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Key enzymes of the glyoxylate cycle, isocitrate lyase (ICL) and malate synthase (MS), have been detected in the liver of alloxan-treated rats. The activity of ICL in rat liver was 0.040 μmol/min/mg protein and the activity of MS was 0.022 μmol/min/mg protein. These enzymes were associated with the peroxisomal fraction. The activities of citrate synthase, malate synthase and malate dehydrogenase detected in the peroxisomal fraction were also increased by alloxan treatment. Isocitrate lyase was partially purified and displayed catalytic and regulatory properties similar to those of the enzyme isolated from the liver of starved rats (Popov, V.N. et al. (1996) FEBS Lett. 391, 87–90).

The occurrence of the glyoxylate cycle has not previously been demonstrated in any of the Archaea. In halophilic archaea, only isocitrate lyase activity has been detected. The halophilic archaeon Haloferax volcanii was tested for the presence of the other key enzyme of this pathway, malate synthase. High activities of this enzyme were detected when the carbon source was acetate. Both glyoxylate cycle key enzymes, isocitrate lyase and malate synthase, from Hf. volcanii were purified and characterized.

We report on the isolation of a gene encoding yeast malate synthase. A yeast genomic library was screened using a probe homologous to the yeast enzyme obtained by the polymerase chain reaction. The nucleotide sequence of the cloned gene was determined. Computer analysis showed that the isolated gene is identical to the one previously described as DAL7, which is involved in allantoin metabolism [Mol. Cell. Biol. 9 (1989) 3231-3243]. Enzymatic activities of multicopy transformants, Southern analysis and disruption mutants predict the existence of two genes encoding malate synthases that are differentially regulated at the transcriptional level. Malate synthase; Glyoxylate pathway; Allantoin metabolism; Gene disruption.

Glyoxysomal 3-ketoacyl-CoA thiolase is the last enzyme in the β-oxidation of fatty acids in plant glyoxysomes. A full-length cDNA of the glyoxysomal 3-ketoacyl-CoA thiolase from Brassica napus and a truncated version, lacking the N-terminal targeting signal were cloned in a T7 promoter-based vector. Both recombinant proteins were expressed in Escherichia coli and activity was measured. Full-length and truncated 3-ketoacyl-CoA thiolase have comparable activity in E. coli. Moreover, full-length 3-ketoacyl-CoA thiolase was purified from E. coli and N-terminal sequencing of the protein confirmed that the precursor form indeed is enzymatically active.

The key enzymes of the glyoxylate cycle, isocitrate lyase and malate synthase, have been detected in liver of food-starved 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 microM 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 microM, a K(m)(glyoxylate) of 3 mM and a pH optimum of 7.6.

A rapid and efficient method for the purification of malate synthase, an enzyme uniquely confined to glyoxysomes, from cotyledons of Brassica napus L. has been developed. The two step purification procedure is based on the consequent utilization of the tendency of malate synthase to form high molecular weight aggregates. Malate synthase was purified 75-fold to apparent homogeneity with a specific activity of 180 nkat/mg protein. The estimated molecular weight of malate synthase subunits was 63 kDa. Polyclonal antibodies raised against malate synthase in rabbits detect on Western blots only one single polypeptide with an identical molecular weight.

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.

Yeasts with disruptions in the genes PYC1 and PYC2 encoding the isoenzymes of pyruvate carboxylase cannot grow in a glucose-ammonium medium (Stucka et al. (1991) Mol. Gen. Genet. 229, 307–315). We have isolated a dominant mutation, BPC1-1, that allows growth in this medium of yeasts with interrupted PYC1 and PYC2 genes. The BPC1-1 mutation abolishes catabolite repression of a series of genes and allows expression of the enzymes of the glyoxylate cycle during growth in glucose. A functional glyoxylate cyle is necessary for suppression as a disruption of gene ICL1 encoding isocitrate lyase abolished the phenotypic effect of BPC1-1 on growth in glucose-ammonium. Concurrent expression from constitutive promoters of genes ICL1 and MLS1 (encoding malate synthase) also suppressed the growth phenotype of pyc1 pyc2 mutants. The mutation BPC1-1 is either allelic or closely linked to the mutation DGT1-1.

Several candidate clones carrying partial cDNAs for yeast peroxisomal enzymes, such as catalase, carnitine acetyltransferase, isocitrate lyase, malate synthase and acyl-CoA oxidase, were efficiently isolated at a single plating from a phage λgt11 recombinant cDNA library prepared with poly(A)-rich RNA from an n -alkane-grown yeast, Candida tropicalis , with a mixture of antibodies against the respective purified enzymes. Among them, one candidate clone carrying partial cDNA for catalase was subcloned and subjected to nucleotide sequence analysis. We succeeded in determining that the amino acid sequence deduced from the nucleotide analysis included the sequences derived from the two peptide fragments obtained from the purified enzyme.

The adenosylcobalamin-dependent enzyme glutamate mutase from Clostridium tetanomorphum catalyses the reversible rearrangement of (2S)-glutamate to (2S,3S)-3-methylaspartate. In this conversion 6 carbon centers are involved. The stereochemistry of 4 has been elucidated whereas the formation of the methyl group from the methylene group remains to be established. To solve this problem, (2S,3R)- and (2S,3S)-[3,3-2H1,3H]glutamates were prepared via the 2-oxo[3,3-2H2 or 3H]glutarates by incubation with isocitrate dehydrogenase in deuterium oxide or tritiated water. The labelled glutamates were fermented with growing cells of C. tetanomorphum to butyrate and acetate. Butyrate was further degraded to acetate in which methyl group over 90% of the tritium of the starting glutamate was retained. The chirality of the acetates was determined with malate synthase and fumarase. In both samples complete racemisation was found. This result confirms the rule that racemisation occurs in all adenosylcobalamin-dependent rearrangements in which methyl groups are formed. A methylene radical as intermediate could explain these observations. In a control experiment inversion of configuration in the formation of the methine group of (2S,3S)-3-methylaspartate from the methylene group of (2S)-glutamate was confirmed. Glutamates stereospecifically labelled at C-4 were synthesized from chiral acetates via citrate.

When glyoxysomes isolated from endosperm of 3-day germinated Ricinus communis L. seedlings were incubated with [γ-32P]ATP, phosphorylation of a small number of endogenous glyoxysomal proteins occurrred. A 64 kDa glyoxysomal matrix protein was predominately phosphorylated. Based upon results of immunoprecipitation, 2D-polyacrylamide gel electrophoresis and Western blotting, the 64 kDa phosphorylated protein is identified as the subunit of malate synthase (EC 4.1.3.2). Phosphorylation of malate synthase was completed within 5 min and the amino acid residue phosphorylated was serine. Phosphorylation was inhibited by EDTA, EGTA and NaF, but enhanced by Triton X-100.

catalyzes the condensation of acetyl-CoA with glyoxylate to form malate [2]. The enzyme requires Mg2’ for activity [3]. It is specific for the substrate glyoxylate and does not show catalytic activity in the presence of the substrate analogues pyruvate, oxaloacetate, cY-ketoglutarate etc. 131. Some enzymological [3-5j and physico-chemical [3,5-81 investigations on the yeast enzyme have been performed earlier. As shown by previous CD- measurements [6] the binding of substrates is accom- panied by structural alterations of the enzyme, while binding of Mg2+ does not cause significant structural effects. In the present experiments sedimentation analysis, as well as CD- and fluorescence-spectroscopy were applied to measure structural changes of the enzyme under the same conditions regarding buffer, temper- ature and specific activity as used for small-angle X-ray experiments [9]. The enzyme shows a concen- tration-dependent decrease of specific activity and dissociation upon high dilution. Binding of the sub- strates to the apoenzyme, at concentrations where no dissociation occurs, causes small but significant con- formational changes of the enzyme. 2.

A cDNA clone (pKMS: insert, 1.9 kbp) for glyoxysomal malate synthase was prepared from a cDNA library constructed by poly(A) + RNA from etiolated pumpkin cotyledons and was recombined into the in vitro transcription plasmid, Bluescript. Malate synthase synthesized in an in vitro transcription-translation system was translocated into isolated glyoxysomes. The translocated malate synthase was protected from digestion by added proteinase K. Malate synthase, a glyoxysome-specific enzyme, could also be translocated into leaf peroxisomes prepared from green pumpkin cotyledons, indicating that the machinery for protein translocation into leaf peroxisomes is similar to that into glyoxysomes. When microbodies prepared from greening pumpkin cotyledons were used in the translocation experiments, the translocated malate synthase was observed to be degraded. This result shows that the glyoxysome-specific enzyme, malate synthase, is degraded in microbodies during the microbody transition from glyoxysomes to leaf peroxisomes.

The phosphorylation of NADP-specific isocitrate dehydrogenase in an isocitrate lyase and in a malate synthase mutant of Escherichia coli has been investigated. The results clearly demonstrate that isocitrate dehydrogenase may undergo an acetate-induced phosphorylation in organisms which do not have a functional glyoxylate cycle. This observation, together with those reported in Salmonella typhimurium, suggest that the current notion concerning the interrelationship between the glyoxylate cycle and the reversible phosphorylation of NADP-isocitrate dehydrogenase in microbial physiology should be reevaluated, and that phosphoenolpyruvate may be a key factor in the regulation of the reversible covalent modification of this enzyme in vivo.

The key catalysts of the glyoxylate cycle, isocitrate lyase (EC 4.1.3.1) and malate synthase (EC 4.1.3.2), have been investigated in a variety of tissues [l-3]. In general, the biological a~im~ation of acetate units or the conversion of fatty acids to carbohydrates requires function of glyoxylate cycle [4-61. For example, the function of this cycle is required for conversion of fat to carbohydrate during germination of fat-rich seedlings [7 1. Moreover, the specific activities of isocitrate lyase and malate synthase are highest in second-stage larvae of the nematode Ascaris lumbricoides at a time when the conversion of lipid to carbohydrate is maximal [ 81. The presence of isocitrate lyase and malate synthase has also been reported in several other nematodes [9&O] including a number of free-living forms [ 1 l-141. Although in recent years scattered information has been published on the biochemistry of larval development of free-living nematodes such as Ykrbatrix aceti [14,15] and Caerzorhabditis ekgans [16], ~formation about the basic bio~ern~t~ is still sketchy. For example, nothing is known about the function of glyoxylate cycle enzymes during embryogenesis, although the presence of high levels of isocitrate lyase in early post-embryonic states of C. elegans [ 161 implies that this enzyme functions during embryogenesis. We now present evidence which supports the function of the glyoxylate cycle during embryogenesis of C. eleguns. AS far as we know, this is the first report implicating the occurrence of the glyoxylate cycle in any embryonic animal tissue.

We have resolved eight chromosomal bands from an n-alkane-assimilating yeast, Candida tropicalis pK 233, by using contour-clamped homogeneous electric field gel electrophoresis (CHEF). From the results of hybridization of DNA probes of yeast peroxisomal enzymes—catalase, acyl-CoA oxidase, carnitine acetyltransferase, isocitrate lyase, malate synthase, acetoacetyl-CoA thiolase, and 3-ketoacyl-CoA thiolase — to Southern transfers of CHEF gels, these genes were proven not necessarily to be located on the same chromosome. This fact shows that the genes encoding the enzymes tested were not distributed to be cistronic, although simultaneous and inducible synthesis of peroxisomal enzymes occurred in harmony with the proliferation of peroxisomes, suggesting that their co-ordinated expression might be mainly regulated by certain trans-acting factors.

The presence of additives during X-irradiation of malate synthase led to radioprotective effects against primary and post-irradiation inactivation. Pronounced effects were provided by typical scavengers, sulfhydryl reagents and specific ligands (substrates, products, analogues). The results show that scavenging and specific protection are responsible for the protective efficiency of additives. Scavengers delete noxious species formed during irradiation or post-radiationem. Sulfhydryl reagents may act as repair substances. Specific ligands protect the active site of the enzyme and the essential sulfhydryls; specific protection is more pronounced post-radiationem. Ligands and sulfhydryl reagents may additionally act as scavengers. A cumulative index for the protective power of additives against both sorts of inactivation was established.