated that the enzymes were compartmentalized within particles increasing in buoyant density with time of development (1.226 to 1.245 grams per cubic centimeter from 22 to 50 DAA). Of particular significance were the observations in 22-day embryos of smooth surfaced membrane dilations of rough endoplasmic reticulum having cytochemical catalase reactivity, and the demonstrations of catalase activities in microsomal fractions isolated throughout the 16- to 50-DAA period. Our data do not allow determination of the mechanism(s) for enzyme activation and/or addition to previously existing or newly formed microbodies, but do show that development and acquisition of enzyme activities within glyoxysomes occur sequentially and thus are not regulated in concert as previously thought.     

Caenorhabditis elegans: Decay of isocitrate lyase during larval development

Changes in the levels of isocitrate lyase, malate synthase, catalase, fumarase, and NADP⁺-isocitrate dehydrogenase have been investigated during larval development of the free-living soil nematode Caenorhabditis elegans in the presence and absence of Escherichia coli. The specific activities of isocitrate lyase, malate synthase, and catalase are maximal at the time of egg hatching and, thereafter, decline during larval development when larvae feed on E. coli, whereas in the absence of E. coli specific activities of the same enzymes increase for 12 hr and subsequently remain constant. There is, however, no change in specific activity of fumarase or NADP⁺-isocitrate dehydrogenase during the same developmental period, in either case. Cycloheximide at 100 μM arrests the decline of isocitrate lyase during development of feeding larvae but has no effect upon the appearance of isocitrate lyase during starvation. The latter is true also for 15 mM itaconate. There is inactivation of isocitrate lyase in crude extracts of frozen worms in comparison to that in analogous extracts prepared from freshly harvested nematodes.     

Comparison of the glyoxysomes and the glyoxysomal enzymes in maize lines with high or low oil content

The developmental profile of the glyoxysomes and their component enzymes catalase, malate synthase, and isocitrate lyase were compared in the scutellum of two maize (Zea mays) lines, Illinois High Oil (IHO, approximately 20% lipid content) and Illinois Low Oil (ILO, less than 0.5% lipid content). The microbodies participate in the catabolism of the seed lipids and are responsible for leading the catabolic products (acetyl-Coenzyme A) into gluconeogenesis. The aim of this study was to determine whether changes in lipid content of the seed resulted in changes in the levels of the glyoxysomal enzymes. Enzyme activity measurements, immunological measurements (in the case of catalase), cell fractionation studies, and electron microscopic observations indicated that the IHO and ILO lines contain similar populations of glyoxysomes and exhibit similar catalase and malate synthase specific activities, despite the significant difference (40-fold) in their lipid content. Only the specific activity of isocitrate lyase was higher (2-fold higher) in the IHO seeds as compared to the ILO.     

Development and regulation of three glyoxysomal enzymes during cotton seed maturation and growth

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).Abbreviations ABA abscisic acid - bp base pairs - DPA days post anthesis - HPI hours post imbibition - kb kilobase (pairs) - M _r relative molecular weight - S Svedberg unit (10^-13s)     

Intact Cell Transformation of Saccharomyces cerevisiae by Polyethylene Glycol

The activities of isocitrate lyase and malate synthase—the key enzymes in the glyoxylate cycle—were found to be fairly high in n-alkane-, acetate-, and propionate-grown cells of Candida tropicalis compared with those in glucose-grown cells. In fact, the results of immunochemical studies showed that the increases in the enzyme levels resulted from increases in the amounts of the enzyme proteins. But the increases in these enzyme activities were not always coincident with the appearance of peroxisomes. Isocitrate lyase and malate synthase were purified from a peroxisome-containing particulate fraction of alkane-grown cells and from whole cells grown on glucose, acetate and propionate. The respective enzymes showed no significant differences in immunochemical properties, specific activities, molecular masses of active forms and subunits, on patterns of limited proteolysis with proteases, but the malate synthases of alkane- and propionate- grown cells showed higher Km values for acetyl-CoA than the enzymes of glucose- and acetate- grown cells. The results indicated that the synthesis of the key enzymes in the glyoxylate cycle did not necessarily have to be coincident with the development of peroxisomes in this yeast.     

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)