Intracellular location of ATP citrate lyase in leaves of Pisum sativum L.

The aim of this work was to discover if there is enough ATP citrate lyase (EC 4.1.3.8) in the cytosol of the leaves of Pisum sativum L. to catalyse the synthesis of the acetyl CoA needed for terpenoid synthesis. Estimates of the maximum catalytic activity of the enzyme in leaves of 7-d-old peas gave values of 113 nmol min^-1 g^-1 fresh weight. The rate of carotenoid accumulation in these leaves corresponded to a requirement for acetyl CoA of 0.7 nmol min^-1 g^-1 fresh weight. The distribution of marker enzymes during fractionation of homogenates of leaves from 7 to 10-d-old peas showed that differential centrifugation led to the isolation in reasonable yields of chloroplasts, mitochondria, peroxisomes and the endomembrane system. None of the above components of the leaf contained appreciable detectable activity of ATP citrate lyase, the distribution of which closely paralleled that of the cytosolic marker. It was concluded that in young leaves of pea most of the ATP citrate lyase is in the cytosol.     

Apparent Inhibition of ATP Citrate Lyase by l-Glutamate In Vitro Is Due to the Presence of Glutamine Synthetase

Preincubation in assay mixture for 30 min at 37 degrees C of ATP citrate lyase from rat brain and liver results in 65-70% inhibition in the presence of 10 mM L-glutamate. This inhibition is specific since none of the known brain metabolites of glutamate shows this effect. ATP and ammonium sulphate-suspended, commercially purified malate dehydrogenase are both important in the generation of inhibition; citrate and NADH are not. The ATP citrate lyase activity in desalted crude extracts and 11% polyethylene glycol-precipitated fractions is inhibited but the enzyme purified by dye affinity chromatography is unaffected. Such purification reveals the presence of a factor responsible for the generation of the inhibition shown to be of Mr 380,000. These lines of evidence implicate endogenous glutamine synthetase, and the involvement of this enzyme is established by the use of its inhibitor L-methionine sulphoximine and by the addition of purified glutamine synthetase to restore the glutamate inhibition of purified ATP citrate lyase. The phenomenon probably arises from the production by glutamine synthetase of ADP, a known product inhibitor of ATP citrate lyase. Therefore contrary to previous reports elsewhere, L-glutamate has no role in the regulation of brain ATP citrate lyase and thus the supply of cytoplasmic acetyl groups for biosynthesis.     

Plastid targeting and transient expression of rat liver ATP: citrate lyase in pea protoplasts

Protoplasts isolated from pea leaves (Pisum sativum L. cv. Hurst Greenshaft) were electroporated in the presence of plasmid pDR#1, which contains the rat liver ATP:citrate lyase gene fused to a duplex 35S cauliflower mosaic virus promoter with a transit peptide sequence of the Rubisco small subunit. The level of enzyme expression and viability of protoplasts were both influenced by polyethylene glycol treatment before electroporation. Under the optimised electroporation conditions, an average increase of ATP:citrate lyase activity of 14% was observed in the transfected cells after 24 h, with a similar magnitude of change in the abundance of the corresponding mRNA. Immunoblot analysis confirmed the correct expression and targeting of ATP:citrate lyase protein in the chloroplasts of pea protoplasts. These results provide a basis for the establishment of a procedure for targeting heterologous protein into pea plastids in the presence of a transit peptide. Received: 14 June 1996 / Revision received: 24 November 1996 / Accepted: 4 January 1997     

Presence and regulation of ATP:citrate lyase from the citric acid producing fungus Aspergillus niger

ATP:citrate lyase (EC 4.1.3.8) has been identified in cell-free extracts from the filamentous fungus Aspergillus niger. The enzyme was located in the cytosol. It exhibits an activity at least ten times that of acetate-CoA-kinase (EC 6.2.1.1) during growth on carbohydrates as carbon sources, and is thus considered responsible for acetyl-CoA formation under these conditions. It is formed constitutively and its biosynthesis does not appear to be controlled by changes in the nitrogen or carbon source or type. ATP:citrate-lyase appears to be very labile during conventional purification procedures; a method involving fast protein liquid anion exchange chromatography was thus developed in order to obtain enzyme preparations sufficiently free of enzymes which could interfere with kinetic investigations. This preparation displays commonly known characteristics of ATP:citrate lyase with respect to substrate affinities and cofactor requirements, with the exception that the affinity for citrate is rather low (2.5 mM). No activator was found. The enzyme is inhibited by nucleoside diphosphates, nucleoside monophosphates and palmitoyl-CoA. Regulation of ATP:citrate lyase be the energy charge of the cytosol in relation to lipid or citric acid accumulation is discussed in view of these findings. Present address: Institut für Allgemeine Biochemie, Universität Wien, Währingerstrasse 38, A-1090 Wien, Austria     

The Contribution of Citrate to the Synthesis of Acetyl Units in Synaptosomes of Developing Rat Brain

Abstract: The activities of pyruvate dehydrogenase, citrate synthase, and choline acetyltransferase in rat brain synaptosomes increased during on-togenesis by 3 and 14 times, respectively. Activity of ATP-citrate lyase decreased by 26% during the same period. Pyruvate consumption by synapto-somes from 1-day-old animals was 40% lower than that found in older rats; however, citrate efflux from intrasynaptosomal mitochondria in immature synaptosomes was over twice as high as that in mature ones. The rates of production of synaptoplasmic acetyl-CoA, ATP-citrate lyase were 1.03, 1.40, and 0.49 nmol/min/mg protein in 1-, 10-day-old, and adult rats, respectively. 3-Bromopyruvate (0.5 m M ) inhibited pyruvate consumption by 70% and caused a complete block of citrate utilization by citrate lyase in every age group. Parameters of citrate metabolism in cerebellar synaptosomes were the same as those in cerebral ones. These data indicate that production of acetyl-CoA. from citrate in synaptoplasm may be regulated either by adaptative, age-dependent changes in permeability and carrier capacity of the mitochondrial membrane or by the inhibition of synthesis of intramitochondrial acetyl-CoA. ATP-citrate lyase activity is not a rate-limiting factor in this process. Metabolic fluxes of pyruvate to cytoplasmic citrate and acetyl-CoA. are presumably the same in both cholinergic and noncholinergic nerve endings. The significance of citrate release from intrasynaptosomal mitochondria as a regulatory step in acetylcholine synthesis is discussed.     

Characterization of citrate lyase from Clostridium sporosphaeroides

Cells of Clostridium sporosphaeroides which were grown on citrate contained citrate lyase and citrate lyase acetylating enzyme, but no detectable citrate synthase and citrate lyase deacetylase activities. Citrate lyase from C. sporosphaeroides was purified to homogeneity as judged by polyacrylamide gel electrophoresis and high performance liquid chromatography. In contrast to the enzyme from Clostridium sphenoides, the addition of l-glutamate was not necessary for activity and stabilization of the enzyme. The purified enzyme had a specific activity of 34 U/mg protein and was comparable to other citrate lyases with respect to its molecular weight and subunit composition. Electron microscopic investigations showed that similar to the lyase from C. sphenoides and in contrast to all other citrate lyases examined so far, the majority of the enzyme molecules was present in star form.     

A proposed biochemical mechanism for citric acid accumulation by Aspergillus niger Yang No. 2 growing in solid state fermentation

Aspergillus niger Yang No. 2 and its mutant strain SL1 were grown in solid state fermentation. Samples were taken after 2, 4 and 6 days of incubation and the mycelia were analysed for their intracellular concentrations of some organic acids and adenylates and the activities of selected enzymes. Strain Yang No. 2 contained high concentrations of citrate with very little oxalate, while strain SL1 contained lower concentrations of citrate but considerably higher concentrations of oxalate. As the fermentation proceeded, strain Yang No. 2 showed a much higher ratio of ATP:AMP than did strain SL1. In addition, the enzyme ATP:citrate lyase became undetectable during citrate accumulation in strain Yang No. 2, while its activity remained high during oxalate accumulation in strain SL1. It is proposed that citrate accumulation by strain Yang No. 2 during solid state fermentation is due to blockage of its metabolism in the mitochondrion via inhibition of isocitrate dehydrogenase by the high ATP:AMP ratio, and in the cytosol by repression of ATP:citrate lyase activity.     

Relationship between fatty acid binding proteins,acetyl-CoA formation and fatty acid synthesis in developing human placenta

The relationship between fatty acid binding proteins, ATP citrate lyase activity and fatty acid synthesis in developing human placenta has been studied. Fatty acid binding proteins reverse the inhibitory efect of palmitoyl-CoA and oleate on ATP citrate lyase and fatty acid synthesis. In the absence of these inhibitors fatty acid binding proteins activate ATP citrate lyase and stimulate [ 1-¹⁴ C] acetate incorporation into placental fatty acids indicating binding of endogenous inhibitors by these proteins. Thus these proteins regulate the supply of acetyl-CoA as well as the synthesis of fatty acids from that substrates. As gestation proceeds and more lipids are required by the developing placenta fatty acid binding protein content, activity of ATP citrate lyase and rate of fatty acid synthesis increase indicating a cause and efect relationship between the demand of lipids and supply of precursor fatty acids during human placental development.     

Citrate metabolism in anaerobic bacteria

Abstract The regulation of anaerobic citrate metabolism is very diverse among different groups of bacteria. In organisms like Streptococcus lactis and Clostridium sporosphaeroides which lack citrate synthase, the activity of its antagonistic enzyme, citrate lyase, need not be regulated. Many anaerobes like Rhodocyclus gelatinosus and Clostridium sphenoides are able to synthesize their own l -glutamate and contain citrate synthase. In these bacteria the activity of citrate metabolizing enzymes which are involved in a cascade system are under strict control. In Rc. gelatinosus activation/inactivation of citrate lyase is controlled by acetylation/deacetylation which is catalyzed by its corresponding regulatory enzymes, citrate lyase ligase and citrate lyase deacetylase. In C. sphenoides inactivation of citrate lyase is accomplished by deacetylation as well as by changing in the enzyme conformation. Activation of citrate lyase is catalyzed by citrate lyase ligase whose activity in addition is modulated by phosphorylation/dephosphorylation. Further, electron transport process also seems to play a role in the inactivation of citrate metabolizing enzymes in enteric bacteria.     

ATP Citrate lyase from Ipomoea batatas root tissue infected with Ceratocystis fimbriata

ATP citrate lyase was detected in sweet potato (Ipomoea batatas) root tissue infected with Ceratocystis fimbriata. The activity increased strik     

ATP-linked citrate lyase activity in the green sulfur bacterium Chlorobium limicola forma thiosulfatophilum

Cell-free extracts of the green sulfur bacterium Chlorobium limicola forma thiosulfatophilum strains 1C and L have been shown to cleave citrate with the formation of oxaloacetate and acetyl-CoA. This capacity was found in autotrophically grown cells as well as in the cells grown on media with acetate or L-glutamate. Citrate lyase activity in cell-free extracts is only measurable in the presence of citrate, adenosine-5-triphosphate, coenzyme A and Mg²⁺ or Mn²⁺. It is concluded on the basis of the obtained data that C. limicola f. thiosulfatophilum contains adenosine-5-triphosphate-linked citrate lyase (E.C.4.1.3.8). In contrast to green bacteria in the purple bacteria Ectothiorhodospira shaposhnikovii, Rhodospirillum rubrum and Thiocapsa roseopersicina citrate lyase activity was not found.     

Noncorrelation of Choline Kinase or ATP Citrate Lyase with Cholinergic Activity in Rat Brain

Abstract: The activity of choline acetyltransferase was used as an index of cholinergic structures in regions of rat brain. The activities of ATP citrate lyase and choline kinase correlated poorly with cholinergic activity in whole tissue fractions, contrasting with the good correlation between acetylcholinesterase and choline acetyltransferase. Choline acetyltransferase was preferentially localised in synaptosomes prepared from regions of high (striatum) or intermediate (cortex, medulla oblongata/pons) cholinergic activity. In general, this was not true for either choline kinase or ATP citrate lyase.     

The Interaction of Dithiothreitol and Acetyl Coenzyme A in a Radiochemical Assay for Rat Brain ATP:Citrate Oxaloacetate Lyase

Abstract: [¹⁴C]Acetyl-CoA was found to react spontaneously with dithiothreitol to give a relatively apolar product which was readily extractable into a butanol-toluene scintillant. This technique was used in a rapid, reproducible assay for rat brain ATP:citrate lyase using [1,5-¹⁴C]citrate as substrate. The tissue extract, a 14,000 g supernatant, exhibited a lyase activity of approximately 7 nmol acetyl-CoA produced/min per mg supernatant protein, and was inhibited ≥79% by α-ketoglutaric acid (10 m m ), Cu²⁺ (1 m m )and Zn²⁺(1 m m ). [¹⁴C]Oxaloacetate, [¹⁴C]malate and endogenous citrate synthase were found not to interfere significantly with lyase estimations, but NADH was required in the reaction mixture to inhibit acetyl-CoA hydrolase activity.     

Inhibition of the synthesis of acetylcholine in rat brain slices by (-)-hydroxycitrate and citrate

Abstract: Slices of rat caudate nucleus were incubated in a solution of 123 mM-NaCl, 5 mM-KCl, 1.2 mM-MgCl₂, 1.2 mM-NaH₂PO₄, 25 mM-NaHCO₃, 0.2 mM-choline chloride, 0.058 mM-paraoxon, 1 mM-EGTA, and oxidizable substrates. (−)-Hydroxycitrate, a specific inhibitor of ATP-citrate lyase (EC 4.1.3.8), used at a concentration of 2.5 mM, inhibited the synthesis of acetylcholine (ACh) from [1,5-¹⁴C]citrate by 82–86%, but that from [U-¹⁴C]glucose by only 33%, from [2-¹⁴C]pyruvate by 24% and from [1-¹⁴C-acetyl]carnitine by 8%; the production of ¹⁴CO₂ from these substrates was not substantially changed. The synthesis of ACh from glucose and pyruvate was in hibited also by citrate; 2.5 mM- and 5 mM-citrate diminished it by 43% and 66%, respectively; the production of from [U-¹⁴C]glucose and from [1-¹⁴C]pyruvate was not affected. The mechanism of the inhibitory effect of citrate on the synthesis of ACh is not clear; the possibility is discussed that citrate alters the intracellular milieu in cholinergic neurons by chelating the intracellular Ca²⁺ and decreases the supply of mitochondrial acetyl-CoA to the cytosol. The results with (−)-hydroxycitrate indicate that the cleavage of citrate by ATP-citrate lyase is not responsible for the supply of more than about one-third of the acetyl-CoA which is used for the synthesis of ACh when glucose or pyruvate are the main oxidizable substrates. This proportion may be even smaller, since (−)-hydroxycitrate possibly affects the synthesis of ACh from glucose and pyruvate by a mechanism (unknown) similar to that of citrate, rather than by the inhibition of ATP-citrate lyase.     

Widespread occurrence of ATP:citrate lyase and carnitine acetyltransferase in filamentous fungi

Ten filamentous fungi, belonging to five different genera of both higher and lower fungi, including oleaginous fungi and fungi known to produce secondary metabolites, all possessed both ATP:citrate lyase (17–84nmol min–1 (mg protein)–1) and carnitine acetyltransferase activity (9–62nmol min–1 (mg protein)–1). The possession of these two enzymes appears to be a common feature in filamentous fungi.     

Distribution of the ability for citrate utilization amongst Clostridia

44 species of the genus Clostridium were examined with regard to their ability to grow on citrate. In addition to Clostridium sphenoides, a known citrate utilizer, the following species were found to utilize citrate: C. sporosphaeroides, C. symbiosum, C. rectum, C. indolis, C. subterminale and C. sporogenes. The major products formed from citrate were acetate, ethanol and carbon dioxide (not measured). Minor products were butyrate, butanol, acetone and acetoin.The enzyme citrate lyase was detectable in cell extracts of C. sporosphaeroides and C. symbiosum using an optical assay. Evidence for the presence of this enzyme in the other species was obtained in immunological experiments and in experiments with [1,5-¹⁴C]citrate.     

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

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

Itaconic acid: An inhibitor of isocitrate lyase in Tetrahymena pyriformis in vitro and in vivo

The succinate analog itaconic acid was observed to be a competitive inhibitor of the glyoxylate cycle specific enzyme isocitrate lyase (EC 4.1.3.1) in cell-free extracts of Tetrahymena pyriformis. Itaconic acid also inhibited net in vivo glycogen synthesis from glyoxylate cycle-dependent precursors such as acetate but not from glyoxylate cycle-independent precursors such as fructose. The effect of itaconic acid on the incorporation of ¹⁴C into glycogen from various ¹⁴C-labeled precursors was also consistent with inhibition of isocitrate lyase by this compound. Another analog of succinate which shares a common metabolic fate with itaconic acid, mesaconic acid, had no effect on isocitrate lyase activity in vitro or on ¹⁴C-labeled precursor incorporation into glycogen in vivo. In addition, itaconic acid did not affect gluconeogenesis from lactate in isolated perfused rat livers, a system lacking the enzyme isocitrate lyase. These results are taken as evidence that itaconic acid is an inhibitor of glyoxylate cycle-dependent glyconeogenesis Tetrahymena pyriformis via specific competitive inhibition of isocitrate lyase activity.     

The structure and computational analysis of Mycobacterium tuberculosis protein CitE suggest a novel enzymatic function

Fatty acid biosynthesis is essential for the survival of Mycobacterium tuberculosis and acetyl-coenzyme A (acetyl-CoA) is an essential precursor in this pathway. We have determined the 3-D crystal structure of M. tuberculosis citrate lyase beta-subunit (CitE), which as annotated should cleave protein bound citryl-CoA to oxaloacetate and a protein-bound CoA derivative. The CitE structure has the (beta/alpha)(8) TIM barrel fold with an additional alpha-helix, and is trimeric. We have determined the ternary complex bound with oxaloacetate and magnesium, revealing some of the conserved residues involved in catalysis. While the bacterial citrate lyase is a complex with three subunits, the M. tuberculosis genome does not contain the alpha and gamma subunits of this complex, implying that M. tuberculosis CitE acts differently from other bacterial CitE proteins. The analysis of gene clusters containing the CitE protein from 168 fully sequenced organisms has led us to identify a grouping of functionally related genes preserved in M. tuberculosis, Rattus norvegicus, Homo sapiens, and Mus musculus. We propose a novel enzymatic function for M. tuberculosis CitE in fatty acid biosynthesis that is analogous to bacterial citrate lyase but producing acetyl-CoA rather than a protein-bound CoA derivative.     

Regional and Subcellular Distribution of ATP-Citrate Lyase and Other Enzymes of Acetyl-CoA Metabolism in Rat Brain

The activities of ATP-citrate lyase in frog, guinea pig, mouse, rat, and human brain vary from 18 to 30 μmol/h/g of tissue, being several times higher than choline acetyltransferase activity. Activities of pyruvate dehydrogenase and acetyl coenzyme A synthetase in rat brain are 206 and 18.4 μmol/h/g of tissue, respectively. Over 70% of the activities of both choline acetyltransferase and ATP-citrate lyase in secondary fractions are found in synaptosomes. Their preferential localization in synaptosomes and synaptoplasm is supported by RSA values above 2. Acetyl CoA synthetase activity is located mainly in whole brain mitochondria (RSA, 2.33) and its activity in synaptoplasm is low (RSA, 0.25). The activities of pyruvate dehydrogenase, citrate synthase, and carnitine acetyltransferase are present mainly in fractions C and B_p. No pyruvate dehydrogenase activity is found in synaptoplasm. Striatum, cerebral cortex, and cerebellum contain similar activities of pyruvate dehydrogenase, citrate synthase, carnitine acetyltransferase, fatty acid synthetase, and acetyl-CoA hydrolase. Activities of acetyl CoA synthetase, choline acetyltransferase and ATP-citrate lyase in cerebellum are about 10 and 4 times lower, respectively, than in other parts of the brain. These data indicate preferential localization of ATP-citrate lyase in cholinergic nerve endings, and indicate that this enzyme is not a rate limiting step in the synthesis of the acetyl moiety of ACh in brain.