Specific Activity Of Isocitrate Lyase Research Articles

Isolation of an oxalate-resistant Ashbya gossypii strain and its improved riboflavin production

An oxalate-resistant strain of Ashbya gossypii was naturally isolated from spores grown on an oxalate-containing medium, and its medium was optimized to improve riboflavin production. Riboflavin production by the resistant strain was three-fold higher than that by the wild-type organism when grown in flask cultures. Medium optimization increased the riboflavin production by the resistant strain to 5 g l−1, which was five-fold higher than that obtained by the wild-type strain. The productivity was reproduced in a 3-l bioreactor. During the early growth phase, the specific activity of isocitrate lyase in the oxalate-resistant strain was slightly higher than that in the wild-type strain. Proteomic analysis of the oxalate-resistant strain revealed that the expression of aldose reductase and cobalamin-independent methionine synthase decreased significantly. This is the first report that describes the natural isolation of a riboflavin producer using an antimetabolite-containing medium to enhance the riboflavin production level. This method should also be useful for improving the productivity of other bioproducts since it does not require any mutations or genetic modifications of the microorganism.

Effect of white light on cell expansion and lipid netabolism in sunflower cotyledons

Summary The growth of the cotyledons of etiolated sunflower seedlings ( Helianthus annuus L.) is promoted by white light (WL). This process is due to an enhancement in the rate of cell expansion. The degradation of lipid reserves is stimulated by WL. An analysis of different fractions of homogenized cotyledons revealed that the enzyme lipase is exclusively localized in the oil bodies (oleosomes) of the cells. In WL-treated seedlings, lipolytic activity was higher than in dark-grown controls. In contrast, the specific activity of isocitrate lyase, a key enzyme of the glyoxylate cycle, was reduced upon exposure to WL. The results are discussed with respect to the relationship between sugar accumulation and cell expansion in the cotyledons and the hypocotyl of the developing sunflower seedling.

Acetate metabolism by Escherichia coli in high-cell-density fermentation

Little is known about the cellular physiology of Escherichia coli at high cell densities (e.g., greater than 50 g [dry cell weight] per liter), particularly in relation to the cellular response to different growth conditions. E. coli W3100 cultures were grown under identical physical and nutritional conditions, by using a computer-controlled fermentation system which maintains the glucose concentration at 0.5 g/liter, to high cell densities at pH values of 6.0, 6.5, 7.0, and 7.5. The data suggest a relationship between the pH of the environment and the amount of acetate excreted by the organism during growth. At pH values of 6.0 and 6.5, the acetate reached a concentration of 6 g/liter, whereas at pH 7.5, the acetate reached a concentration of 12 g/liter. Furthermore, at pH values of 6.0 to 7.0, the E. coli culture undergoes a dramatic metabolic switch in which oxygen and glucose consumption and CO2 evolution all temporarily decreased by 50 to 80%, with a concomitant initiation of acetate utilization. After a 30-min pause in which approximately 50% of the available acetate is consumed, the culture recovers and resumes consuming glucose and oxygen and producing acetate and CO2 at preswitch levels. During the switch period, the specific activity of isocitrate lyase typically increases approximately fourfold.

Characterization of Saccharomycopsis lipolytica mutants that express temperature-sensitive synthesis of isocitrate lyase.

Four mutants specifically deficient in the activity of isocitrate lyase were independently isolated in the alkane yeast Saccharomycopsis lipolytica. Genetic analysis by means of protoplast fusion and mitotic haploidization revealed that the mutations were recessive and non-complementary at a single genetic locus, icl. icl is a structural gene for isocitrate lyase, because some revertants from icl-1 and icl-3 mutants produced thermolabile isocitrate lyase in comparison with the wild-type enzyme, and also because the gene dosage effect was observed on the specific activity of isocitrate lyase in icl+/icl-1 and icl+/icl-3 heterozygotes. The icl-3 mutation also gave rise to temperature-sensitive revertants that could grow on acetate at 23 degrees C but not at 33 degrees C, exhibiting temperature-sensitive synthesis as well as thermostable activity of isocitrate lyase. Studies on purified isocitrate lyase showed that this enzyme is tetrameric and that the enzyme synthesized at 23 degrees C by a temperature-sensitive synthesis mutant was indistinguishable from the wild-type enzyme with respect to the subunit molecular weight (59,000), the isoelectric pH (5.3), the thermostability, and the Km value for threo-Ds-isocitrate (0.2 mM). When induced by acetate at 33 degrees C, the temperature-sensitive synthesis mutant did not express isocitrate lyase activity but did synthesize polypeptides whose electrophoretic mobilities were equal to that of the purified mutant enzyme. Hence, the temperature-sensitive mutation assumed in the structural gene for isocitrate lyase might have prevented the maturation of the polypeptide chains synthesized at the restrictive temperature.

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.

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.

Itaconate, an isocitrate lyase-directed inhibitor in Pseudomonas indigofera.

Enzymes catalyzing steps from ethanol to acetyl-coenzyme A, from malate to pyruvate, and from pyruvate to glucose 6-phosphate were identified in ethanol-grown Pseudomonas indigofera. Enzymes catalyzing the catabolism of glucose to pyruvate via the Entner-Doudoroff pathway were identified in glucose-grown cells. Phosphofructokinase could not be detected in Pseudomonas indigofera. Itaconate, a potent inhibitor of isocitrate lyase, abolished growth of P. indigofera on ethanol at concentrations that had little effect upon growth on glucose. The date obtained through enzyme analyses and studies of itaconate inhibition with both extracts and toluene-treated cells suggest that itaconate selectively inhibits and reduces the specific activity of isocitrate lyase.

Changes in metabolic reserves and enzyme activities during zoospore motility and cyst germination in Phytophthora palmivora

Lipids measured as acyl glycerides and free fatty acids provided the major energy source during a 6-h motile and a 2-h germination period in zoospores and cysts, respectively, of Phytophthora palmivora. Carbohydrates and proteins decreased slightly during the 6-h motile period but increased significantly during germination. Specific activity of isocitrate lyase decreased both during zoospore motility and cyst germination. Only trace amounts of malate synthase activity were detected in zoospores and cysts. The activities of both NAD-isocitrate and malate dehydrogenases increased slightly, while those of NADP-isocitrate and succinate dehydrogenases decreased during the 6-h motile period. During the 2-h germination period the specific activities of NAD- and NADP-isocitrate, malate, and succinate dehydrogenases increased. It appears that during the motile stage the glyoxylate cycle provided more metabolites for the Krebs cycle than it did during germination.

The Regulatory Role of the Embryo on the Development of Isocitrate Lyase Activity during Germination of Ponderosa Pine Seeds

AbstractThe specific activity of isocitrate lyase rapidly increased in the megagametophytic tissue of cold‐stratified seeds of ponderosa pine (Pinus ponderosa Laws) prior to and after germination. When the embryo was removed at germination, isocitrate lyase activity continued to develop. However, in the total absence of the embryo, only a small increase in the specific activity of the enzyme was observed. The development of the enzyme was inhibited by cycloheximide, actinomycin D and abscisic acid.The embryo produced an unidentified factor which enhanced the development of isocitrate lyase activity in the megagametophytic tissue. This embryo factor could not be replaced by the hormones indoleacetic acid (IAA), gibberellic acid (GA3) or benzylaminopurine (BA). Indoleacetic acid had little effect upon enzyme development. Gibberellic acid and benzylaminopurine inhibited isocitrate lyase development in the megagametophytic tissue of the seed.

Evidence of glyoxysomes in germinating pine seeds

By means of differential centrifugation, assay of enzyme activity and electron microscopy, the occurrence of glyoxysomes was demonstrated in germinating Pinus pinea seeds. All the enzyme activities of the glyoxylate cycle were found in the glyoxysome fraction. The specific activity of isocitrate lyase, located in this organelle, increased during germination of the seed. Electron microscopy of three different zones of the endosperm showed a gradual disappearance of lipid bodies and aleurone grains while glyoxysomes, mitochondria and membrane systems increased during germination. The most abundant organelles in vacuolated cells were the glyoxysomes, which seemed to be closely associated with the rough endoplasmic reticulum.

Isocitrate Lyase and Adenosine Triphosphate Malate Lyase as Key Enzymes for the Methylotrophic Growth of Bacterium 5H2

During an investigation of the carbon-assimilation pathway of C1 compounds by the facultative methylotroph bacterium 5H2 (Hampton & Zatman, 1973) isocitrate lyase (EC 4.1.3.1) activities in cell-free extracts of organisms grown on a variety of substrates were measured by the method of Dixon & Kornberg (1959). The method of Kramer et a/. (1959) was used to confirm that a product of the reaction was glyoxylate, the phenylhydrazone of the latter producing the intensely red-coloured 1,Sdiphenylformazancarboxylic acid on treatment with ferricyanide. The isocitrate lyase specific activities of bacterium 5H2 grown on tetramethylammonium chloride, trimethylamine, dimethylamine, methylamine, and on acetate, were respectively, 72, 84, 103, 95 and 150munits/mg of protein, whilst the values for extracts of cells grown on fructose, glutamate and on glycerol were respectively 1.9, I .5 and 2.0munits/mg of protein. These results suggest that isocitrate lyase is important for the inethylotrophic growth of bacterium 5H2 and, not unexpectedly, for growth on acetate. High specific activities of malate synthase (EC 4.1.3.2), measured by method of Dixon & Kornberg (1959), were found in cell-free extracts of bacterium 5H2 grown on CI as well as non-C1 substrates. The results of the following induction experiment confirmed the importance of isocitrate lyase for the growth of bacterium 5H2 on trimethylamine. Glycerol-grown cells, shaken at 30°C in a mineral medium to which 0.2% trimethylarnine had been added, showed no significant growth for the first 6h but the specific activity of isocitrate lyase increased from 2.0 to approximately 45 munits/mg of protein during this period. The specific activity of isocitrate lyase reached a maximum value of approximately lOOmunits/mg of protein about 3 h later, by which time the bacteria were growing exponentially. In a simultaneous experiment in which glycerol-grown cells were shaken at 30°C in the mineral medium containing 0.2% glycerol, exponential growth occurred immediately and there was no increase in the specific activity of the isocitrate lyase throughout the experiment. Present evidence (Salem et al., 1972; Dunstan el a[., 1972; Quayle, 1972) suggests that, in organisms which use the serine pathway of C1 assimilation, regeneration of the glycine acceptor for C1 units arises via the cleavage of a C4 metabolite. During a search for such a C, cleavage reaction, it was observed that crude cell-free extracts of trimethylamine-grown bacterium 5H2 (French press, 35000g supernatant) catalyse, in the presence of hydroxylamine, an ATP-, CoAand Mgz+-dependent formation of acetylhydroxamate from L-malate. Further investigation indicated that the reaction is mediated by a malate cleavage enzyme (ATP malate lyase) which splits L-malate to acetylCoA and glyoxylate and which is similar to the malate lyase cleavage activity described by Tuboi & Kikuchi (1963) in Rhodopseirclomorias spheroicks grown phototrophically with malate. Our reaction mixture ( I ml total volume) contained (pniol): Tris-HC1 buffer pH7.8, 200; ATP, 10; CoA, 0.1; MgCI2, 5 ; L-malate, 100; NH20H,HC1 freshly neutralized, 500; crude cell-free extract (I-2mg of protein). This was incubated without shaking at 30°C, usually for 30min, the reaction being stopped by heating at 100°C for 2min, and a sample of the supernatant being chromatographed on paper by the method of Tuboi & Kikuchi (1962) to identify the hydroxarnates. Acetyl-, succinyland malyl-hydroxamic acids for marker spots on the chromatograms were prepared from their respective anhydrides by the method of Lipmann & Tuttle (1945). A measure of the total hydroxamic acids in the reaction mixture was obtained by the FeCI, method of Lipmann & Tuttle (1945). The major FeCI,-positive spot on the chromatogranis was identified as acetylhydroxamic acid, and a minor spot accounting for approximately 5 % of the total hydroxamate, was identified as malylhydroxamic acid. Glyoxylate production was determined i n reaction mixtures in which the hydroxylamine was

The promotion of isocitrate lyase activity in hazel cotyledons by exogenous gibberellin

Treatment of intact dormant hazel seeds with gibberellin for 96 hours, after an initial 24 hour immersion in sterile distilled water, resulted in a substantially greater increase in the specific activity of isocitrate lyase than that observed in the corresponding water controls. The fresh weights of embryonic axes increased over the same period, but were not appreciably greater after exposure to gibberellin than after sterile distilled water treatment.