Subcellular localization and properties of glyoxylate cycle enzymes in the liver of rats with alloxan diabetes.

Key enzymes of the glyoxylate cycle (isocitrate lyase and malate synthetase) were found in the liver and kidney of rats suffering from alloxan diabetes. The activities of these enzymes in the liver were 0.080 and 0.0430 U/mg protein, respectively. Isocitrate lyase activity in the kidney was 0.030 U/mg protein, and that of the malate synthetase was 0.018 U/mg protein. Peroxisomal localization of the enzymes was shown. A novel malate dehydrogenase isoform was found in a liver of rats suffering from the alloxan diabetes. The isocitrate lyase was isolated by selective (NH4)2SO4 precipitation and DEAE-Toyopearl chromatography. The resulting enzyme preparation had specific activity 6.1 U/mg protein, corresponding to 76.25-fold purification with 32.6% yield. The isocitrate lyase was found to follow the Michaelis--Menten kinetic scheme (Km for isocitrate, 0.08 mM) and to be competitively inhibited by glucose 1-phosphate (Ki = 1. 25 mM), succinate (Ki = 1.75 mM), and citrate (Ki = 1.0 mM); the pH optimum of the enzyme was 7.5 in Tris-HCl buffer.     

Induction of glyoxylate cycle enzymes in rat liver upon food starvation

The key enzymes of the glyoxylate cycle, isocitrate lyase and malate synthase, have been detected in liver of foodstarved rats. Activities became measurable 3 days and peaked 5 days after the beginning of starvation. Both enzymes were found in the peroxisomal cell fraction after organelle fractionation by isopycnic centrifugation. Isocitrate lyase was purified 112-fold by ammonium sulfate precipitation, and chromotography on DEAE-cellulose and Toyopearl HW-65. The specific activity of the purified enzyme was 9.0 units per mg protein. The K_m(isocitrate) was 68 μM and the pH optimum was at pH 7.4. Malate synthase was enriched 4-fold by ammonium sulfate precipitation. The enzyme had a K_m(acetyl-CoA) of 0.2 μM, a K_m(glyoxylate) of 3 mM and a pH optimum of 7.6.     

Comparative Analysis of Glyoxylate Cycle Key Enzyme Isocitrate Lyase from Organisms of Different Systematic Groups

Isocitrate lyase and malate synthase are the key enzymes of glyoxylate cycle that represents the most important stage on the pathway of conversion of fatty acids to carbohydrates. Until now, induction of enzymes of this metabolic pathway was considered to take place only in cells of prokaryotes, plants, fungi, and nematodes in response to arising demands in carbohydrates. However, the isocitrate lyase activities have been detected in the liver of food-starved rats in our previous work and in pupas of the butterfly Papilio machaon in the present study. The enzymes from both studied objects were purified to homogeneous condition. The main kinetic and physicochemical properties of isocitrate lyase were studied. Organisms of evolutionary distant taxa—mammal, insect, and plant—were chosen for comparative analysis of properties of the studied enzyme. A substantial similarity of kinetic and physicochemical properties of plant and animal isocitrate leases has been found. At the same time, the absence of specific for prokaryotic, plant, and nematode isocitrate lyase nucleotide sequences has been established in mRNA from liver of starved rats and swallowtail pupa. These results are completely confirmed by analysis of the complete genome sequences of the mouse, Drosophila, and human. The obtained data raise the question about the pathway of evolution of genes of the glyoxylate cycle key enzymes.     

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 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.     

2-Methylisocitrate lyases from the bacterium Escherichia coli and the filamentous fungus Aspergillus nidulans: characterization and comparison of both enzymes.

In Escherichia coli and Aspergillus nidulans, propionate is oxidized to pyruvate via the methylcitrate cycle. The last step of this cycle, the cleavage of 2-methylisocitrate to succinate and pyruvate is catalysed by 2-methylisocitrate lyase. The enzymes from both organisms were assayed with chemically synthesized threo-2-methylisocitrate; the erythro-diastereomer was not active. 2-Methylisocitrate lyase from E. coli corresponds to the PrpB protein of the prp operon involved in propionate oxidation. The purified enzyme has a molecular mass of approximately 32 kDa per subunit, which is lower than those of isocitrate lyases from bacterial sources ( approximately 48 kDa). 2-Methylisocitrate lyase from A. nidulans shows an apparent molecular mass of 66 kDa per subunit, almost equal to that of isocitrate lyase of the same organism. Both 2-methylisocitrate lyases have a native homotetrameric structure as identified by size-exclusion chromatography. The enzymes show no measurable activity with isocitrate. Starting from 250 mM pyruvate, 150 mM succinate and 10 microM PrpB, the enzymatically active stereoisomer could be synthesized in 1% yield. As revealed by chiral HPLC, the product consisted of a single enantiomer. This isomer is cleaved by 2-methylisocitrate lyases from A. nidulans and E. coli. The PrpB protein reacted with stoichiometric amounts of 3-bromopyruvate whereby the activity was lost and one amino-acid residue per subunit became modified, most likely a cysteine as shown for isocitrate lyase of E. coli. PrpB exhibits 34% sequence identity with carboxyphosphoenolpyruvate phosphonomutase from Streptomyces hygroscopicus, in which the essential cysteine residue is conserved.     

Evidence for an operative glyoxylate cycle in the thermoacidophilic crenarchaeon Sulfolobus acidocaldarius

Both key enzymes for the glyoxylate cycle, isocitrate lyase (EC 4.1.3.1) and malate synthase (EC 4.1.3.2), were purified and characterized from the thermoacidophilic crenarchaeon Sulfolobus acidocaldarius. Whereas the former enzyme was copurified with the aconitase, the latter enzyme could be enriched to apparent homogeneity. Amino acid sequencing of three internal peptides of the isocitrate lyase revealed the presence of highly conserved residues. With respect to cofactor requirement and quarternary structure the crenarchaeal malate synthase might represent a novel type of this enzyme family. High activities of both glyoxylate cycle enzymes could already be detected in extracts of glucose grown cells and both increased about two-fold in extracts of acetate grown cells.     

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 properties of NADP-isocitrate dehydrogenase from the unicellular cyanobacterium Synechocystis sp. PCC 6803.

NADP-dependent isocitrate dehydrogenase activity has been screened in several cyanobacteria grown on different nitrogen sources; in all the strains tested isocitrate dehydrogenase activity levels were similar in cells grown either on ammonium or nitrate. The enzyme from the unicellular cyanobacterium Synechocystis sp. PCC 6803 has been purified to electrophoretic homogeneity by a procedure that includes Reactive-Red-120-agarose affinity chromatography and phenyl-Sepharose chromatography as main steps. The enzyme was purified about 600-fold, with a yield of 38% and a specific activity of 15.7 U/mg protein. The native enzyme (108 kDa) is composed of two identical subunits with an apparent molecular mass of 57 kDa. Synechocystis isocitrate dehydrogenase was absolutely specific for NADP as electron acceptor. Apparent Km values were 125, 59 and 12 microM for Mg2+, D,L-isocitrate and NADP, respectively, using Mg2+ as divalent cation and 4, 5.7 and 6 microM for Mn2+, D,L-isocitrate and NADP, respectively, using Mn2+ as a cofactor. The enzyme was inhibited non-competitively by ADP (Ki, 6.4 mM) and 2-oxoglutarate, (Ki, 6 mM) with respect to isocitrate and in a competitive manner by NADPH (Ki, 0.6 mM). The circular-dichroism spectrum showed a protein with a secondary structure consisting of about 30% alpha-helix and 36% beta-pleated sheet. The enzyme is an acidic protein with an isoelectric point of 4.4 and analysis of the NH2-terminal sequence revealed 45% identity with the same region of Escherichia coli isocitrate dehydrogenase. The aforementioned data indicate that NADP isocitrate dehydrogenase from Synechocystis resembles isocitrate dehydrogenase from prokaryotes and shows similar molecular and structural properties to the well-known E. coli enzyme.     

Tricarboxylic acid and glyoxylate cycle enzyme activities in Thiobacillus versutus,an isocitrate lyase negative organism

An analysis was made of the specific enzyme activities of the TCA and glyoxylate cycle in Thiobacillus versutus cells grown in a thiosulphate- or acetate-limited chemostat. Activities of all enzymes of the TCA cycle were detected, irrespective of the growth substrate and they were invariably lower in the thiosulphate-grown cells. Of the glyoxylate cycle enzymes, isocitrate lyase was absent but malate synthase activity was increased from 15 nmol·min^-1·mg^-1 protein in thiosulphate-grown cells to 58 nmol·min^-1·mg^-1 protein in acetate-grown cells. Suspensions of cells grown on thiosulphate were able to oxidize acetate, although the rate was 3 times lower than that observed with acetate-grown cells. The respiration of acetate was completely inhibited by 10 mM fluoroacetate or 5 mM arsenite. Partially purified citrate synthase from both thiosulphate- and acetate-grown cells was completely inhibited by 0.5 mM NADH and was insensitive to inhibition by 1 mM 2-oxoglutarate or 1 mM ATP. The specific enzyme activities of the TCA and glyoxylate cycle in T. versutus were compared with those of Pseudomonas fluorescens, an isocitrate lyase positive organism, after growth in a chemostat limited by acetate, glutarate, succinate or glutamate. The response of the various enzyme activities to a change in substrate was similar in both organisms, with the exception of isocitrate lyase.Abbreviations TCA tricarboxylic acid - DNTB 2,2-dinitro-5,5-dithiobenzoic acid - APAD acetylpyridine adenine dinucleotide - PMS phenazine methosulphate - DCPIP 2,6-dichlorophenol-indophenol - DOC dissolved organic carbon     

Leaf peroxisomes are directly transformed to glyoxysomes during senescence of pumpkin cotyledons

Summary After the functional transition of glyoxysomes to leaf peroxisomes during the greening of pumpkin cotyledons, the reverse microbody transition of leaf peroxisomes to glyoxysomes occurs during senescence. Immunocytochemical labeling with protein A-gold was performed to analyze the reverse microbody transition using antibodies against a leaf-peroxisomal enzyme, glycolate oxidase, and against two glyoxysomal enzymes, namely, malate synthase and isocitrate lyase. The intensity of labeling for glycolate oxidase decreased in the microbodies during senescence whereas in the case of malate synthase and isocitrate lyase intensities increased strikingly. Double labeling experiments with protein A-gold particles of different sizes showed that the leaf-peroxisomal enzymes and the glyoxysomal enzymes coexist in the microbodies of senescing pumpkin cotyledons, indicating that leaf peroxisomes are directly transformed to glyoxysomes during senescence.     

Glyoxylate cycle in Mucor racemosus.

The dimorphic phycomycete Mucor racemosus was grown in media containing acetate, glutamate, and peptone as carbon sources. The component enzymes of the glyoxylate bypass, isocitrate lyase and malate synthase, were present under these conditions throughout the growth cycles. Highest specific activities for each enzyme were found in media with acetate as the carbon source. In an enriched peptone medium containing glucose, neither activity was detected until glucose was exhausted from the medium. Treatment of acetate-grown cells with glucose resulted in a rapid decline in the specific activities of both enzymes. The importance of this cycle in acetate-grown cells was indicated by the ability of itaconic acid (100 mM) to inhibit the growth of M. racemosus in acetate but not glutamate media. Itaconate was also shown to be a potent inhibitor of isocitrate lyase activity in vitro.     

Translational or post-translational processes affect differentially the accumulation of isocitrate lyase and malate synthase proteins and enzyme activities in embryos and seedlings of Brassica napus

We have analyzed the accumulation of the glyoxylate cycle enzymes isocitrate lyase and malate synthase in embryos and seedlings of Brassica napus L. The two enzyme activities and proteins begin to accumulate during late embryogeny, reach maximal levels in seedlings, and are not detected in young leaves of mature plants. We showed previously that mRNAs encoding the two enzymes exhibit similar qualitative patterns of accumulation during development and that the two mRNAs accumulate to different levels in both embryos and seedlings (L. Comai et al., 1989, Plant Cell 1, 293-300). In this report, we show that the relative accumulation of the proteins and activities do not correspond to these mRNA levels. In embryos and seedlings, the specific activities of isocitrate lyase and malate synthase are approximately constant. By contrast, the ratio of malate synthase protein to mRNA is 14-fold higher than that of isocitrate lyase. Differences in the translational efficiencies of the two mRNAs in vitro do not appear to account for the discrepancy between mRNA and protein levels. Our results suggest that translational and/or post-translational processes affect differentially the accumulation of the proteins.     

Purification and Characterization of Isocitrate Lyase from Ethanol-grown Euglena gracilis

Isocitrate lyase was purified to homogeneity from ethanol-grown Euglena gracilis. The specific activity was 0.26 μmol/min/mg protein. The molecular mass of the enzyme was calculated to be 380 kDa by gel filtration on a Superose 6 column. The subunit molecular mass of the enzyme was 116 kDa as determined by SDS-polyacrylamide gel electrophoresis. These results showed that the native form of this enzyme was a trimer composed of three identical subunits. The pH optimum for cleavage and condensation reactions was 6.5 and 7.0, respectively. The K_m values for isocitrate, glyoxylate and succinate were 3.8, 1.3 and 7.7 mM, respectively. Isocitrate lyase absolutely required Mg for enzymatic activity. This is the first report of the purification of isocitrate lyase to homogeneity from Euglena gracilis.     

DEVELOPMENTAL STUDIES ON GLYOXYSOMES IN RICINUS ENDOSPERM

The development of glyoxysomes and their associated enzymes, isocitrate lyase and malate synthetase, was studied in the endosperm of castor bean seeds during germination and early growth in darkness. The protein content of the glyoxysome fraction, separated by sucrose density centrifugation, increased linearly from day 2 to day 4 and declined subsequently, while maximum enzyme activities were reached at day 5. The specific activities of the enzymes in the glyoxysomes increased until day 5 and remained constant thereafter. At all stages of germination the only organelle with isocitrate lyase activity was the glyoxysome, but at the earlier stages a greater portion of the total activity was recovered in the soluble form. Malate synthetase was found primarily in the glyoxysomes after day 4, but at earlier stages part of the activity appeared at regions of lower density on the sucrose gradient. It was shown that this particulate malate synthetase activity was due to glyoxysomes broken during preparation, and that, as a result of this breakage, isocitrate lyase was solubilized. We conclude that both enzymes are housed in the glyoxysome in vivo throughout the germination period, and that the rise and fall in enzyme activities in phase with fat breakdown correspond to the net production and destruction of this organelle.     

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.     

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.     

Characterization of the isocitrate lyase gene from Corynebacterium glutamicum and biochemical analysis of the enzyme.

Isocitrate lyase is a key enzyme in the glyoxylate cycle and is essential as an anaplerotic enzyme for growth on acetate as a carbon source. It is assumed to be of major importance in carbon flux control in the amino acid-producing organism Corynebacterium glutamicum. In crude extracts of C. glutamicum, the specific activities of isocitrate lyase were found to be 0.01 U/mg of protein after growth on glucose and 2.8 U/mg of protein after growth on acetate, indicating tight regulation. The isocitrate lyase gene, aceA, was isolated, subcloned, and characterized. The predicted gene product of aceA consists of 432 amino acids (M(r), 47,228) and shows up to 57% identity to the respective enzymes from other organisms. Downstream of aceA, a gene essential for thiamine biosynthesis was identified. Overexpression of aceA in C. glutamicum resulted in specific activities of 0.1 and 7.4 U/mg of protein in minimal medium containing glucose and acetate, respectively. Inactivation of the chromosomal aceA gene led to an inability to grow on acetate and to the absence of any detectable isocitrate lyase activity. Isocitrate lyase was purified to apparent homogeneity and subjected to biochemical analysis. The native enzyme was shown to be a tetramer of identical subunits, to exhibit an ordered Uni-Bi mechanism of catalysis, and to be effectively inhibited by 3-phosphoglycerate, 6-phosphogluconate, phosphoenolpyruvate, fructose-1,6-bisphosphate, and succinate.