Isocitrate lyase from Pseudomonas indigofera I. Preparation, amino acid composition and molecular weight

doi:10.1016/0005-2787(65)90615-5

Biochimica et Biophysica Acta (BBA) - Nucleic Acids and Protein Synthesis

Volume 96, Issue 1, January 1965, Pages 114-122

https://doi.org/10.1016/0005-2787(65)90615-5 Get rights and content

Abstract

An improved method for the purification of isocitrate lyase (D_s-isocitrate glyoxalate-lyase, EC 4.1.3.1) from Pseudomonas indigofera is described. The enzyme preparation is homogeneous by sedimentation and diffusion criteria and virtually homogeneous by gel-electrophoretic analysis.

The sedimentation coefficient (s_{20, w}), diffusion coefficient (D_{20, w}), partial specific volume, molecular weight, extinction coefficient at 280 mμ, absorbancy ratio at 280 to 260 mμ, and turnover number of crystalline isocitrate lyase are respectively, 9.49·10⁻¹³ sec, 3.87·10⁻⁷ cm²/sec, 0.730, 2.22·10⁵, 1.710 cm²/g, 2.03, and 7300 moles glyoxylate formed per min per mole enzyme.

The amino acid composition of the enzyme was determined by ion-exchange chromatography. All of the common amino acids are present in the enzyme. The enzyme contains 21 half-cystine residues, 255 basic amino acid residues, 197 aspartic plus asparagine residues, and 240 glutamic plus glutamine residues per mole.

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Metabolic adaptations to environmental changes were studied in Caenorhabditis elegans. To assess adjustments in enzyme function, maximum activities of key enzymes of main metabolic pathways were determined. After a 12 h incubation at varying temperatures (10, 20°C) and oxygen supplies (normoxia or anoxia), the activities of the following enzymes were determined at two measuring temperatures in tissue extracts: lactate dehydrogenase (LDH; anaerobic glycolysis), 3-hydroxyacyl-CoA-dehydrogenase (HCDH; fatty acid oxidation), isocitrate dehydrogenases (NAD-IDH, NADP-IDH; tricarboxylic acid cycle) and isocitrate lyase (ICL; glyoxylate cycle). Incubation at 20°C induced a strong increase in maximum LDH activity. Anoxic incubation caused maximum HCDH and NADP-IDH activities and, at 10°C incubation, LDH activity to increase. Maximum NAD-IDH and ICL activities were not influenced by any type of incubation. In order to study the time course of metabolic adaptations to varying oxygen supplies, relative quantities of free and protein-bound NADH were determined in living C. elegans using time-resolved fluorescence spectroscopy. During several hours of anoxia, free and protein-bound NADH showed different time courses. One main result was that just at the moment when the protein-bound NADH had reached a constant level, and the free NADH started to increase rapidly, the worms fell into a rigor state. The data on enzyme activity and NADH fluorescence can be interpreted on the basis of a two-stage model of anaerobiosis.
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In order to facilitate the purification of isocitrate lyase from Aspergillus nidulans, the isocitrate lyase overexpressing strain JCB4a was derived. Isocitrate lyase was purified to homogeneity by the criterion of polyacrylamide gel electrophoresis and anti-isocitrate lyase polyclonal antibodies were raised. Stabilization of purified enzyme, when stored at – 20 °C, required the addition of 1 mM dithiothreitol (DTT) plus 1 mM ethylenediaminetetraacetate (EDTA). Aspergillus nidulans isocitrate lyase is a multimeric enzyme with a native molecular weight of 240 kDa and composed of four monomers of 59 kDa. The enzyme required 5 mM Mg and 1 mM DTT or cysteine for full activity. EDTA at 1 mM replaced the requirement of a thiol compound for activity. The K_m for threo-D_s-isocitrate was 0·050 mM, and the enzyme activity was inhibited by succinate, itaconate and structural analogs of glyoxylate as well as by fructose-1,6-bisphosphate.
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A purification scheme is described for the glyoxylate cycle enzyme isocitrate lyase from maize scutella. Purification involves an acetone precipitation and a heat denaturation step, followed by ammonium sulfate precipitation and chromatography on DEAE-cellulose and on blue-Sepharose. The latter step results in the removal of the remaining malate dehydrogenase activity, and of a high molecular mass (62 kDa) but inactive degradation product of isocitrate lyase. Catalase can be completely removed by performing the DEAE-cellulose chromatography in the presence of Triton X-100. Pure isocitrate lyase can be stored without appreciable loss of activity at −70 °C in 5 mm triethanolamine buffer containing 6 mm MgCl₂, 7 mm 2-mercaptoethanol, and 50% ( $v v$ ) glycerol, pH 7.6. Maize isocitrate lyase is a tetrameric protein with a subunit molecular mass of 64 kDa. Purity of the enzyme preparation was demonstrated by polyacrylamide gel electrophoresis in the presence of dodecylsulfate, in acid (pH 3.2) urea and by isoelectric focusing (pI = 5.1). Maize isocitrate lyase is devoid of covalently linked sugar residues. From circular dichroism measurements we estimate that its structure comprises 30% α-helical and 15% β-pleated sheet segments. The enzyme requires Mg²⁺ ions for activity, and only Mn²⁺ apparently is able to replace this cation to a certain extent. The kinetics of the isocitrate lyase-catalyzed cleavage reaction were investigated, and the amino acid composition of the maize enzyme was determined. Finally the occurrence of an association between maize isocitrate lyase and catalase was observed. Such a multienzyme complex may be postulated to play a protective role in vivo.
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The glyoxylate cycle was first discovered during studies on bacteria and fungi with the ability to grow on acetate or ethanol as the sole carbon source. Isocitrate lyase, the first enzyme unique to the glyoxylate cycle, has been studied in numerous prokaryotic and eukaryotic organisms. However, information on this enzyme from Escherichia coli is limited. We have recently reported the purification and in vitro phosphorylation of this enzyme. In this present study we have examined and characterized a variety of inhibitors, the divalent cation requirement and the amino acid composition of E. coli isocitrate lyase and compared these results to those obtained with other organisms.
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A proteinase was copurified with Pinus pinea isocitrate lyase. Its proteolytic action, consisting of isocitrate lyase irreversible inactivation and the appearance of enzyme forms with lower M_rs, became evident when Mg²⁺ was removed from pure preparations or when EDTA or SDS was added. This action was greatly reduced by Mn²⁺ or oxalate, and partially by PMSF, but not by several other protease inhibitors. Some data suggest that the proteolytic activity may be adsorbed on isocitrate lyase. A model for isocitrate lyase inactivation, involving Mg²⁺ dissociation, is proposed.

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^∗: On leave of absence from the Central Research Laboratory, Ajinomoto Co., Inc., Kawasaki, Japan.

^∗∗: On leave of absence from the Institute of Applied Microbiology, University of Tokyo, Japan.

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Isocitrate lyase from Pseudomonas indigofera I. Preparation, amino acid composition and molecular weight

Abstract

J. Biol. Chem.

Anal. Biochem.

Anal. Biochem.

J. Biol. Chem.

J. Bacteriol.