Showing papers on "ATP citrate lyase published in 1997"

Anaerobic citrate metabolism and its regulation in enterobacteria.

Abstract: Several species of enterobacteria are able to utilize citrate as carbon and energy source. Under oxic conditions in the presence of a functional tricarboxylic acid cycle, growth on this compound solely depends on an appropriate transport system. During anaerobiosis, when 2-oxoglutarate dehydrogenase is repressed, some species such as Klebsiella pneumoniae and Salmonella typhimurium, but not Escherichia coli, are capable of growth on citrate by a Na+-dependent pathway forming acetate, formate, and CO2 as products. During the last decade, several novel features associated with this type of fermentation have been discovered in K. pneumoniae. The biotin protein oxaloacetate decarboxylase, one of the key enzymes of the pathway besides citrate lyase, is a Na+ pump. Recently it has been shown that the proton required for the decarboxylation of carboxybiotin is taken up from the side to which Na+ ions are pumped, and a membrane-embedded aspartate residue that is probably involved both in Na+ and in H+ transport was identified. The Na+ gradient established by oxaloacetate decarboxylase drives citrate uptake via CitS, a homodimeric carrier protein with a simultaneous-type reaction mechanism, and NADH formation by reversed electron transfer involving formate dehydrogenase, quinone, and a Na+-dependent NADH:quinone oxidoreductase. All enzymes specifically required for citrate fermentation are induced under anoxic conditions in the presence of citrate and Na+ ions. The corresponding genes form a cluster on the chromosome and are organized as two divergently transcribed operons. Their co-ordinate expression is dependent on a two-component system consisting of the sensor kinase CitA and the response regulator CitB. The citAB genes are part of the cluster and are positively autoregulated. In addition to CitA/CitB, the cAMP receptor protein (Crp) is involved in the regulation of the citrate fermentation enzymes, subjecting them to catabolite repression.

Malonyl-CoA regulation in skeletal muscle: its link to cell citrate and the glucose-fatty acid cycle.

Abstract: Malonyl-CoA is an inhibitor of carnitine palmitoyltransferase I, the enzyme that controls the oxidation of fatty acids by regulating their transfer into the mitochondria. Despite this, knowledge of how malonyl-CoA levels are regulated in skeletal muscle, the major site of fatty acid oxidation, is limited. Two- to fivefold increases in malonyl-CoA occur in rat soleus muscles incubated with glucose or glucose plus insulin for 20 min [Saha, A. K., T. G. Kurowski, and N. B. Ruderman. Am. J. Physiol. 269 (Endocrinol. Metab. 32): E283-E289, 1995]. In addition, as reported here, acetoacetate in the presence of glucose increases malonyl-CoA levels in the incubated soleus. The increases in malonyl-CoA in all of these situations correlated closely with increases in the concentration of citrate (r2 = 0.64) and to an even greater extent the sum of citrate plus malate (r2 = 0.90), an antiporter for citrate efflux from the mitochondria. Where measured, no increase in the activity of acetyl-CoA carboxylase (ACC) was found. Inhibition of ATP citrate lyase with hydroxycitrate markedly diminished the increases in malonyl-CoA in these muscles, indicating that citrate was the major substrate for the malonyl-CoA precursor, cytosolic acetyl-CoA. Studies with enzyme purified by immunoprecipitation indicated that the observed increases in citrate could have also allosterically activated ACC. The results suggest that in the presence of glucose, insulin and acetoacetate acutely increase malonyl-CoA levels in the incubated soleus by increasing the cytosolic concentration of citrate. This novel mechanism could complement the glucose-fatty acid cycle in determining how muscle chooses its fuels. It could also provide a means by which glucose acutely modulates signal transduction in muscle and other cells (e.g., the pancreatic beta-cell) in which its metabolism is determined by substrate availability.

The reductive tricarboxylic acid cycle of carbon dioxide assimilation: initial studies and purification of ATP-citrate lyase from the green sulfur bacterium Chlorobium tepidum.

Abstract: Carbon dioxide is fixed largely by the reductive tricarboxylic acid (RTCA) cycle in green sulfur bacteria. One of the key enzymes, ATP-citrate lyase, was purified to apparent homogeneity from the moderately thermophilic green sulfur bacterium Chlorobium tepidum. The molecular weight of the native enzyme was about 550,000, and the preponderance of evidence indicated that the protein is composed of identical subunits (Mr of approximately 135,000) which degraded to two major proteins with Mrs of approximately 65,000 and approximately 42,000. Western immunoblots and in vitro phosphorylation experiments indicated that these two species could have been the result of proteolysis by an endogenous protease, similar to what has been observed with mammalian, yeast, and mold ATP-citrate lyase. In addition to apparent structural similarities, the catalytic properties of C. tepidum ATP-citrate lyase showed marked similarities to the eukaryotic enzyme, with significant differences from other prokaryotic ATP-citrate lyases, including the enzyme from the closely related organism Chlorobium limicola. Phosphorylation of C. tepidum ATP-citrate lyase occurred, presumably on a histidine residue at the active site, similar to the case for the mammalian enzyme. In contrast to the situation observed for other prokaryotic ATP-citrate lyase enzymes, the C. tepidum enzyme was not able to replace ATP and GTP for activity or use Cu2+ to replace Mg2+ for enzyme activity. Given the apparent structural and catalytic similarities of the enzyme from C. tepidum and its eukaryotic counterpart, the C. tepidum system should serve as an excellent model for studies of the enzymology and regulation of this protein.

Antimycins, inhibitors of ATP-citrate lyase, from a Streptomyces sp.

Abstract: A related group of compounds belonging to the antimycin class of antibiotics was found in culture broth produced by a Streptomyces species. The group includes known antimycins A1, A2, A3 and A4, and new antimycins A7 and A8. These compounds inhibit ATP-citrate lyase with Ki values of 4 to 60 μM against the substrate magnesium citrate. The structures of the new antimycins were determined by spectroscopic analyses.

Correlation of ATP/citrate lyase activity with lipid accumulation in developing seeds of Brassica napus L.

Abstract: The temporal distribution of ATP/citrate lyase (ACL) activity in developing seeds of Brassica napus L. closely paralleled both that of acetyl-CoA carboxylase and the overall rate of lipid biosynthesis. Maximum ACL activities (250 nmol acetyl-CoA formed min−1·g fresh seed) were recorded between 35 to 42 d after pollination and, if the in vitro data could be extrapolated to the situation in vivo, could account for half of the acetyl-CoA required for the measured rate of fatty acid biosynthesis during seed development. The enzyme appeared to be localized in a subcellular compartment, which was clearly separated from mitochondria on a sucrose gradient and by differential centrifugation, and which corresponded to the chloroplast organelle.

Bioelectrochemical Determination of Citric Acid in Real Samples Using a Fully Automated Flow Injection Manifold

Abstract: An enzymic method for the determination of citric acid in fruits, juices and sport drinks is proposed. The method is based on the action of the enzymes citrate lyase, oxaloacetate decarboxylase and pyruvate oxidase, which convert citric acid into H2O2 with the latter being monitored amperometrically with a H2O2 probe. The enzymes pyruvate oxidase and oxaloacetate decarboxylase were immobilized. A multi-membrane system, consisting of a cellulose acetate membrane for the elimination of interferants, an enzymic membrane and a protective polycarbonate membrane were placed on a Pt electrode and used with a fully automated flow injection manifold. Several parameters were optimized, resulting in a readily constructed and reproducible biosensor. Interference from various compounds present in real samples was minimized. Calibration graphs were linear over the range 0.01–0.9 mM pyruvate, 0.015–0.6 mM oxaloacetate and 0.015–0.5 mM citrate. The throughput was 30 samples h–1 with an RSD of 1.0% (n = 8); the mean relative error was 2.4% compared with a standard method. The recovery was 96–104%. A 8–10% loss of the initial activity of the sensor was observed after 100–120 injections.

Transcriptional regulatory region for expression of the rat ATP citrate-lyase gene.

Abstract: We previously mapped the sequences responsive to insulin/glucose stimulation and polyunsaturated fatty-acid suppression in the proximal promoter region between positions –104 and –20 of the ATP citrate-lyase (ACL) gene [Fukuda, H., Iritani, N., Katsurada, A. & Noguchi, T. (1996) FEBS Lett. 380, 204–207]. To investigate further the regulatory DNA sequences required for stimulation and suppression of this gene, primary cultured hepatocytes were transfected with plasmids containing the 5′-flanking sequences of the rat ACL gene fused to the chloramphenicol acetyltransferase (CAT) gene. When two copies of the sequences spanning –64 to –41 (linked to ACLcat20) were used for transfection, CAT activity significantly increased in response to insulin/glucose treatment. This increase was inhibited by addition of polyunsaturated fatty acid. Mutational analysis of this region showed that sequences between –55 and –51 are essential for recognition and interaction with trans-acting factors. Gel mobility shift assays using the sequence from –64 to –41 as a probe revealed nuclear factor(s) from rat liver that specifically complexed with the sequences. In addition, by antibody supershift assays, we have detected the binding of the transcriptional factor Sp1 at the G+C-rich region located within –64 to –41 of the ACL promoter. On the other hand, the formations of DNA-protein complexes with Sp1 binding site or ACL(–64 to –41) were decreased in rats fed a high-carbohydrate diet in comparison with those in rats fasted or fed a polyunsaturated fatty-acid-rich diet. Cotransfection studies in rat hepatocytes, with the Sp1 expression vector and ACLcat constructs, showed the inactivation of the promoter. These results demonstrated that the region from –64 to –41 of the ACL gene was responsible for stimulation due to insulin/ glucose, the stimulation was suppressed by polyunsaturated fatty acid, and Sp1 may be involved in the regulation.

Purification and properties of citrate synthase from Acetobacter europaeus

Abstract: Citrate synthase (EC 4.1.3.7) was purified from the acidophilic bacterium Acetobacter europaeus to electrophoretic homogeneity. The specific activity was 228 units/mg of protein during the exponential ethanol-oxidation growth phase. The enzyme has a molecular mass of 280 kDa and is a hexamer with a subunit size of 46 kDa. The apparent Km values were 20 μM for oxaloacetate and 51 μM for acetyl-CoA. Unlike citrate synthase from other Gram-negative bacteria, the activity of the enzyme was inhibited by ATP, slightly enhanced by ADP and not effected by NADH. Acetate caused activation of the enzyme. The pH optimum on the citrate synthase activity in vitro was 8.1. The amino-terminal amino acid sequence of the purified enzyme was ENGKSATISLNGKDVALPVL.

Synthesis of citrate from phosphoenolpyruvate and acetylcarnitine by mitochondria from rabbit enterocytes: implications for lipogenesis.

Abstract: Enterocytes from fasted rabbits make glucose from exogenous fructose and dihydroxyacetone at rates of 180 and 91 nmol/min/108 cells but do not make glucose from glycerol, aspartate, malate, lactate, α-ketoglutarate, glutamate or glutamine. Total activities of phosphoenolpyruvate carboxykinase, fructose 1,6-bisphosphatase and glucose 6-phosphatase in isolated enterocytes are 0.44, 0.60 and 1.90 μmol/min/108 cells, and ≥95% of carboxykinase activity is intramitochondrial. Enterocytes contain marginal glycerol kinase (0.05 μmol/min/108 cells) and essentially no pyruvate carboxylase activities. Enterocyte mitochondria synthesize citrate from exogenous phosphoenolpyruvate and acetylcarnitine at a rate of 2.40 nmol/min/mg protein. Citrate formation is highly dependent on exogenous HCO3− and inhibited strongly by 3-mercaptopicolinate and 1,2,3-benzenetricarboxylate. Citrate synthesis is stimulated consistently by GDP and significantly so by GTP. Citrate production is unaffected by ADP or ATP. Enterocytes from fasted-refed rabbits contain activities of 0.05, 0.12, 0.39 and 0.56 μmol/min/mg cytosolic protein of ATP:citrate lyase, NADP:malate dehydrogenase, glucose 6-phosphate dehydrogenase and NADP:isocitrate dehydrogenase. Activities of NADP:malate dehydrogenase, glucose 6-phosphate dehydrogenase and NADP:isocitrate dehydrogenase are significantly higher in enterocytes from fasted-refed rabbits than those from fasted rabbits. Mitochondrial phosphoenolpyruvate carboxykinase in enterocytes in vivo could convert glycolysis-derived phosphoenolpyruvate to oxaloacetate that, with acetyl CoA, could form citrate for export to support cytosolic lipogenesis as an activator of acetyl CoA carboxylase, a source of carbon via ATP:citrate lyase and of NADPH via NADP:malate dehydrogenase or NADP:isocitrate dehydrogenase.

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

Abstract: 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.