Regulation of Glyoxysomal Enzymes during Germination of Cucumber: 2. Isolation and Immunological Detection of Isocitrate Lyase and Catalase

The glyoxysomal enzymes isocitrate lyase and catalase have been isolated from etiolated cucumber (Cucumis sativus) cotyledons. The enzymes co-purified through polyethyleneimine precipitation and (NH₄)₂SO₄ precipitation, and were resolved by gel filtration on Sepharose 6B followed by chromatography on diethylaminoethyl-cellulose (isocitrate lyase) or hydroxylapatite (catalase). Purity of the isolated enzymes was assessed by sodium dodecyl sulfate-polyacrylamide electrophoresis, isoelectric focusing, and immunoelectrophoresis. Antibodies raised to both enzymes in rabbits and in tumor-bearing mice were shown to be monospecific by immunoelectrophoresis against total homogenate protein. Isocitrate lyase and catalase represent about 0.56% and 0.1%, respectively, of total extractable cotyledonary protein. Both enzymes appear to be present in a single form. Molecular weights of the native enzymes and its subunits are 225,000 and 54,500 for catalase, and 325,000 and 63,500 for isocitrate lyase. The pH optimum for isocitrate lyase is about 6.75 in morpholinopropane sulfonic acid buffer, but varies significantly with buffer used. The K_m for d-isocitrate is 39 micromolar. A double antibody technique (rabbit anti-isocitrate lyase followed by ¹²⁵I-labeled goat anti-rabbit immunoglobulin G) has been used to visualize isocitrate lyase subunit protein on sodium dodecyl sulfate-polyacrylamide with high specificity and sensitivity.     

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     

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.     

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.     

Messenger RNA for isocitrate lyase from Chlorella fusca var. vacuolata

Chlorella fusca cultures growing in the light and adapting to acetate in the dark were labelled with adenine-³H and adenine-¹⁴C, respectively. Poly(A)-containing RNA from the mixed cultures was analysed for ¹⁴C/³H ratio after polyacrylamide gel electrophoresis in 98% formamide. The RNA from acetateadapting C. fusca cells contained excess label migrating in the gels at a position equivalent to about 0.85×10⁶ mol.wt. Partially purified anti-isocitrate lyase serum linked to p-aminobenzoyl-cellulose bound 3.5–13% of polysomes from acetate-adapting C. fusca, containing 5–10% of polysomal poly(A)-containing RNA. The antibody-bound poly(A)-containing RNA fraction showed a unimodal size distribution with a mean size of about 0.85×10⁶ mol.wt. after electrophoresis on 4% polyacrylamide gels in 98% formamide. Cell-free translation assays showed a three-fold enrichment of isocitrate lyase mRNA after antibody selection of polysomes and indicated that isocitrate lyase mRNA was abundant in acetate-adapting C. fusca cells.Abbreviations A ₂₆₀ unit The amount of material in 1.0 ml giving an absorbance of 1.0 at 260 nm in a 1 cm light path - PAB-cellulose p-aminobenzoyl-cellulose - SDS sodium lauryl sulphate To whom offprint requests are to be sent     

Control of isocitrate lyase synthesis in Chlorella fusca var. vacuolata. Rate of enzyme synthesis in the presence and absence of acetate measured by [35S]methionine labelling and immunoprecipitation.

The rate of increase of isocitrate lyase activity was measured in darkened Chlorella fusca var. vaculoata cultures in the presence and absence of acetate and compared with the rate of incorporation of [35S]methionine into isocitrate lyase enzyme protein under the same conditions. Isocitrate lyase enzyme protein was isolated for this purpose by specific immunoprecipitation and sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. After 4h in the dark, in the presence of acetate the rate of increase of isocitrate lyase activity was 75 times that in the absence of acetate. Incorporation of [35S]methionine into isocitrate lyase was 140 times greater in the presence of acetate. Incorporation of [35S]methionine into the trichloroacetic acid-insoluble fraction overall was about five times as fast in the presence of acetate. These data are not consistent with an increased turnover of isocitrate lyase enzyme molecules, sufficient to account for the low rate of increase of isocitrate lyase activity in the absence of acetate. The greater rate of enzyme synthesis in the presence of acetate must therefore be due to some effect of this metabolite on the processing or translation of isocitrate lyase mRNA.     

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.     

Immunological Characterization of Plant Ornithine Transcarbamylases

Pea (Pisum sativum L.) ornithine transcarbamylase (OTC) antisera were used to investigate the immunological relatedness of several plant and animal OTC enzymes. The antisera immunoprecipitated OTC activity in all monocot and dicot species tested, and sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis of immunoprecipitated protein revealed monomeric proteins ranging from 35,200 to 36,800 daltons in size. Pea OTC antisera did not recognize mammalian OTC protein. OTC activity and protein levels detected on sodium dodecyl sulfate polyacrylamide gel electrophoresis immunoblots from homogenates of green leaf, etiolated epicotyl and cotyledon, and root tissues of pea were poorly correlated. This might result from differences in amounts of enzymatically active OTC protein in the homogenates. Alternatively, the antisera may fail to recognize different isozyme forms of OTC, which have been reported for some plant species. A putative cytosolic precursor OTC (pOTC) polypeptide exhibiting and M_r = 39,500 to 40,000 daltons was immunoprecipitated from in vitro translation mixtures of total pea leaf poly(A)⁺ RNA. The size of the pOTC polypeptide, as compared with mature OTC monomer (36,000 daltons), suggests that a 4 kilodalton N-terminal leader sequence, like that responsible for mitochondrial targeting of the mammalian enzyme, may be involved in organellar import of the plant enzyme.     

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

The 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.     

Regulation of isocitrate lyase inRhodobacter capsulatus E1F1

InRhodobacter capsulatus E1F1, isocitrate lyase (ICL) (EC 4.5.3.1) is a regulatory enzyme whose levels are increased in the presence of acetate as the sole carbon source. Acetate activated isocitrate lyase in a process dependent on energy supply and de novo protein synthesis. In contrast to isocitrate lyase, isocitrate dehydrogenase (ICDH) activity was independent of the carbon source used for growth and significantly increased in darkened cells. Pyruvate or yeast extract prevented in vivo activation of isocitrate lyase in cells growing on acetate. The enzyme was reversibly inactivated to a great extent in vitro by pyruvate and other oxoacids presumably involved in acetate metabolism. These results suggest that, inR. capsulatus E1F1, isocitrate lyase is regulated by both enzyme synthesis and oxoacid inactivation.     

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.     

Plant microbody proteins. Purification and glycoprotein nature of glyoxysomal isocitrate lyase from cucumber cotyledons.

1. Isocitrate lyase from cotyledons of cucumber seedlings (Cucumis sativus) has been purified 100-fold. Two methods of preparing the soluble glyoxylate cycle enzyme are described: an elaborated method which used crude extracts of cucumber cotyledons, and another procedure which started with purified glyoxysomes from 4-day-old cotyledons and included a separation of glyoxysomal matrix enzymes by zonal centrifugation. The product behaved as a single species when tested by (a) polyacrylamide gel electrophoresis in the presence of dodecyl sulfate, (b) zonal centrifugation, and (c) double immunodiffusion against rabbit antibody to isocitrate lyase. 2. Isocitrate lyase of cucumber glyoxysomes exhibited a molecular weight of 255,000 and was composed of four apparently identical subunits of Mr 64,000. An isoelectric point of 5.9 was determined. 3. It was shown that isocitrate lyase is a glycoprotein, (a) by Schiff stain on polyacrylamide gels, (b) by periodate oxidation of the enzyme, subsequent reduction with NaB[3H]4 and electrophoretic analysis of the labelled glycoprotein, and (c) by incorporation of [3H]glucosamine in vivo into a protein which could be precipitated with antibodies to isocitrate lyase and revealed a 64,000-Mr band upon electrophoresis.     

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.     

Purification and characterization of the first archaeal aconitase from the thermoacidophilic Sulfolobus acidocaldarius.

The first archaeal aconitase was isolated from the cytosol of the thermoacidophilic Sulfolobus acidocaldarius. Interestingly, the enzyme was copurified with an isocitrate lyase. This enzyme, directly converting isocitrate, the reaction product of the aconitase reaction, was also unknown in crenarchaeota, thus far. Both proteins could only be separated by SDS gel electrophoresis yielding apparent molecular masses of 96 kDa for the aconitase and 46 kDa for the isocitrate lyase. Despite of its high oxygen sensitivity, the aconitase could be enriched 27-fold to a specific activity of approximately 55 micromol x min(-1) x mg(-1), based on the direct aconitase assay system. Maximal enzyme activities were measured at pH 7.4 and the temperature optimum for the archaeal enzyme was recorded at 75 degrees C, slightly under the growth optimum of S. acidocaldarius around 80 degrees C. Thermal inactivation studies of the aconitase revealed the enzymatic activity to be uninfluenced after one hour incubation at 80 degrees C. Even at 95 degrees C, a half-life of approximately 14 min was determined, clearly defining it as a thermostable protein. The apparent K(m) values for the three substrates cis-aconitate, citrate and isocitrate were found as 108 microM, 2.9 mM and 370 microM, respectively. The aconitase reaction was inhibited by the typical inhibitors fluorocitrate, trans-aconitate and tricarballylate. Amino-acid sequencing of three internal peptides of the S. acidocaldarius aconitase revealed the presence of highly conserved residues in the archaeal enzyme. By amino-acid sequence alignments, the S. acidocaldarius sequence was found to be highly homologous to either other putative archaeal or known eukaryal and bacterial sequences. As shown by EPR-spectroscopy, the enzyme hosts an interconvertible [3Fe--4S] cluster.     

The Kinetics of Isocitrate Lyase Formation in Chlorella: Evidence for the Promotion of Enzyme Synthesis by Photophosphorylation

The addition of acetate to aerobic Chlorella pyrenoidosa indarkness was followed by the formations of isocitrate lyaseactiity. After a lag period of 40 minutes the formation proceededat a constant rate. By use of actylamide gel electrophoresisit was shown that the increase in enzyme activity was accompaniedby the formation of a new protein which, after separation byelectrophoresis, contained isocitrate lyase activity. The formationof isocitrate lyase was repressed by glucose; it was repressedby light in the presence of carbon dioxide, but not when DCMUwas added. In light, plus DCMU, isocitrate lyase was formedanaerobically and the capacity for photo-formation of isocitratelyase was saturated at 500 ergs/cm²/sec. In this respect theprocess resembled the photo-conversion of glucose to polysaccharidebut differed from the photo-assimilation of carbon dioxide whichbecame saturated at a heigher light intensity. Monochromaticlight of 706 mµ wavelength supported both isocitrate layseformation and the conversion of glucose to polysaccharide butnot carbon dioxide fixation. It is concluded that ATP generatedby cyclic photophosphorylatin can provide the energy for isocitratelyase synthesis in Chlorella.     

Die zeitliche Dauer der Isocitrat-Lyase-Synthese in Kotyledonen von Wassermelonenkeimlingen

Summary Previously, it was deduced from inhibitor experiments that isocitrate lyase (EC 4.1.3.1.) is synthesized de novo in watermelon cotyledons during the first 3 days of germination, which explains the sharp increase of activity during this period. The following decrease of activity was interpreted as the result of a limited half life of the enzyme molecule (Hock and Beevers, 1966).This hypothesis has been confirmed now by density labeling experiments of isocitrate lyase with deuterium. Seedlings grown from day 0 on D₂O (80 vol. %) contained a heavier enzyme at the time of maximum activity than control seedlings grown on H₂O (Fig. 6). No incorporation of deuterium into isocitrate lyase, however, was detectable when the cotyledons were labeled only from day 3 1/2 on, i.e. after the stage of maximum activity had been passed (Fig. 10), in spite of the fact that D₂O was taken up from the cotyledons in considerable quantities. —These results prove at the same time that density labeling of the isocitrate lyase during early stages of germination was a result of de novo synthesis rather than a mere artifact produced by isotopic exchange.An improved method for the purification of isocitrate lyase from higher plants is introduced.     

Glucose-stimulated phosphorylation of yeast isocitrate lyase in vivo

Incorporation of 32P into Saccharomyces cerevisiae isocitrate lyase was observed after addition of glucose to a culture incubated with [32P]orthophosphoric acid. A band of 32P-labelled protein was coincident with the enzyme band when immunoprecipitates were subjected to SDS-PAGE and autoradiography. No label was found in the band corresponding to the isocitrate lyase when immunoprecipitation was done with a control pre-immune serum or in the presence of excess pure unlabelled enzyme. The incorporation of phosphate was associated with a decrease in enzyme activity. Phosphorylated isocitrate lyase was not proteolytically degraded when cells were cultured in mineral medium. The loss of protein antigenicity only took place when the yeast was grown in a complex medium containing glucose.     

The Estimation of the Quantity of Isocitrate Lyase Protein in Acetate-Adapted Cells of Chlorella pyrenoidosa

The quantity of isocitrate lyase protein was estimated, as apercentage of cell dry weight, by three different electrophoreticmethods: (a) direct collection and determination of proteinsafter electrophoresis; (b) separation and estimation of ₃₅S-labelledproteins; (c) estimation from the density of stained bands onacrylamide gels. The possibility that protein-protein interactionduring electrophoresis might interfere with the results wasconsidered and discounted. The average result from the threemethods is that, in acetate-adapted cells of Chlorella pyrenoidosa,isocitrate lyase protein constitutes about 7.0 per cent of totalsoluble proteins (100,000 g supernatant), that is 1.0 per centof cell dry weight. The estimate agrees well with one basedon the increase in specific activity of the enzyme during purification.     

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.     

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.