Purification and characterization of isocitrate lyase fromEscherichia coli

Isocitrate lyase has been purified to homogeneity, as determined by SDS-polyacrylamide gel electrophoresis and subsequent silver staining, fromEscherichia coli D₅H₃G₇. The enzyme was found to have a subunit molecular weight of 48,000 and a native molecular weight of 188,000 as determined by gel filtration chromatography. Thus, the enzyme appears to have tetrameric structure. The isoelectric point was determined to be 4.6, and the enzyme displayed a pH optimum at 7.3. The K_m of isocitrate lyase forthreo-Ds-isocitrate was determined to be 8 M. The purification procedure is highly reproducible and results in a 39% net yield of purified protein.     

Characterization of activity and expression of isocitrate lyase in Mycobacterium avium and Mycobacterium tuberculosis

Analysis by two-dimensional gel electrophoresis revealed that Mycobacterium avium expresses several proteins unique to an intracellular infection. One abundant protein with an apparent molecular mass of 50 kDa was isolated, and the N-terminal sequence was determined. It matches a sequence in the M. tuberculosis database (Sanger) with similarity to the enzyme isocitrate lyase of both Corynebacterium glutamicum and Rhodococcus fascians. Only marginal similarity was observed between this open reading frame (ORF) (termed icl) and a second distinct ORF (named aceA) which exhibits a low similarity to other isocitrate lyases. Both ORFs can be found as distinct genes in the various mycobacterial databases recently published. Isocitrate lyase is a key enzyme in the glyoxylate cycle and is essential as an anapleurotic enzyme for growth on acetate and certain fatty acids as carbon source. In this study we express and purify Icl, as well as AceA proteins, and show that both exhibit isocitrate lyase activity. Various known inhibitors for isocitrate lyase were effective. Furthermore, we present evidence that in both M. avium and M. tuberculosis the production and activity of the isocitrate lyase is enhanced under minimal growth conditions when supplemented with acetate or palmitate.     

Molecular characterization of a bifunctional glyoxylate cycle enzyme, malate synthase/isocitrate lyase, in Euglena gracilis

Euglena gracilis induced glyoxylate cycle enzymes when ethanol was fed as a sole carbon source. We purified, cloned and characterized a bifunctional glyoxylate cycle enzyme from E. gracilis (EgGCE). This enzyme consists of an N-terminal malate synthase (MS) domain fused to a C-terminal isocitrate lyase (ICL) domain in a single polypeptide chain. This domain order is inverted compared to the bifunctional glyoxylate cycle enzyme in Caenorhabditis elegans, an N-terminal ICL domain fused to a C-terminal MS domain. Purified EgGCE catalyzed the sequential ICL and MS reactions. ICL activity of purified EgGCE increased in the existence of acetyl-CoA at a concentration of micro-molar order. We discussed the physiological roles of the bifunctional glyoxylate cycle enzyme in these organisms as well as its molecular evolution.     

In vitro phosphorylation ofEscherichia coli isocitrate lyase

The in vitro phosphorylation of isocitrate lyase was demonstrated in partially purified sonic extracts ofEscherichia coli. Extracts were incubated with [gamma³²P]-ATP and subsequently analyzed by two-dimensional polyacrylamide gel electrophoresis. Isocitrate lyase was determined to be phosphorylated by autoradiography and Western blot analyses of the gels. Purified isocitrate lyase comigrates with the phosphorylated form of the enzyme; this suggests that the enzyme may become catalytically active concomitant with phosphorylation.     

Purification and characterization of isocitrate lyase from the wood-destroying basidiomycete Fomitopsis palustris grown on glucose

Isocitrate lyase (EC 4.1.3.1), a key enzyme in the glyoxylate cycle, was purified 76-fold with 23% yield as an electrophoretically homogeneous protein from the wood-destroying basidiomycete Fomitopsis palustris grown on glucose. The native enzyme has a molecular mass of 186 kDa, consisting of three identical subunits of 60 kDa. The K(m) for DL-isocitrate was found to be 1.6 mM at the optimum pH (7.0). The enzyme required Mg(2+) (K(m) 92 microM) and sulfhydryl compounds for optimal activity. The enzyme activity was strongly inhibited by oxalate and itaconate with a K(i) of 37 and 68 microM, respectively. The inhibition by the glycolysis and tricarboxylic acid cycle intermediates and related compounds suggested that the isocitrate lyase was a regulatory enzyme playing a crucial role in the fungal growth.     

An isocitrate lyase of higher plants: analysis and comparison of some molecular properties

A new purification procedure for isocitrate lyase from Pinus pinea is reported. The final preparation shows charge homogeneity and a purity degree higher than 95%. It is possible to remove catalase completely by exploiting the high hydrophobicity of isocitrate lyase. The enzyme has a Mr of 264,000 and is likely composed of four subunits, each with a Mr of 66,000. The binding of radioactively labeled oxalate revealed four catalytic sites per oligomer. These data suggest that isocitrate lyase subunits are similar, if not identical. The Michaelis constant for isocitrate is equal to 33 microM; molecular activity is about 2670 mol X min-1 X mol of enzyme-1. The amino acid composition of the enzyme was also determined. Isocitrate lyase appears resistant to proteolysis by carboxypeptidase A. Hydrazinolysis, Edman degradation, and dansyl chloride treatment indicate that both carboxy and amino terminals are probably inaccessible or blocked.     

Purification and properties of isocitrate lyase from flax seedlings.

Isocitrate lyase has been purified from flax (Linum usitatissimum) seedlings. The final preparation was homogeneous by the criteria of polyacrylamide disc gel electrophoresis, immunodiffusion, and immunoelectrophoresis. From exclusion chromatography on Sephadex G-200, the molecular weight and Stoke's radius of the enzyme were 264,000 and 5.28 × 10^?7 cm, respectively. The subunit molecular weight was 67,000. Thus, the enzyme appears to be tetrameric. The enzyme required Mg²⁺ and cysteine for activity. The optimal pH of the enzyme was 7.5 both in Tris and in phosphate buffers. There are three disulfide bridges and two of eight cysteine residues are buried. Inactivation of isocitrate lyase resulted from short-term modification of enzymatic thiols but this could be reversed by added thiols. The enzyme was competitively inhibited by glyoxylate, l-tartrate, and malonate in catalysis of isocitrate cleavage.     

Characterization of methylmalonyl-CoA mutase involved in the propionate photoassimilation of Euglena gracilis Z

Significant accumulation of the methylmalonyl-CoA mutase apoenzyme was observed in the photosynthetic flagellate Euglena gracilis Z at the end of the logarithmic growth phase. The apoenzyme was converted to a holoenzyme by incubation for 4 h at 4°C with 10 μM 5′-deoxyadenosylcobalamin, and then, the holoenzyme was purified to homogeneity and characterized. The apparent molecular mass of the enzyme was calculated to be 149.0 kDa ± 5.0 kDa using Superdex 200 gel filtration. SDS–polyacrylamide gel electrophoresis of the purified enzyme yielded a single protein band with an apparent molecular mass of 75.0 kDa ± 3.0 kDa, indicating that the Euglena enzyme is composed of two identical subunits. The purified enzyme contained one mole of prosthetic 5′-deoxyadenosylcobalamin per mole of the enzyme subunit. Moreover, we cloned the full-length cDNA of the Euglena enzyme. The cDNA clone contained an open reading frame encoding a protein of 717 amino acids with a calculated molecular mass of 78.3 kDa, preceded by a putative mitochondrial targeting signal consisting of nine amino acid residues. Furthermore, we studied some properties and physiological function of the Euglena enzyme.     

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.     

Isolation and properties of watermelon isocitrate lyase

The glyoxylate cycle enzyme, isocitrate lyase (EC 4.1.3.1) was purified from cotyledons of Citrullus vulgaris (watermelon). The final preparation, which had been 97-fold purified with a specific activity of 16.1 units/mg protein in a yield of 36%, was homogeneous by gel- and immunoelectrophoretic criteria. The tetrameric enzyme had: a molecular weight of 277 000, a sedimentation coefficient of 12.4 s, and a K_m for D_s-isocitrate equal to 0.25 mM. Isocitrate lyase from this source is not a glycoprotein as shown by total carbohydrate content after precipitation by trichloroacetic acid of the purified enzyme. Reduction of the enzyme with thiols increased activity and maximal activity was obtained with at least 5 mM dithiothreitol. EDTA partially substituted for thiol in freshly isolated enzyme. Watermelon isocitrate lyase was also protected against thermal denaturation at 60° for at least 1 hr by 5 mM Mg²⁺ plus 5 mM oxalate. Oxalate was a competitive inhibitor with respect to isocitrate (K_i: 1.5 μM, pH 7.5, 30°).     

Purification and Characterization of a Cold-adapted Isocitrate Lyase and a Malate Synthase from Colwellia maris,a Psychrophilic Bacterium

Isocitrate lyase (ICL) and malate synthase (MS) of a psychrophilic marine bacterium, Colwellia maris, were purified to electrophoretically homogeneous state. The molecular mass of the ICL was found to be 240 kDa, composed of four identical subunits of 64.7 kDa. MS was a dimeric enzyme composed of 76.3 kDa subunits. N-Terminal amino acid sequences of the ICL and MS were analyzed. Purified ICL had its maximum activity at 20°C and was rapidly inactivated at the temperatures above 30°C, but the optimum temperature for the activity of MS was 45°C. NaCl was found to protect ICL from heat inactivation above 30°C, but the salt did not stabilize MS. Effects of temperatures on the kinetic parameters of both the enzymes were examined. The K_m for the substrate (isocitrate) of ICL was decreased with decreasing temperature. On the other hand, the K_m for the substrate (glyoxylate) of MS was increased with decreasing temperature. The calculated value of free energy of activation of ICL was on the same level as that of MS.     

The purification and physicochemical characterization of maize (Zea mays L.) isocitrate lyase.

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 degrees 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% alpha-helical and 15% beta-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.     

The action of exogenous gibberellic acid on isocitrate lyase —mRNA in germinating castor bean seeds

Gibberellic acid (GA₃) stimulates isocitrate lyase activity of the endosperm during germination of castor bean seeds. Isocitrate lyase from castor bean was purified and an antibody to it was prepared from rabbit serum. This antibody was used to measure the amounts of isocitrate lyase-mRNA using an in vitro translation system. No specific stimulation of isocitrate lyase-mRNA by application of GA₃ was detected. The stimulation of isocitrate lyase activity by exogenous GA₃ may be accounted for by the action of the growth substance in advancing the overall production of rRNA and mRNA which accelerates the rate of total protein synthesis during germination. The application of Amo 1618 retards the production of isocitrate lyase activity but also retards protein synthesis in general. This suggests that endogenous gibberellins also act non-specifically in the regulation of protein synthesis during castor bean germination.Abbreviations SDS sodium dodecyl sulphate - GA₃ gibberellic acid - PAGE polyacrylamide gel electrophoresis     

GLYOXYLATE CYCLE ENZYME ACTIVITIES IN THE CYANOBACTERIUM ANACYSTIS NIDULANS1

The enzymes of the glyoxylate cycle, isocitrate lyase (EC.4.1.3.1) and malate synthase (EC.4.1.3.2), were measured in cell-free extracts from the cyanobacterium Anacystis nidulans Drouet during photoautotrophic growth in medium aerated with ordinary air (0.03% CO₂). Isocitrate lyase had an average specific activity of 112 nmoles·min^?1·mg protein^?1 whereas malate synthase had an average specific activity of 12.5 nmoles·min^?1·mg protein^?1. Unpurified isocitrate lyase showed classical Michaelis kinetics with a K_m of 8 mM. Isocitrate lyase activity was strongly inhibited by numerous cellular metabolites at 10 mM concentration. The previously reported low specific activity for isocitrate lyase may be due to metabolite inhibition caused by growth in high CO₂ concentrations. The activities reported for isocitrate lyase and malate synthase suggest the operation of the glyoxylate cycle in Anacystis nidulans under CO₂-limiting growth conditions.     

The disappearance of isocitrate lyase from the green alga Chlorella fusca studied by immunoprecipitation

When acetate-adapted cultures of Chlorella fusca were transferred to nitrogen-free medium containing glucose, isocitrate lyase activity was lost over a period of about 25 h. Using a combination of in vivo isotope labelling and immunoprecipitation with anti-isocitrate lyase IgG it was shown that: 1. The onset of loss of enzyme activity preceeded the complete cessation of enzyme synthesis. 2. Disappearance of isocitrate lyase activity was accompanied by loss of enzyme protein, without accumulation of antigenic protein distinguishable from the normal subunit polypeptide of the enzyme, as judged by SDS gel electrophoresis of immunoprecipitated samples from supernatant cell-free extracts. 3. SDS gel electrophoresis of immunoprecipitated isocitrate lyase revealed the presence of antigenic protein bands of M_r about twice that of the normal subunit polypeptide, but the appearance of these apparent dimer forms did not obviously correlate with enzyme degradation. 4. Isoelectric focusing of immunoprecipitated isocitrate lyase showed that the enzyme became progressively more oxidised during the period of its degradation in vivo. 5. By titrating crude broken cell suspensions with anti-isocitrate lyase antibody, preliminary evidence was obtained for transfer of the enzyme from the soluble fraction to an insoluble form as part of the process of disappearance.     

Purification and Properties of Isocitrate Lyase from Pupas of the Butterfly Papilio machaon L.

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 (Ki = 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).     

Regulation of isocitrate lyase in a mutant of Rhodopseudomonas capsulata adapted to growth on acetate

Studies on acetate utilization by Rhodopseudomonas capsulata strain St. Louis indicated that the wild type grew poorly on acetate and made little if any of the glyoxylate cycle enzyme isocitrate lyase. A spontaneous mutant, Ac-l, capable of vigorous and immediate growth on acetate and exhibiting high levels of isocitrate lyase activity, was isolated in the course of those studies.Isocitrate lyase was not formed when the mutant was grown on malate. Addition of malate to cultures of Ac-l growing on acetate resulted in loss of the enzyme by dilution through growth.Starvation of acetate-grown Ac-l for acetate resulted in a rapid and complete loss of isocitrate lyase activity which was shown to be energy dependent. Readdition of acetate to a starved culture previously grown on acetate resulted in a rapid recovery of enzyme activity. The recovery required energy and was sensitive to chloramphenicol inhibition at any time during the recovery phase.     

Sugar Synthesis in Leptospira

The presence and some properties of the key enzymes of the glyoxylate cycle, isocitrate lyase (threo-D_s-isocitrate glyoxylate-lyase, EC 4.1.3.1) and malate synthase (L-malate glyoxylate-lyase (CoA-acetylating) EC 4.1.3.2), were investigated in Leptospira biflexa. Isocitrate lyase activity was found for the first time in the organism. The enzyme was induced by ethanol but not by acetate. The optimum pH was 6.8. The activity was inhibited by phosphoenolpyruvate, a specific inhibitor of isocitrate lyase. The optimum pH of malate synthase of L. biflexa was about 8.5. The K_m value for glyoxylate was 3.0 × 10^?3 M and the activity was inhibited by glycolate, the inhibitor. The results strongly suggested the presence of a glyoxylate cycle in Leptospira. The possibility that the glyoxylate cycle plays an essential role in the synthesis of sugars, amino acids and other cellular components as an anaplerotic pathway of the tricarboxylic acid cycle in Leptospira was discussed.     

Caenorhabditis elegans: purification of isocitrate lyase and the isolation and cell-free translation of poly(A+)RNA

Isocitrate lyase (EC 4.1.3.1) from mixed larval populations of Caenorhabditis elegans was stabilized in crude extracts by centrifugation over a 0.2-0.6 M sucrose gradient for 2.5 hr in a vertical rotor (VTi 50) at 210,000g. The peak fractions from this sucrose gradient showed a half-life of 33 hr at 30 C and 225 hr at 4 C in contrast to 2.5 and 52 hr, respectively, for the crude extract. A purification scheme involving (NH4)2SO4 precipitation and chromatography on Sepharose 6B and diethylaminoethyl-cellulose yielded isocitrate lyase that gave one band after electrophoresis in a sodium dodecyl sulfate-gel polymerized from 12% acrylamide. The purified enzyme with a molecular weight of 250,000 and subunit molecular weight of 61,600, had a specific activity of 2 mumoles glyoxylate formed min-1 mg protein-1, and was obtained in a 4% yield. Isocitrate lyase from C. elegans lost 80-85% of its activity in the precipitation by 33-55% (NH4)2SO4, but this step appeared to be necessary for purification to homogeneity. The use of fast protein liquid chromatography appeared to be promising in that it provided an enzyme preparation that was about 50% pure with a specific activity as high as 3 mumoles glyoxylate formed min-1 mg protein-1. Poly(A+)RNA was isolated from C. elegans and translated in wheat germ cell-free system. Analysis on a 10% sodium dodecyl sulfate-polyacrylamide gel showed varied translation products including one or more 60,000-Da polypeptides.     

Purification and regulatory properties of isocitrate lyase from Escherichia coli ML308.

Isocitrate lyase was purified to homogeneity from Escherichia coli ML308. Its subunit Mr and native Mr were 44,670 +/- 460 and 17,000-180,000 respectively. The kinetic mechanism of the enzyme was investigated by using product and dead-end inhibitors of the cleavage and condensation reactions. The data indicated a random-order equilibrium mechanism, with formation of a ternary enzyme-isocitrate-succinate complex. In an attempt to predict the properties of isocitrate lyase in intact cells, the effects of pH, inorganic anions and potential regulatory metabolites on the enzyme were studied. The Km of the enzyme for isocitrate was 63 microM at physiological pH and in the absence of competing anions. Chloride, phosphate and sulphate ions inhibited competitively with respect to isocitrate. Phosphoenolpyruvate inhibited non-competitively with respect to isocitrate, but the Ki value suggested that this effect was unlikely to be significant in intact cells. 3-Phosphoglycerate was a competitive inhibitor. At the concentration reported to occur in intact cells, this metabolite would have a significant effect on the activity of isocitrate lyase. The available data suggest that the Km of isocitrate lyase for isocitrate is similar to the concentration of isocitrate in E. coli cells growing on acetate, about one order of magnitude higher than the Km determined in vitro in the absence of competing anions.