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Summary Alkaloids cannot be supposed to be waste products of general metabolism. They are formed only for a relatively brief time during the lifespan of the culture and under conditions of reduced proliferation. Living rather than dying cells are required for alkaloid synthesis. The alkaloid yield and differentiated cells were observed to be inversely proportional. This pattern is correlated with formation of secondary shunt products. The district between the cell-pool tryptophan “down” and “up” phases is a promising target for the investigation of regulation of the alkaloid formation and continuous fermentation of these compounds. Considering the general phenomenon of secondary metabolism we may guess that alkaloid formation includes derepression of the appropriate portion of the genome to obtain transcription and translation of specific synthetases and subsequent activity of these enzymes for a relatively brief period. The ability to produce alkaloids is not only easily lost genetically, but is also extraordinarily susceptible to suppression by rather minor environmental manipulations. The production of alkaloids occurs only in nutrient media with high osmotic pressure. Ergoline alkaloids do not definitely belong to a group of physiologically inert secondary metabolites. They are, in fact, able to influence primary shunt metabolism, and may reflect a regulatory device of the producing cell. Alkaloids increase the activities of tryptophan synthetase and acetyl-CoA carboxylase of the ergot fungus and exert a negative effect on citrate synthase, isocitrate lynse, and malate synthase. Also inhibition of Claviceps peptidases by peptide alkaloids is of peculiar interest. Addition of ergometrine (10 -4 M) to the submerged culture of C. paspali increased the total-alkaloid yield by 120%. However, the stimulatory effect was inversely proportional to the concentration of the supplied ergometrine. Implicit in these data is that in alkaloid-producing cultures accumulating alkaloids may suppress the attainment of the yield that would be achieved by continuous control of the alkaloid level.

Summary: Hyphomicrobium sp. were grown on ethanol, acetate, 3-hydroxybutyrate and methanol. The specific activities of the following enzymes were measured in cell-free extracts of Hyphomicrobium strain x: ethanol dehydrogenase, acetaldehyde dehydrogenase, acetothiokinase, 3-hydroxybutyrate dehydrogenase, β-keto thiolase, citrate synthase, aconitate hydratase, isocitrate dehydrogenase, α-oxoglutarate dehydrogenase, succinate dehydrogenase, fumarate hydratase, malate dehydrogenase, isocitrate lyase, malate synthase, malate dehydrogenase (decarboxylating), phosphoenolpyruvate carboxylase, pyruvate kinase, phosphoenolpyruvate carboxykinase, phosphopyruvate hydratase and phosphoglycerate mutase. From a comparison of the specific activities of these enzymes in the different cell-free extracts, it was concluded that during growth of Hyphomicrobium sp. on either ethanol, acetate or 3-hydroxybutyrate, energy and reducing power is generated mainly by the tricarboxylic acid (TCA) cycle. Carbon is assimilated via the glyoxylate cycle with phosphoenolpyruvate carboxykinase functioning as the main gluconeogenic enzyme. The conversion of ethanol, acetate and 3-hydroxybutyrate to acetyl-CoA occurs by pathways previously reported in other micro-organisms.

Malate synthase has a molecular weight of about 170 000 as shown by ultracentrifugation, sucrose gradient centrifugation, and thin layer gel-chromatography. High dilution, extremes of pH, succinylation, and treatment with sodium dodecylsulfate suggest the enzyme to be a tetramer. The CD spectrum is typical for a globular protein with moderate helical content (∼30 %), and shows anomalous Cotton effects at 250–290 nm. Binding of substrates (acetyl-CoA, glyoxylate) or the substrate analog pyruvate causes slight conformational changes which are reflected in alterations of the CD bands in the range of aromatic absorption; binding of Mg2+ causes no structural effects, suggesting the metal ion to be involved in enzymatic catalysis rather than structural alterations.

Cytochemical staining techniques for microbodies (peroxisomes) are limited at present to the enzymes catalase and α-hydroxy acid oxidase, and neither technique can distinguish glyoxysomes from other microbodies. Described here is a procedure using ferricyanide for the cytochemical demonstration by light and electron microscopy of malate synthase activity in glyoxysomes of cotyledons from fat-storing cucumber and sunflower seedlings. Malate synthase, a key enzyme of the glyoxylate cycle, catalyzes the condensation of acetyl CoA with glyoxylate to form malate and release free coenzyme A. Localization of the enzyme activity is based on the reduction by free CoA of ferricyanide to ferrocyanide, and the visualization of the latter as an insoluble, electron-opaque deposit of copper ferrocyanide (Hatchett's brown). The conditions and optimal concentrations for the cytochemical reaction mixture were determined in preliminary studies using a colorimetric assay developed to measure disappearance of ferricyanide at 420 nm. Ultrastructural observation of treated tissue reveals electron-opaque material deposited uniformly throughout the matrix portion of the glyoxysomes, with little background deposition elsewhere in the cell. The reaction product is easily visualized in plastic sections by phase microscopy without poststaining. Although the method has been applied thus far only to cotyledons of fat-storing seedlings, it is anticipated that the technique will be useful in localizing and studying glyoxylate cycle activity in a variety of tissues from both plants and animals.

It is shown that the generation of the insoluble precipitate, copper ferrocyanide, provides a distinct, rapid, and sensitive method for localizing malate synthase in polyacrylamide gels. Both enzymes of the glyoxylate cycle, isocitrate lyase and malate synthase, can now be specifically stained using the same polyacrylamide gel system. This can contribute substantially to elucidating the developmental or adaptive patterns of the glyoxylate cycle and in determining the degree of homology of glyoxylate cycle enzymes from different organisms or organelles. These techniques also provide a simple method for establishing the occurrence of isoenzymes. Finally, the ability to detect a reaction capable of reducing ferricyanide to form cooper ferrocyanide suggests that this technique may be useful for localization of many of the enzymes which generate CoASH (18,19).

The isocitrate lyase from a thermophilic Bacillus is activated about threefold by a variety of salts. Such strong stimulation of activity is not seen with isocitrate lyase from the mesophiles, Bacillus licheniformis, Bacillus megaterium, Escherichia coli , and Aspergillus nidulans . The salt activation is markedly pH-dependent. At pH values above 8.6, salt (KCl) indeed inhibits the enzyme activity. Potassium chloride also causes a significant shift of the pH optimum of the enzyme towards the acid side. As the temperature of the enzyme reaction is raised, activation becomes progressively weaker. Potassium chloride also affords considerable protection against enzyme denaturation at 55 C. The activation and the stabilization, however, appear to be independent effects. Of six other enzymes in the thermophile that were examined, isocitrate dehydrogenase was equally strongly activated by KCl and malate synthase was less strongly, but significantly, activated; citrate synthase, malate dehydrogenase, glutamate dehydrogenase, and lactate dehydrogenase were unaffected or slightly inhibited by KCl. The property of being strongly activated by salt appears to be a peculiar characteristic of the thermophile isocitrate lyase and possibly evolved concomitantly with its thermostability.

The glucose-utilizing mutantHydrogenomonas strain H16G+ differs from the original strain H16 in having a higher specific activity of glucose-6-phosphate dehydrogenase. During incubation of the original strain or of the mutant H16G+ in a mineral salts/fructose-medium under an atmosphere of 80% H2 + 20% O2, neither growth nor formation of the enzymes of the Entner-Doudoroff system occur. Molecular hydrogen represses the formation of these enzymes even in the presence of carbon dioxide, peptone, or lactate. Under air, the formation of the enzymes of the Entner-Doudoroff pathway is not repressed by lactate nor by acetate, glutamate or pyruvate. In strain H16G+ fructose suppresses the adaptation to glucose; glucose does not repress the formation of a fructose permease. Fructose also suppresses adaptation to and utilization of glutamate and aspartate, but not of lactate. In cells grown either chemolithotrophically or on fructose acetyl-CoA kinase, malate synthase and isocitrate lyase are rapidly formed under air after addition of acetate; the formation of these enzymes is also completely suppressed by molecular hydrogen or fructose.

Tetrahymena pyriformis cells were grown axenically, harvested in the stationary phase of the cultures when the organism is glyconeogenetically active, homogenized, and fractionated either by isopycnic density gradient centrifugation in a sucrose gradient or by zonal differential sedimentation through a sucrose gradient. Protein and enzyme determinations carried out on the fractions led to the following conclusions. (a) The mitochondria, characterized by a median equilibrium density in aqueous sucrose of 1.21 to 1.22 and by a median sedimentation coefficient in 0.25 m sucrose at 0° of the order of 2 x 104 S, contain about 20% of the total protein, and account for the bulk of the respiratory activity of the homogenates with all tricarboxylic acid cycle intermediates as substrates. They contain most or all of the citrate synthase, succinate dehydrogenase, fumarase, and malate dehydrogenase activities of the cells, half or more of the aconitase activity, and small but significant amounts (about 5%) of the NADP-linked isocitrate dehydrogenase. (b) The peroxisomes, with a median equilibrium density in aqueous sucrose of 1.24 to 1.25 and a median sedimentation coefficient in 0.25 m sucrose at 0° of 4 to 5 x 103 S, contain the bulk of the catalase, d-amino acid oxidase (previous work), l-α-hydroxy acid oxidase, glyoxylate oxidase, isocitrate lyase, and malate synthase activities, as well as about half of the NADP-linked isocitrate dehydrogenase, in association with less than 10% of the total protein. (c) The lysosomes are the main bearers of acid phosphatase and other acid hydrolases, contain a small proportion of the total protein, have a median equilibrium density in aqueous sucrose of about 1.14, and comprise a fast moving component sedimenting more rapidly than the mitochondria and a slowly moving component sedimenting together with the peroxisomes. Of the enzymes mentioned, only aconitase and the NADP-linked isocitrate dehydrogenase occur definitely in the soluble phase of the cytoplasm, being recovered to the extent of up to 50% with the supernatant fraction of the homogenates. Small amounts of several other enzymes and larger amounts of acid hydrolases also are present in this fraction, but breakage of particles could be responsible for this finding. The significance of these results is discussed in reference to the partition of important metabolic pathways between the mitochondria and the peroxisomes.

Two malate synthases are known to occur in Escherichia coli: one of them malate synthase A allows, with isocitritase, the growth on acetate, the other malate synthase G, the growth on glycolate as sole carbon sources. Mutants impaired in their growth on acetate (aceA, aceB) or on glycolate (glc) are characterized by their, growth rate on various carbon sources, by specific activities of isocitritase and malate synthase and by their relative content in each type of the two malate synthases. aceA and aceB mutants are deficient in isocitritase and malate synthase A respectively; mutant glc lacks the malate synthase G. The aceB mutation impairs growth on acetate only when present in a gle mutated strain. In strains of wild type for the genes concerned, growing on acetate, malate synthase A constitutes approximately 60% of the total activity of malate synthase, the rest being malate synthase G; on glycolate, the activity is nearly entirely represented by malate synthase G. Isocitritase and malate synthase A activities appear in similar relative amounts through different growth conditions, particularly, during growth on limited carbon source (chemostat); in such conditions, they allow the growth of a glucose negative strain (glu) even on glucose as sole carbon source. The location of aceA, aceB and glc mutations on the genetic map are established by sexual crosses and by phage 363 transductions. The frequencies of cotransduction between aceA or aceB and known loci (metB, glu-1, argH2, purD, metA, malB) completed by three point test analysis, lead to a detailed genetic map of this region (Fig. 3); aceA and aceB are located close together, between metA and malB. The glc mutation (deficiency in malate synthase G) happened in the strain HfrDV21 simultaneously with a translocation of the sexual factor of the parent strain Hfr 30SOMA4. The glc locus and the sexual factor of this new Hfr are located between the known loci argG and his, close to the marker serA with which glc is cotransducible. HfrDV21 transfers the genetic markers pheB, purC and his in the same order as the Hfr KL16 but approximately 8 minutes earlier. The results, together with the properties of the strain constitutive for isocitritase and malate synthase A, point to the occurrence of an ace operon (aceA, aceB) regulated by repression-derepression. Malate synthase G seems to be so easily inducible (by glyoxylate?) that it allows growth on acetate of a strain deficient in malate synthase A. The regulatory properties and physiological roles of the two isoenzymes suggest that the inducible malate synthase G would be more primitive than the repressible malate synthase A.

Summary: A study has been made in methanol- and ethanol-grown Hyphomicrobium x of the specific activities of the following enzymes: serine hydroxymethyltransferase, serine-glyoxylate aminotransferase, hydroxypyruvate reductase, glycerate kinase, phosphopyruvate hydratase, phosphopyruvate carboxylase, serine dehydratase, malate dehydrogenase (decarboxylating), phosphopyruvate synthase, phosphopyruvate carboxykinase, glycerate phosphomutase, malate dehydrogenase, malate lyase (CoA acetylating-ATP cleaving), citrate synthase, aconitate hydratase, isocitrate lyase and malate synthase. It was concluded that during growth on ethanol the glyoxylate cycle operated, while during growth on methanol, C1 units and glyoxylate were converted to malate by the serine pathway. The glyoxylate was regenerated by cleavage of malate into glyoxylate and acetyl-CoA. The acetyl-CoA was itself oxidized to glyoxylate by some of the reactions of the tricarboxylic acid cycle and isocitrate lyase, thus permitting net synthesis of C3 and C4 compounds from methanol and carbon dioxide.

Wegener, Warner S. (Albert Einstein Medical Center, Philadelphia, Pa.), Henry C. Reeves, and Samuel J. Ajl . Heterogeneity of the glyoxylate-condensing enzymes. J. Bacteriol. 90: 594–598. 1965.—Evidence is presented that the enzymatic condensations of glyoxylate with acetyl-CoA (malate synthase), propionyl-CoA (α-hydroxyglutarate synthase), butyryl-CoA (β-ethylmalate synthase), and valeryl-CoA (β- n -propylmalate synthase) are catalyzed by different enzymes. The possibility that these activities resulted from a single enzyme possessing a broad fatty acid acyl-CoA substrate specificity was ruled out. The latter was suggested by the fact that cells grown on a number of short-chain fatty acids exhibited all the above activities. The conclusion that these reactions are catalyzed by different enzymes is based on the following considerations: (i) the enzymes can be differentially inactivated by heat; (ii) under various growth conditions, where all the condensing enzymes are present, their respective activities do not show a constant ratio, as would be expected if they were catalyzed by a single enzyme; and (iii) under appropriate growth conditions, one or more of these enzymes has been shown to be present to the exclusion of others.

A study was made on the effect of gibberellic acid on photosynthesis, respiration, gluconeogenesis, and development of some enzymes involved in these process in dwarf pea plants. Gibberellic acid-treated shoots have higher rates of photosynthesis and respiration, and the hormone stimulated activities of phosphopyruvate carboxylase and ribulose diphosphate carboxylase in stems and leaves. The small gibberellic acid effect on both enzymes in leaves and the large effect in stems, implies a redistribution of CO 2 -fixing capacity towards internodes of treated plants. Hormonal stimulation of amylase activity was confined to internodes, and cotyledons seem only to play a passive role in development of the growth response. Malate synthase could not be detected in the plant, and this, together with the low levels of isocitrate lyase found suggest that the glyoxylate cycle is inoperative in peas. The hypothesis is advanced that gibberellic acid actively directs glucose supply toward elongating internodes.

The relative contributions of the dicarboxylic acid and the tricarboxylic acid cycles to the oxidative catabolism of glyoxylate in Escherichia coli K-12 were deduced by analysis of mutant strains that were blocked in the formation of glyoxylate carboligase and of malate synthase G (the “glycolate form” of malate synthase). Mutant strains unable to form malate synthase G were unimpaired in their ability to oxidize glyoxylate. Hence, the dicarboxylic acid cycle does not appear to play an essential role in this process. Organisms blocked in the synthesis of glyoxylate carboligase did not oxidize glyoxylate at a detectable rate, indicating that wild-type organisms convert glyoxylate to acetyl-coenzyme A and oxidize it via the tricarboxylic acid cycle. The foregoing evidence indicates that malate synthase G plays an anaplerotic role during growth with glycolate or acetate as the carbon source. The in vivo activity of malate synthase G was not detectable when the intracellular concentration of acetyl-coenzyme A was low, suggesting that this substrate or a closely related metabolite exerts a sensitive positive control over the enzyme. The synthesis of malate synthase G appears to be induced directly by glycolate which may be formed by a constitutive reduced nicotinamide adenine dinucleotide phosphate-dependent glyoxylate reductase in glyoxylate- or acetate-grown cells.

The role of isocitrate lyase during growth on propionate was investigated using three strains (E-26, K-12, and W) of Escherichia coli . The activity of this enzyme during growth on propionate varied with respect to the strain employed, the concentration of propionate added to the mineral salts culture medium, and the phase of growth at which cells were harvested. The growth behavior of E-26 and of K-12 mutants indicates that in these strains of E. coli , the formation of C 4 acids from propionate can be accomplished either by the phosphoenolpyruvate (PEP) carboxylase pathway or by the glyoxylate bypass. In the early phase of growth, pyruvate formed from the oxidation of propionate is metabolized primarily via the PEP carboxylase pathway. In such cultures, the activity of isocitrate lyase and malate synthase is low. As growth proceeds, there occurs a shift in metabolism, such that the carboxylase pathway is inhibited and the decarboxylation of pyruvate to form acetyl-CoA is stimulated. In such cultures, the formation of the enzymes of glyoxylate bypass is derepressed. In contrast to E-26 and K-12, the formation of isocitrate lyase is not derepressed in E. coli W cultured on propionate.

SUMMARY: The derepression and localization of the glyoxylate cycle enzymes in Coprinus lagopus (sensu Buller) have been studied. Differential and sucrose density gradient centrifugation of extracts indicated a particulate localization for isocitrate lyase (EC. 4.1.3.1) and malate synthase (EC. 4.1.3.2). The particle is distinct from, and less dense than, the mitochondrion and is considered to be a glyoxysome.

Microbodies from rat liver and a variety of plant tissues were osmotically shocked and subsequently centrifuged at 40,000 g for 30 min to yield supernatant and pellet fractions. From rat liver microbodies, all of the uricase activity but little glycolate oxidase or catalase activity were recovered in the pellet, which probably contained the crystalline cores as many other reports had shown. All the measured enzymes in spinach leaf microbodies were solubilized. With microbodies from potato tuber, further sucrose gradient centrifugation of the pellet yielded a fraction at density 1.28 g/cm3 which, presumably representing the crystalline cores, contained 7% of the total catalase activity but no uricase or glycolate oxidase activity. Using microbodies from castor bean endosperm (glyoxysomes), 50–60% of the malate dehydrogenase, fatty acyl CoA dehydrogenase, and crotonase and 90% of the malate synthetase and citrate synthetase were recovered in the pellet, which also contained 96% of the radioactivity when lecithin in the glyoxysomal membrane had been labeled by previous treatment of the tissue with [14C]choline. When the labeled pellet was centrifuged to equilibrium on a sucrose gradient, all the radioactivity, protein, and enzyme activities were recovered together at peak density 1.21–1.22 g/cm3, whereas the original glyoxysomes appeared at density 1.24 g/cm3. Electron microscopy showed that the fraction at 1.21–1.22 g/cm3 was comprised of intact glyoxysomal membranes. All of the membrane-bound enzymes were stripped off with 0.15 M KCl, leaving the "ghosts" still intact as revealed by electron microscopy and sucrose gradient centrifugation. It is concluded that the crystalline cores of plant microbodies contain no uricase and are not particularly enriched with catalase. Some of the enzymes in glyoxysomes are associated with the membranes and this probably has functional significance.

SYNOPSIS. The glyoxylate cycle operates at a high level in Euglena gracilis when acetate is the only carbon source, and at a low level when glucose is the only carbon source, as indicated by activities of malate synthase. Altho glucose causes a moderate repression of some of the enzymes of the glyoxylate cycle, it neither represses nor inhibits malate synthase. The specific activity of the malic enzyme was about 5-fold greater in acetate-grown Euglena than in glucose-grown cells, but the absolute rate of CO2 fixation was about twice as great in cells grown on glucose. The respiratory quotient was unity regardless of substrate.

An investigation was undertaken to assess the formation of several glyoxylate condensing enzymes during growth on a series of short-chain fatty acids. When Escherichia coli was grown on acetate, high malate synthase and low β- n -propylmalate synthase activities were observed. Cells grown on either propionate or butyrate possessed high malate synthase activity, and in addition formed low levels of α-hydroxyglutarate, β-ethylmalate and β- n -propylmalate synthase activity. In contrast, growth on valerate effected high activity of all these glyoxylate condensing enzymes. Evidence is presented that valerate does not act to induce or derepress enzyme formation, but rather acts to select a mutant population which is constitutive for the formation of these enzymes. Such mutants also are able to grow with a much shorter lag on propionate, butyrate, valerate, caproate and heptanoate. The possible relationship between formation of these enzymes and growth on short-chain fatty acids is discussed.

Malate synthetase has been purified some 300-fold from crude extracts of germinating castor-bean seedlings. The enzyme was completely inactive in the absence of divalent cations and was maximally activated by Mg++. The Km for acetyl coenzyme A was 6.7 · 10−5M and that for glyoxylate was 8.5 · 10−5M. None of a variety of aldehydes substituted for glyoxylate. No evidence was obtained that the enzyme could catalyze the breakdown of malate.

Reeves, Henry C. (Albert Einstein Medical Center, Philadelphia, Pa.) and Samuel J. Ajl . Function of malate synthetase and isocitritase in the growth of bacteria on two-carbon compounds. J. Bacteriol. 83: 597–601. 1962.— Escherichia coli , grown in a glucose-citrate medium under anaerobic conditions, is devoid of the enzymes malate synthetase and isocitritase. It has been demonstrated that the adaptation of such cells to aerobic growth in a medium containing acetate as the sole carbon source involves at least two separate events. The first appears to be an adaptation which permits the oxidative dissimilation of acetate and involves primarily the synthesis of condensing enzyme. The second, which also precedes all division, is the synthesis of isocitritase and malate synthetase. Finally, a definite correlation has been found to exist between the synthesis of isocitritase and malate synthetase and the lag period prior to cell proliferation.

1. 1. The presence of iso citrate lyase and of malate synthase has been demonstrated in adult Fasciola hepatica . All enzymes of the glyoxylate cycle are therefore present, as those enzymes common with the tricarboxylic acid cycle have been previously demonstrated in this organism. 2. 2. Fructose-1,6-diphosphatase is present in the adult fluke and as phosphoenolpyruvate carboxykinase and the necessary glycolytic enzymes are also present, the complete complement of enzymes required for hexose phosphate formation from dicarboxylic acids is available in F. hepatica . 3. 3. As is the case with the mammalian fructose-1,6-diphosphatase, the fluke enzyme with maximal activity at pH 9 requires the presence of Mg 2+ and EDTA for optimal activity and AMP inhibits this activity. 4. 4. C 14 -2,3-succinate is incorporated into glycogen. The possible significance of such glyconeogenic activity, and of the glyoxylate cycle in F. hepatica are discussed.

The levels of Krebs cycle, glyoxylate cycle, and certain other enzymes were measured in a wild-type strain and in seven groups of acetate-nonutilizing ( acu ) mutants of Neurospora crassa , both after growth on a medium containing sucrose and after a subsequent 6-hr incubation in a similar medium, containing acetate as the sole source of carbon. In the wild strain, incubation in acetate medium caused a rise in the levels of isocitrate lyase, malate synthase, phosphoenolpyruvate carboxykinase, acetyl-coenzyme A synthetase, nicotinamide adenine dinucleotide phosphate-linked isocitrate dehydrogenase, citrate synthase, and fumarate hydratase. Isocitrate lyase activity was absent in acu-3 mutants; acu-5 mutants lacked acetyl-coenzyme A synthetase activity; and no oxoglutarate dehydrogenase activity (or only low levels) could be detected in acu-2 and acu-7 mutants. In acu-6 mutants, phosphoenolpyruvate carboxykinase activity was either very low or absent. No specific biochemical deficiencies could be attributed to the acu-1 and acu-4 mutations. The role of several of these enzymes during growth on acetate is discussed.

A procedure was developed for the isolation of membranes of glyoxysomes from the endosperm of germinating castor beans (Ricinus communis L. var. zanzibariensis). These membranes were prepared by osmotic-shock treatment of isolated glyoxysomes. In some cases this was followed by mild sonication. They were isolated by centrifugation on to a 57% (w/w) sucrose layer and purified by isopycnic sucrose gradient centrifugation. The membrane preparations were characterized by electronmicroscopy and assayed for enzyme activities. They consisted mainly of ghost-type vesicles and were found to contain a major part of fatty acid β-oxidation, malate synthase, citrate synthase and antimycin-A-insensitive NADH-cytochrome c reductase activities of the whole glyoxysomes. Malate dehydrogenase may also be bound to the membrane to a significant extent. A tentative scheme is postulated describing the structural organization of fatty acid β-oxidation and of the glyoxylate cycle in glyoxysomes.

Results are presented on the intracellular localization of some of the enzymes of gluconeogenesis, of the tricarboxylic acid cycle and of related enzymes in Astasia and Euglena grown with various substrates. The results indicate the particulate nature of at least part of the malate synthase of Astasia and of part of the malate synthase and isocitrate lyase in Euglena. However, the presence of glyoxysomes (microbodies) in Astasia and Euglena is still open to question, since it has not, so far, been possible to separate the enzymes of the glyoxylate cycle from succinate dehydrogenase in the particulate fraction.

The presence of isocitrate lyase and malate synthase was detected in cell-free extracts ofAcetobacter aceti, grown in a mineral medium with acetate as sole carbon source. The presence of these enzymes explains the ability of this strain to grow with ethanol or acetate as sole carbon source, which is an important characteristic in Frateur's classification system forAcetobacter. In addition to isocitrate lyase and malate synthase, these cell-free extracts were found to contain glyoxylate carboligase, tartronicsemialdehyde reductase and glycerate kinase. The induction of these enzymes during growth on acetate is thought to be caused by the very high activity of isocitrate lyase, which may lead to an accumulation of glyoxylate. The importance of this pathway in cells growing with acetate as sole carbon source for the synthesis of their carbohydrate components is discussed. The presence of the enzymes from the pathway from glyoxylate to 3-phosphoglycerate explains the ability of this strain to grow with ethyleneglycol and glycollate as sole carbon source.

Peroxisomes from spinach leaves contain three aminotransferases, a glutamate-glyoxylate, a serine-pyruvate and an aspartate aminotransferase. Five other enzymes previously described in leaf peroxisomes were measured, namely, catalase, glycolate oxidase, NADH-hydroxypyruvate reductase, and NAD-malate dehydrogenase. A NADPisocitrate dehydrogenase was also associated with isolated leaf peroxisomes, but its specific activity was much less than the other enzyme. Most of the NADP-isocitrate dehydrogenase activity was soluble. Serine-pyruvate aminotransferase activity in the particulate fraction was localized entirely in the peroxisomes. Isoenzymes of asparate aminotransferase were found in chloroplasts, mitochondria, and peroxisomes. The activities of the chloroplast and mitochondrial forms of this aminotransferase were greater than the peroxisomal isoenzyme. The two peroxisomal serine-pyruvate and aspartate aminotransferases were separated by disc electrophoresis and isoelectric focusing. The aminotransferases had different substrate specificity. Other enzymes of the tricarboxylic acid cycle, with the exception of an isoenzyme of NAD-malate dehydrogenase, were detected in mitochondria but not in the leaf peroxisomal fractions. Enzymes for the reductive amination of α-keto acids, the interconversion of C4 and C3 acids, hydroxyaspartate dehydratase, and the α and β oxidation systems for fatty acids could not be detected in leaf peroxisomes. Preliminary evidence is presented for a malate synthase in leaf mitochondria.

Howes, William V. (Washington State University, Pullman) and Bruce A. McFadden . Isocitrate lyase and malate synthase in Pseudomonas indigofera . II. Enzyme changes during the phase of adjustment and the early exponential phase. J. Bacteriol. 84: 1222–1227. 1962.—The differential rates of synthesis (DRS) of malate synthase and isocitrate lyase were investigated at 30 C during the phase of adjustment (lag) and the early exponential phase in Pseudomonas indigofera . Cells grown on ethanol-acetate-yeast extract or succinate-yeast extract were added to a medium containing ethanol or succinate, and the DRS of each enzyme was followed. A reproducible decline in both enzymatic activities occurred after inoculation when a lag in growth was observed. To define the DRS occurring after this decline, a new parameter, ε, was defined and calculated for linear segments of a DRS plot. Certain variations of ε during DRS suggested that a balance between induction (or derepression) and repression controls, in part at least, malate synthase and isocitrate lyase levels. Effects of chloramphenicol upon enzyme synthesis after the period of expected decline were studied. No degradation or synthesis of malate synthase occurred in the presence of chloramphenicol, although, after degradation, slight synthesis of isocitrate lyase occurred. Acetate, butyrate, ethanol, glyoxylate, and isocitrate did not stimulate production of either enzyme in nongrowing cultures of high cell density. In fact, some degradation of each enzyme occurred.

The high level of lipids in seeds of some species of conifers suggested that the glyoxylate cycle might have a role in conifer seed metabolism.Six species (Pinus pinea, Pinus pinaster, Pinus canariensis, Pinus strobus, Abies alba, and Cupressus sempervirens) were investigated for their lipid content and malate synthase and isocitrate lyase level. The fatty acid composition of the triglyceride fraction was also investigated. The correlation between lipid content of germinating seed with the presence of the cycle was confirmed. The enzymes of the glyoxylate cycle were not detected in Cupressus sempervirens where the lipid content is very low.

1 By chemical synthesis starting from phenylacetylene and utilizing stereospecific reactions, two specimens of acetic acid were prepared in which the following conditions were closely approached: (a) some of the methyl groups consisted of one hydrogen, one deuterium, and one tritium atom attached to carbon; (b) all such methyl groups in one specimen had the R configuration, and all such methyl groups in the other specimen had the S configuration, and the correct absolute configuration was assignable to each specimen from the method of synthesis used; (c) all methyl groups containing a tritium atom also contained a deuterium atom. 2 Each specimen of acetic acid was converted enzymically into acetyl-coenzyme A and (in the same incubation) this product was condensed with glyoxylic acid on malate synthase to form two specimens of S-malic acid. 3 Each specimen of malic acid was incubated with fumarase until the tritium content of the organic acids (fumarate + malate) had ceased to fall. 4 Malic acid synthesized from R-acetic acid retained 69% of its carbon-bound tritium (in fumarate + malate) on incubation with fumarase. Malic acid synthesised from S-acetic acid retained 31% of its carbon-bound tritium. 5 The enzymic assay described in paragraphs 2–4 constitutes a method for determining the absolute configuration of asymmetric methyl groups, provided that a substantial proportion of the tritium in the specimen examined is present in such groups. 6 The method thus established can be used to study the mechanism of enzymic reactions in which a methylene group is converted stereospecifically into a methyl group. With one logical proviso–that it is necessary to known whether protium or deuterium is preferentially removed by the enzyme from methyl groups also containing tritium–the method is also applicable to enzymic reactions in which a methyl group is converted stereospecifically into a methylene group. 7 Subject to the above proviso, it is indicated that on malate synthase the removal of hydrogen and the attachment of the glyoxylate residue occurs with inversion of configuration.

The kinetics of derepression of isocitrate lyase, malate synthase, phosphoenolpyruvate carboxykinase, citrate synthase, fumarate hydratase, and malate dehydrogenase were studied following transfer of mycelium grown on a medium containing sucrose to one containing acetate as sole source of carbon. The maximum rate of isocitrate lyase synthesis was proportional to the number of isocitrate lyase structural genes. Comparisons of the repressed levels of isocitrate lyase and malate synthase on sucrose medium and the derepressed levels in acetate medium showed an over-all coordination between the levels of these two enzymes. A similar relationship existed between the levels of fumarate hydratase, citrate synthase and malate dehydrogenase. The Krebs cycle enzymes also showed coordinate repression on sucrose medium under conditions of enhanced catabolite repression. Other results, however, showed that neither for the glyoxylate shunt enzymes nor for the Krebs cycle enzymes was this coordination invariable. Under conditions in which some of the Krebs cycle enzymes assumed different roles no coordination was observed. It is suggested that this type of coordination, which is frequently observed in the pathways of central carbon metabolism in eucaryotes, results from the metabolic repressors of each enzyme being in equilibrium or in a constant ratio to one another. The coordination does not arise because the structural genes of each enzyme belong to the same operon.

Summary In Saccharomyces cerewsiae the synthesis of alcohol dehydrogenase (EC 1.1.1.1), malate dehydrogenase (EC 1.1.1.37), isocitrate lyase (EC 4.1.3.1) and malate synthase (EC 4.1.3.2) is repressed by glucose. All these enzymes participate in the formation of carbohydrates from alcohol. Therefore the “glucose effect” is useful for repressing ghiconeogenesis, which is not necessary with glucose in the growth-medium. In the case of malate dehydrogenase, glucose effects not only a repression of the synthesis but also a rapid decrease of enzyme activity. 2-Deoxyglucose and glucosamine have the same repressing effect on enzyme synthesis as glucose. Neither substance is catabolized in yeast; they are only phos-phorylated by adenosine 5′-triphosphate. Therefore we can assume that repression of the synthesis of alcohol dehydrogenase, malate dehydrogenase, isocitrate lyase and malate synthase is not caused by metabolites but by glucose itself, glucose 6-phos-phate, or perhaps by changes in the concentrations of adenosine 5'-triphosphate or -diphosphate.

SUMMARY: The respiratory physiology of Euglena gracilis grown heterotrophically on a defined medium was examined as a function of culture age when growth was supported by glucose, acetate or ethanol as sole carbon source. The endogenous rate of oxygen consumption in general paralleled that of total protein content, usually showing a steady decline through log. and stationary phases of growth. The rate of oxygen consumption stimulated by ethanol or acetate remained fairly constant during log. phase but. decreased sharply in early stationary phase. The specific activities of representative respiratory enzymes remained essentially proportional to one another and to total oxygen consumption, but the activities of malate synthase and the malic enzyme increased greatly in stationary phase glucose-grown organisms. In general, respiratory growth was out of balance with the rate of cell division but in essential balance with the rate of biosynthesis.

1. 1. Carbohydrate and lipid reserves decreased when Panagrellus redivivus and Turbatrix aceti were starved. 2. 2. The enzymes of β-oxidation and the tricarboxylic acid cycle were present, and their functional activity was indicated by the ability of both species to oxidize absorbed 14C-U-palmitic acid to CO2. 3. 3. T. aceti, and to a lesser extent P. redivivus, also incorporated fatty acid carbon into glycogen. The presence of the glyoxylate cycle enzymes, isocitrate lyase and malate synthase, and of the gluconeogenic enzymes phosphoenolpyruvate carboxykinase and fructose-1,6-diphosphatase, constitute a possible mechanism for this conversion.

Evidence for the presence of phosphate transacetylase and malate synthase in Azotobacter vinelandii is presented. The latter enzyme completes the requirements for the operation of the glyoxylate cycle in this organism. Malate synthase and phosphate transacetylase did not show a requirement for thiol activation, while EDTA substituted for cysteine as an activator for acetate kinase. The oxygen sensitivity of acetate kinase, phosphate transacetylase, isocitrate lyase and malate synthase has been examined with crude cell-free extracts. In 6 h, these enzymes lost 45, 94, 32 and 42% of their activity, respectively, when exposed to 1.0 amt oxygen. The relation of these losses to the oxygen inhibition of acetate oxidation in A. vinelandii has been discussed.

1. The enzymes in ultrasonically prepared extracts of Chloropseudomonas ethylicum were studied to elucidate how this organism assimilates acetate and carbon dioxide and why it cannot grow with either of these two compounds alone. 2. Such extracts can (i) convert acetate and oxaloacetate into α-oxoglutarate, (ii) convert oxaloacetate into succinyl-CoA, (iii) convert phosphopyruvate into 3-phosphoglyceraldehyde and (iv) interconvert phosphopyruvate and pyruvate via oxaloacetate. 3. Pyruvate kinase, α-oxoglutarate dehydrogenase, ribulose diphosphate carboxylase, isocitrate lyase and malate synthase were not detected. 4. It is difficult to detect aconitate hydratase, fumarate hydratase and citrate synthase in extracts of the organism ultrasonically treated in tris buffer; to demonstrate these enzymes extracts should be prepared in phosphate buffer containing 2-mercaptoethanol. 5. Provided that this organism can synthesize pyruvate from acetate and carbon dioxide, the enzymes detected are sufficient to account for the nutritional requirements of this organism.

Ergot alkaloids are formed only for arelatively brief time during the lifespan of the culture and under conditions of reduced proliferation. They cannot be taken for waste products of general metabolism. Ergot strains are capable of carrying out all the simple steps of the anthranilic acid—tryptophan cycles. Alkaloids influence activities of certain enzymes of primary metabolism in the ergot mycelium,e.g. tryptophan synthetase, acetyl-CoA carboxylase, citrate synthase, isocitrate lyase, and malate synthase. Ergot alkaloids do not belong to a group of physiologically inert secondary metabolites. A tentative scheme of the enzymic assembly of the ergoline nucleus is presented. The increased yield of ergoline alkaloids may be attributed to the following points: (1) Unbalnced growth of the culture. (2) Support of competition of fatty acids and alkaloid biosynthesis for acetyl-CoA. (3) Decreased activities of tricarboxylic acid and glyoxylate cycles. (4) Positive association between the rate of protein turnover and alkaloid formation. (5) Stimulation of both tryptophan synthesis and degradation via kynurenine—anthranilate. (6) Regulation of tryptophan-histidine cross-pathway. (7) Continuous control of the alkaloid level during fermentation.

The metabolism of propane and propionate by a soil isolate (Brevibacteriumsp. strain JOB5) was investigated. The presence of isocitrate lyase in cells grown on isopropanol, acetate, or propane and the absence of this inducible enzyme inn-propanol- and propionate-grown cells suggested that propane is not metabolized via C-terminal oxidation. Methylmalonyl coenzyme A mutase and malate synthase are constitutive in this organism. The incorporation of14CO2into pyruvate accumulated during propionate utilization suggests that propionate is metabolized via the methyl-malonyl-succinate pathway. These results were further substantiated by radiorespirometric studies with propionate-1-14C, -2-14C, and -3-14Cas substrate. Propane -2-14Cwas shown, by unlabeled competitor experiments, to be oxidized to acetone; acetone and isopropanol are oxidized in this organism to acetol. Cleavage of acetol to acetate and CO2would yield the inducer for the isocitrate lyase present in propane-grown cells.

1. 1. It is known that stored trehalose and glycogen are metabolized during the first 10 days of development of Ascaris lumbricoides eggs and are completely resynthesized from triglyceride fatty acids during the next 15 days. It has now been found that changes in the specific activities of the glyoxylate cycle enzymes, isocitrate lyase and malate synthase closely parallel changes in the rate of carbohydrate resynthesis as well as changes in the rate of incorporation of label from absorbed U-14C-palmitate in both trehalose and glycogen. 2. 2. The gluconeogenic enzymes phosphoenolpyruvate carboxykinase and fructose-1,6-diphosphatase are present throughout development. 3. 3. The glyoxylate cycle and the conversion of th eresulting 4-carbon dicarboxylic acids to trehalose and glycogen account for the unique synthesis of carbohydrate from higher fatty acids. 4. 4. Neither isocitrate lyase nor malate synthase could be detected in homogenates of adult A. lumbricoides muscle.

A simple procedure is reported for the purification of the two malate synthases of Escherichia coli involved in the use of acetate (A) and glycolate (G), respectively, as carbon source. Both isoenzymes have been isolated simultaneously and in equally high amounts from a strain constitutive for the glyoxylate cycle, after growth on glycolate. After separation from each other by column chromatography on DEAE-cellulose, the malate synthases have been purified by heating, ammonium sulfate precipitation and molecular sieve chromatography on a Sephadex G-200 column, in the constant presence of their specific protectors, glycolate for malate synthase A, and oxalate for malate synthase G. The purified preparations so obtained appeared to be homogeneous on the basis of analytical ultracentrifugation and moving-boundary electrophoresis. The sedimentation coefficient at infinite dilution was 4.6 S for malate synthase A and 5.2 S for malate synthase G; their electrophoretic mobilities at pH 8 were 4.3 X 10−5 cm2 V−1 s−1 and 4.1 × 10−5 cm2 V−1 s−1, respectively. The apparent molecular weight of malate synthase A, estimated by Archibald's method, was 54000; that of malate synthase G, estimated by light scattering, was 52000. Threonine and serine have been found, by Sanger's method, to be N-terminal residues of malate synthases A and G, respectively.

When cells of E. coli 64 were harvested in their exponential phase of growth in an acetate medium and incubated aerobically with sodium acetate-2-C14, about 33% of the label appeared in CO2 after 1 hr. Of the radioactivity in the cells, 72% was recovered in the protein hydrolysate, 8% in the nucleic acid, 6% in the lipid and 14% in the ethanol-soluble fractions. The radioactivity in the protein hydrolysate of cells incubated with sodium acetate-2-C14 was approximately 20 times that in the hydrolysate of cells incubated with C14O2 as the carbon source. By spectrophotometric methods, it was demonstrated that cell-free extracts of cells grown on acetate contained acetate kinase and phosphate acetyltransferase, plus, as demonstrated by spectrophotometric and isotopic methods, isocitrate lyase and malate synthase which are characteristic of the glyoxylate cycle. The enzymes of the glyoxylate cycle could not be demonstrated in cell-free extracts of E. coli 64 grown on glucose under either aerobic or anaerobic conditions. Possible functions that E. coli 64 may have in the maintenance of anaerobiosis in the rumen and utilization of acetate through the glyoxylate pathway are discussed.

During an investigation of the carbon-assimilation pathway of C1 compounds by the facultative methylotroph bacterium 5H2 (Hampton & Zatman, 1973) isocitrate lyase (EC 4.1.3.1) activities in cell-free extracts of organisms grown on a variety of substrates were measured by the method of Dixon & Kornberg (1959). The method of Kramer et a/. (1959) was used to confirm that a product of the reaction was glyoxylate, the phenylhydrazone of the latter producing the intensely red-coloured 1,Sdiphenylformazancarboxylic acid on treatment with ferricyanide. The isocitrate lyase specific activities of bacterium 5H2 grown on tetramethylammonium chloride, trimethylamine, dimethylamine, methylamine, and on acetate, were respectively, 72, 84, 103, 95 and 150munits/mg of protein, whilst the values for extracts of cells grown on fructose, glutamate and on glycerol were respectively 1.9, I .5 and 2.0munits/mg of protein. These results suggest that isocitrate lyase is important for the inethylotrophic growth of bacterium 5H2 and, not unexpectedly, for growth on acetate. High specific activities of malate synthase (EC 4.1.3.2), measured by method of Dixon & Kornberg (1959), were found in cell-free extracts of bacterium 5H2 grown on CI as well as non-C1 substrates. The results of the following induction experiment confirmed the importance of isocitrate lyase for the growth of bacterium 5H2 on trimethylamine. Glycerol-grown cells, shaken at 30°C in a mineral medium to which 0.2% trimethylarnine had been added, showed no significant growth for the first 6h but the specific activity of isocitrate lyase increased from 2.0 to approximately 45 munits/mg of protein during this period. The specific activity of isocitrate lyase reached a maximum value of approximately lOOmunits/mg of protein about 3 h later, by which time the bacteria were growing exponentially. In a simultaneous experiment in which glycerol-grown cells were shaken at 30°C in the mineral medium containing 0.2% glycerol, exponential growth occurred immediately and there was no increase in the specific activity of the isocitrate lyase throughout the experiment. Present evidence (Salem et al., 1972; Dunstan el a[., 1972; Quayle, 1972) suggests that, in organisms which use the serine pathway of C1 assimilation, regeneration of the glycine acceptor for C1 units arises via the cleavage of a C4 metabolite. During a search for such a C, cleavage reaction, it was observed that crude cell-free extracts of trimethylamine-grown bacterium 5H2 (French press, 35000g supernatant) catalyse, in the presence of hydroxylamine, an ATP-, CoAand Mgz+-dependent formation of acetylhydroxamate from L-malate. Further investigation indicated that the reaction is mediated by a malate cleavage enzyme (ATP malate lyase) which splits L-malate to acetylCoA and glyoxylate and which is similar to the malate lyase cleavage activity described by Tuboi & Kikuchi (1963) in Rhodopseirclomorias spheroicks grown phototrophically with malate. Our reaction mixture ( I ml total volume) contained (pniol): Tris-HC1 buffer pH7.8, 200; ATP, 10; CoA, 0.1; MgCI2, 5 ; L-malate, 100; NH20H,HC1 freshly neutralized, 500; crude cell-free extract (I-2mg of protein). This was incubated without shaking at 30°C, usually for 30min, the reaction being stopped by heating at 100°C for 2min, and a sample of the supernatant being chromatographed on paper by the method of Tuboi & Kikuchi (1962) to identify the hydroxarnates. Acetyl-, succinyland malyl-hydroxamic acids for marker spots on the chromatograms were prepared from their respective anhydrides by the method of Lipmann & Tuttle (1945). A measure of the total hydroxamic acids in the reaction mixture was obtained by the FeCI, method of Lipmann & Tuttle (1945). The major FeCI,-positive spot on the chromatogranis was identified as acetylhydroxamic acid, and a minor spot accounting for approximately 5 % of the total hydroxamate, was identified as malylhydroxamic acid. Glyoxylate production was determined i n reaction mixtures in which the hydroxylamine was

SYNOPSIS. Cell-free extracts of encysting Acanthamoeba were assayed for the key enzymes of the glyoxylate pathway, viz., isocitrate lyase and malate synthase. Both enzymes were present at the onset of encystment but their activities changed as cyst-wall formation proceeded to completion. Isocitrate lyase activity decreased during the first 4 hr of encystment to a minimum at 4 hr which was 70% of its initial activity. Activity then increased reaching a maximum at 9 hr which was 144% of its initial activity. After 9 hr a decrease in isocitrate lyase activity began which reached 70% of its initial activity at 35 hr. Malate synthase activity slowly decreased throughout encystment to 50% of its initial activity after 35 hr. From these data and others cited, it is concluded that this small soil amoeba has a functional glyoxylate pathway.

1. 1. Malate synthase has been identified in insects. 2. 2. Malate formation depended on the presence of both glyoxylate and acetyl-CoA and was linear with time. 3. 3. In the mosquito, activity was very weak at emergence, but developed within 6–8 days in unfed, blood-fed and sugar-fed groups. 4. 4. In the adult roach, fed on high carbohydrate diet, activity was very weak, but was enhanced by starvation and a high fat diet.

Marked differences in the rates of synthesis of several microbody (peroxisome) enzymes have been observed during the growth of Tetrahymena pyriformis (strain E) in shaking or static peptone medium. The specific activities of catalase and NADP + -isocitrate dehydrogenase, and of the mitochondrial enzyme fumarase, remained virtually constant during exponential growth under all conditions examined, whereas that of glycollate oxidase decreased dramatically in the course of growth. In contrast, isocitrate lyase and malate synthase were synthesized at increasing rates during growth on peptone medium with or without added acetate; highest specific activities were reached in late exponential to early stationary phase. The synthesis of these two enzymes, which appears to be coordinately regulated, was repressed in medium supplemented with glucose. Despite these variations in specific activity, glycollate oxidase as well as the key enzymes of the glyoxylate cycle remained associated with microbodies under all growth conditions tested.

Microbodies were observed in the hyphal tips of all 14 fungi investigated. Their morphology varied among the fungi and their numbers were influenced by the growth medium. Microbodies were closely associated with mitochondria in one fungus and with the endoplasmic reticulum in several fungi. Catalase was not detected in microbodies with the diaminobenzidine cytochemical procedure even though catalase activity was present in extracts of these fungi. The activities of the glyoxylate-cycle enzymes isocitrate lyase and malate synthase were affected by the growth medium and were particulate in the two fungi studied by differential centrifugation. Microbodies are abundant, and they are ubiquitous among the fungi and in some cases they may contain glyoxylate-cycle enzymes.