Showing papers on "ATP citrate lyase published in 2001"

Polyunsaturated fatty acid-rich diets: effect on adipose tissue metabolism in rats

Abstract: The aim of the present study was to evaluate the effect of diets rich in n-6 and n-3 fatty acids on adipose tissue metabolism. Starting at weaning, male Wistar rats were fed ad libitum, for 8 weeks with one of the following diets: C, rat chow; S, rat chow containing 15 % (w/w) soyabean oil; F, rat chow containing 15 % (w/w) fish oil; SF, rat chow containing 15 % (w/w) soyabean and fish oil (5:1, w/w). Casein was added to the fat diets to achieve the same 20 % (w/w) protein content as in the control chow. Food intake and body weight were measured weekly. The rats were killed by decapitation and the retroperitoneal (RET) and epididymal (EPI) white adipose tissues were removed and weighed. Tissue lipid and protein content, in vivo lipogenesis rate, uptake of diet-derived lipids, in vitro lipolytic rate, adipocyte area, lipoprotein lipase, ATP citrate lyase, and malic enzyme activities were evaluated. Carcass lipid and protein contents were also measured. Energy intake was reduced while carcass lipid content was increased in the three fat-fed groups. However, carcass protein and body weight gains were elevated only with diets F and SF. Lipolysis rate was diminished by diets F and SF, while the uptake of diet-derived lipids was elevated by the diet S in both RET and EPI tissues. These metabolic alterations may have contributed to the increase in in vivo lipogenesis rate in the presence of decreased ATP citrate lyase and malic enzyme activities induced by the three lipid diets. These results indicate that enrichment of the diet with polyunsaturated fatty acids causes changes in adipose tissue metabolism that favour fat deposition. Different metabolic pathways were preferentially affected by each type of fatty acid used.

ATP-citrate lyase from the green sulfur bacterium Chlorobium limicola is a heteromeric enzyme composed of two distinct gene products

Abstract: The reductive tricarboxylic acid cycle functions as a carbon dioxide fixation pathway in the green sulfur bacterium, Chlorobium limicola. ATP-citrate lyase, one of the key enzymes of this cycle, was partially purified from C. limicola strain M1 and the N-terminal sequence of a 65-kDa protein was found to show similarity toward eukaryotic ATP-citrate lyase. A DNA fragment was amplified with primers designed from this sequence and an internal sequence highly conserved among eukaryotic enzymes. Using this fragment as a probe, we isolated a DNA fragment containing two adjacent open reading frames, aclB (1197 bp) and aclA (1827 bp), whose products showed significant similarity to the N- and C-terminal regions of the human enzyme, respectively. Heterologous expression of these genes in Escherichia coli showed that both gene products were essential for ATP-citrate lyase activity. The recombinant enzyme was purified from the cell-free extract of E. coli harboring aclBA for further characterization. The molecular mass of the recombinant enzyme was determined to be approximately 532--557 kDa by gel-filtration. The enzyme catalyzed the cleavage of citrate in an ATP(-), CoA- and Mg(2+)-dependent manner, where ATP and Mg(2+) could be replaced by dATP and Mn(2+), respectively. ADP and oxaloacetate inhibited the reaction. These properties suggested that ATP-citrate lyase from C. limicola controlled the cycle flux depending on intracellular energy conditions. This paper provides the first direct evidence that a bacterial ATP-citrate lyase is a heteromeric enzyme, distinct from mammalian enzymes.

Catabolite Repression of the Citrate Fermentation Genes in Klebsiella pneumoniae: Evidence for Involvement of the Cyclic AMP Receptor Protein

Abstract: Klebsiella pneumoniae is able to grow anaerobically with citrate as a sole carbon and energy source by a fermentative pathway involving the Na(+)-dependent citrate carrier CitS, citrate lyase, and oxaloacetate decarboxylase The corresponding genes are organized in the divergent citC and citS operons, whose expression is strictly dependent on the citrate-sensing CitA-CitB two-component system Evidence is provided here that the citrate fermentation genes are subject to catabolite repression, since anaerobic cultivation with a mixture of citrate and glucose or citrate and gluconate resulted in diauxic growth Glucose, gluconate, and also glycerol decreased the expression of a chromosomal citS-lacZ fusion by 60 to 75%, whereas a direct inhibition of the citrate fermentation enzymes was not observed The purified cyclic AMP (cAMP) receptor protein (CRP) of K pneumoniae bound to two sites in the citC-citS intergenic region, which were centered at position -415 upstream of the citC and citS transcriptional start sites Binding was apparently stimulated by the response regulator CitB These data indicate that catabolite repression of the citrate fermentation genes is exerted by CRP and that in the absence of repressing carbon sources the cAMP-CRP complex serves to enhance the basal, CitB-dependent transcription level

Covariance of tricarboxylate carrier activity and lipogenesis in liver of polyunsaturated fatty acid (n-6) fed rats.

Abstract: The mitochondrial tricarboxylate (citrate) carrier plays an important role in hepatic intermediary metabolism because, among other functions, it supplies the cytosol with acetyl units for fatty-acid synthesis. In this study, the effect of polyunsaturated fatty acids (PUFA, n-6) on the function of this mitochondrial transporter and on lipogenic enzyme activities was investigated by feeding rats for 4 weeks with a 15%-fat diet composed of high linoleic safflower oil. Citrate transport was strongly reduced in liver mitochondria isolated from PUFA-treated rats. A reduced transport activity was also observed when solubilized mitochondrial citrate carrier from PUFA-treated rats was reconstituted into liposomes. In the same animals, a decrease of cytosolic lipogenic enzyme activities was observed. These results indicate a coordinated modulation of citrate carrier and of lipogenic enzyme activities by PUFA feeding. Kinetic analysis of the carrier activity showed that only Vmax decreased, whereas Km was almost virtually unaffected. The PUFA-mediated effect is most likely due to the reduced mRNA level and lower content of the citrate carrier protein observed in the safflower oil-fed rats.

Nitrate respiratory metabolism in an obligately autotrophic hydrogen-oxidizing bacterium, Hydrogenobacter thermophilus TK-6.

Abstract: Hydrogenobacter thermophilus strain TK-6 was observed to grow anaerobically on nitrate as an electron acceptor when molecular hydrogen was used as an energy source. Nitrite was detected as the product of a respiratory reaction. 15NO, 15N2O, and 15N2 were detected with Na15NO3 as an electron acceptor. Western immunoblot analysis showed that cell-free extracts from cells grown on nitrate reacted with antibodies against heme cd1-type nitrite reductase from Pseudomonas aeruginosa. The positive bands, which had molecular masses similar to that of the heme cd1-type nitrite reductase, were also stained by heme staining. These results indicate that nitrite reductase of strain TK-6 is a heme cd1-type enzyme. Activity of ATP:citrate lyase, one of the key enzymes of the reductive TCA cycle, was detected in cell-free extract of cells cultivated on nitrate, which indicates that the cycle operates during anaerobic growth.

The Citric Acid Cycle and Fatty Acid Biosynthesis

Abstract: This chapter talks about fatty acid biosynthesis, linked to the citric acid cycle (CAC) through the utilization of acetyl-coenzyme A (CoA) as its starting point. The oxidative decarboxylation of pyruvate is an important reaction in archaea, bacteria, and eukaryotes alike, generating acetyl-CoA necessary for CAC reactions, fatty acid biosynthesis, and many other reactions requiring acyl-CoA. Citrate synthase catalyzes the first step in the oxidative branch of the CAC in which acetyl-CoA and oxaloacetate are condensed to generate citrate and CoA. Aconitase activity has been detected in the cytosolic fraction of Helicobacter pylori cells both by nuclear magnetic resonance (NMR) and spectrophotometric assays. In Escherichia coli isocitrate dehydrogenase acts as a critical branch point between the CAC reactions and the glyoxylate bypass during growth on C2 compounds like acetate. The study of the lipid and fatty acid profiles of eight Helicobacter species has revealed some characteristic features of the Helicobacter genus. Malonyl-acylcarrier protein (ACP) is required not only for initiation of fatty acid biosynthesis, but also for each subsequent round of elongation of the fatty acid chain. To function in fatty acid biosynthesis, the apo-ACP protein must first be activated by transfer of the 4'-phospho-pantotheine from CoA, and this reaction is predicted to be catalyzed by holo-ACP synthase, encoded by acpS in H. pylori.

ATP citrate lyase in the glaucocystophyte alga Cyanophora paradoxa is a cytosolic enzyme: characterisation of the gene for the large subunit at the cDNA and genomic levels.

Abstract: The single-copy gene for the large subunit of ATP citrate lyase (ACL) from the alga Cyanophora paradoxa was characterized at the cDNA and genome levels. The gene product showed high sequence similarity to its mammalian counterparts, but is smaller in size, as is also found for the fungal subunit Acl1 and, most probably, for the corresponding subunit from Arabidopsis thaliana. The C. paradoxa gene is interrupted by at least 12 introns of 53–65 bp with conserved border and branchpoint sequences, and the product lacks a stroma-targeting peptide. Enzyme activity was found in the cytosol of C. paradoxa but not in the plastid (cyanelle) fraction. This is in contrast to the subcellular distribution of ATP citrate lyases in higher plants, where both chloroplast and cytosolic enzymes have been reported.

Methods for production of recombinant Holo-Cytrate Lyase

Abstract: Recombinant production (M1) of a protein (I) with citrate lyase (CL) activity comprises expressing in a host organism, a plasmid that includes a cluster of at least 6 genes and an inducible promoter, then isolation of (I) in active form. Independent claims are also included for the following: (1) recombinant soluble (I) of molecular weight 14-15 kD, produced by (M1); and (2) test kit for determination of citrate comprises: (a) CL; (b) a protein (II) with hydrogen-transfer activity; (c) reduced NAD (nicotinamide-adenine dinucleotide), or its derivative; (d) optionally, stabilizers, activators and/or substances that eliminate or reduce interferences; and (e) buffer solutions.

Methods for the production of recombinant citrate lyase holo-enzyme

Abstract: Preparing a protein (I) with citrate lyase (CL) activity by expressing, in a host organism, a suitable plasmid and isolating (I) in active form, is new. The plasmid contains the information from a cluster containing at least 6 genes, plus an inducible promoter. Independent claims are also included for the following: (1) recombinant soluble protein (Ia) with CL activity and of molecular weight 14-15 kDa, produced by the new method; and (2) a test kit for determining citrate containing essentially (I), a protein (II) with hydrogen-transfer activity, nicotinamide-adenine dinucleotide (NAD), or its derivative, in reduced form, optionally also stabilizers, activators, substances that eliminate/reduce interferences and/or buffer solutions.