Purification and properties of isocitrate lyase from Aspergillus nidulans, a model enzyme to study catabolite inactivation in filamentous fungi

Lucas, J. Ramón De; Amor, Cristina; Díazl, M.; Turner, Geoffrey; Laborda, F.

doi:10.1017/s0953756296002699

Cited by 11 publications

(8 citation statements)

References 24 publications

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“…Proteins (10 µg) from cell extracts of samples taken at 0, 2, 4 and 6 h after the shift were separated in native 7.5% polyacrylamide gels, blotted onto nitrocellulose membranes and colored with specific antibodies against A. nidulans isocitrate lyase Similar amounts of protein (10 µg) from cells extracts of acetate-induced mycelia and mycelia transferred to N-free minimal medium and N-free glucose minimal medium were separated by PAGE under non-denaturating conditions and blotted onto nitrocellulose membranes. In Western blots labelled with antibodies against A. nidulans isocitrate lyase, a single broad band, corresponding to isocitrate lyase separated by native electrophoresis, was identified (De Lucas et al 1997a). A clear correlation between the intensity of this band, recognised by the antiserum (Fig.…”

Section: Resultsmentioning

confidence: 89%

“…Protein concentration was determined by the method of Lowry et al (1951) using the BioRad DC protein assay (Bio-Rad Laboratories, USA) and bovine serum albumin as the standard. Activity of isocitrate lyase (EC 4.1.3.1) was measured by the method described by Dixon and Kornberg (1959) with minor variations (De Lucas et al 1997a).…”

Section: Strain and Growth Conditionsmentioning

confidence: 99%

“…Proteins were transferred onto nitrocellulose membranes using a semi-dry electroblotter according to Kynhse-Andersen (1984). For detection of isocitrate lyase, polyclonal antibodies against A. nidulans isocitrate lyase (De Lucas et al 1997a) were used at dilution 1:10,000 in TBST buffer (30 mM NaCl, 0.01% Tween-20, 2 mM Tris HCl, pH 8.0) (Valenciano et al 1998). Alkaline phosphatase-conjugated goat anti-rabbit IgG (Boehringer, Mannheim, Germany) was utilised as a secondary antibody at the same dilution.…”

Section: Strain and Growth Conditionsmentioning

confidence: 99%

“…Isocitrate lyase in A. nidulans hyphae was localised by the immunogold labelling technique using the method described by Valenciano et al (1998).and specific antibodies raised against A. nidulans isocitrate lyase (De Lucas et al 1997a). …”

Section: Electron Microscopy and Immunocytochemistrymentioning

confidence: 99%

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The catabolite inactivation of Aspergillus nidulans isocitrate lyase occurs by specific autophagy of peroxisomes

Amor

Domínguez

Lucas

et al. 2000

Archives of Microbiology

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In Aspergillus nidulans, activity of the glyoxylate cycle enzyme isocitrate lyase is finely regulated. Isocitrate lyase is induced by growth on C2 compounds and long-chain fatty acids and repressed by glucose. In addition, activity of isocitrate lyase is subject to a second mechanism of catabolite control, glucose-induced inactivation. Here, we demonstrate that the catabolite inactivation of A. nidulans isocitrate lyase, a process that takes place during glucose adaptation of cells grown under gluconeogenic conditions, occurs by proteolysis of the enzyme. Ultrastructural analyses were carried out in order to investigate the cellular processes that govern the catabolite inactivation of this peroxisomal enzyme. Addition of glucose to oleate-induced cells triggered the specific engulfment and sequestration of peroxisomes by the vacuoles. Sequestration of various peroxisomes by a single vacuole was a frequently observed phenomenon. Results obtained by immunoelectron microscopy using antibodies against A. nidulans isocitrate lyase showed that degradation of this peroxisomal enzyme occurred inside the vacuole. In addition, ultrastructural studies demonstrated that microautophagy was the autophagic pathway involved in degradation of redundant peroxisomes during glucose adaptation of oleate-induced cells of A. nidulans.

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Section: Resultsmentioning

confidence: 89%

Section: Strain and Growth Conditionsmentioning

confidence: 99%

Section: Strain and Growth Conditionsmentioning

confidence: 99%

Section: Electron Microscopy and Immunocytochemistrymentioning

confidence: 99%

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The catabolite inactivation of Aspergillus nidulans isocitrate lyase occurs by specific autophagy of peroxisomes

Amor

Domínguez

Lucas

et al. 2000

Archives of Microbiology

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“…The subunit molecular weights of the prokaryotic enzymes are around Mr--48 000, whilst the Mr values of plant ICEs vary between 62 000 and 67 000 and the nematode enzyme appears to have a much larger subunit of 120 000 (Vanni et al, 1990). The ICL from A. nidulans is a homo-tetramer with a subunit Mr of 59 000 (De Lucas et al, 1997).…”

Section: Introductionmentioning

confidence: 99%

Isocitrate lyase fromAspergillus nidulans: crystallization and X-ray analysis of a glyoxylate cycle enzyme

Langridge¹,

Baker²,

Lucas³

et al. 1997

Acta Cryst D

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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.

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Purification and Characterization of Isocitrate Lyase from the Wood-Destroying Basidiomycete Fomitopsis palustris Grown on Glucose

Munir

Hattori

Shimada

2002

Archives of Biochemistry and Biophysics

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Purification and properties of isocitrate lyase from Aspergillus nidulans, a model enzyme to study catabolite inactivation in filamentous fungi

Cited by 11 publications

References 24 publications

The catabolite inactivation of Aspergillus nidulans isocitrate lyase occurs by specific autophagy of peroxisomes

The catabolite inactivation of Aspergillus nidulans isocitrate lyase occurs by specific autophagy of peroxisomes

Isocitrate lyase fromAspergillus nidulans: crystallization and X-ray analysis of a glyoxylate cycle enzyme

Purification and Characterization of Isocitrate Lyase from the Wood-Destroying Basidiomycete Fomitopsis palustris Grown on Glucose

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