Determination of citrate with citrate lyase

Moellering, H; Gruber, Wolfgang

doi:10.1016/0003-2697(66)90172-2

Cited by 423 publications

(124 citation statements)

References 6 publications

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“…The total lactate released in the medium was measured as described previously (23) using the lactate oxidase assay kit from Sigma. Citrate was assayed enzymatically according to the method of Moellering and Gruber (24). Briefly, following the 1-h incubation period either in the absence or presence of insulin (100 nM), AICAR (2 mM), and AICAR plus insulin, cells were disrupted in perchloric acid (0.6 N) by mechanical agitation (vortexing).…”

Section: Western Blot Determination Of Ampk␣1 and -␣2 P-ampk And P-mentioning

confidence: 99%

“…An aliquot of the lower phase was collected, neutralized with KOH (2 N), and placed on ice for 15 min to precipitate the potassium chlorate formed. Tubes were centrifuged again (10,000 rpm, 30 s), and the supernatant was removed and used for subsequent spectrophotometric analysis of citrate (24).…”

Section: Western Blot Determination Of Ampk␣1 and -␣2 P-ampk And P-mentioning

confidence: 99%

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5-Aminoimidazole-4-carboxamide-1-β-D-ribofuranoside-induced AMP-activated Protein Kinase Phosphorylation Inhibits Basal and Insulin-stimulated Glucose Uptake, Lipid Synthesis, and Fatty Acid Oxidation in Isolated Rat Adipocytes

Gaidhu

Fediuc

Ceddia

2006

Journal of Biological Chemistry

103

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The objective of this study was to investigate the effects of 5-aminoimidazole-4-carboxamide-1-␤-D-ribofuranoside (AICAR)-induced AMP-activated protein kinase (AMPK) activation on basal and insulin-stimulated glucose and fatty acid metabolism in isolated rat adipocytes. AICAR-induced AMPK activation profoundly inhibited basal and insulin-stimulated glucose uptake, lipogenesis, glucose oxidation, and lactate production in fat cells. We also describe the novel findings that AICAR-induced AMPK phosphorylation significantly reduced palmitate (32%) and oleate uptake (41%), which was followed by a 50% reduction in palmitate oxidation despite a marked increase in AMPK and acetyl-CoA carboxylase phosphorylation. Compound C, a selective inhibitor of AMPK, not only completely prevented the inhibitory effect of AICAR on palmitate oxidation but actually caused a 2.2-fold increase in this variable. Compound C also significantly increased palmitate oxidation in the presence of inhibitory concentrations of malonyl-CoA and etomoxir indicating an increase in CPT1 activity. In contrast to skeletal muscle in which AMPK stimulates fatty acid oxidation to provide ATP as a fuel, we propose that AMPK activation inhibits lipogenesis and fatty acid oxidation in adipocytes. Inhibition of lipogenesis would conserve ATP under conditions of cellular stress, although suppression of intra-adipocyte oxidation would spare fatty acids for exportation to other tissues where their utilization is crucial for energy production. Additionally, the stimulatory effect of compound C on long chain fatty acid oxidation provides a novel pharmacological approach to promote energy dissipation in adipocytes, which may be of therapeutic importance for obesity and type II diabetes.

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Section: Western Blot Determination Of Ampk␣1 and -␣2 P-ampk And P-mentioning

confidence: 99%

Section: Western Blot Determination Of Ampk␣1 and -␣2 P-ampk And P-mentioning

confidence: 99%

5-Aminoimidazole-4-carboxamide-1-β-D-ribofuranoside-induced AMP-activated Protein Kinase Phosphorylation Inhibits Basal and Insulin-stimulated Glucose Uptake, Lipid Synthesis, and Fatty Acid Oxidation in Isolated Rat Adipocytes

Gaidhu

Fediuc

Ceddia

2006

Journal of Biological Chemistry

103

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“…The purified enzyme was converted completely into its deacetyl form after incubation with 10 mM dithioerythritol for 60 min at 30°C. Protein concentration was determined according to [I61 and the amount of citrate remaining in the medium according to [17].…”

Section: Enzyme Assaysmentioning

confidence: 99%

Covalent modification of citrate lyase ligase from Clostridium sphenoides by phosphorylation/dephosphorylation

Antranikian

Herzberg

Gottschalk

1985

European Journal of Biochemistry

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Citrate lyase ligase was shown to be present in Clostridium sphenoides actively degrading citrate. In contrast to citrate lyase ligase from C. sporosphaeroides and Streptococcus lactis, the enzyme from C. sphenoides was under stringent regulatory control. The alteration of the kinetic properties of the enzyme after depletion of citrate suggested the presence of two different enzyme species in different phases of growth: active and partially active citrate lyase ligase. These enzymes were purified from in vivo 32P-labeled C. sphenoides cells, which were grown on low-phosphate medium containing 40 mM citrate and 1 mCi [32P]orthophosphate. During enzyme purification only the active form of citrate lyase ligase was shown to be radioactively labeled. Growth experiments with 14C-labeled precursors of purines and pyrimidines and subsequent purification of active citrate lyase ligase indicated that the 32P labeling of the enzyme was not due to the incorporation of a nucleotide. Inactivation of the ligase after its treatment with acid phosphatase also suggested that the active form of the enzyme is phosphorylated. Citrate lyase ligase, therefore, is the first known enzyme in an anaerobic bacterium whose activity is modulated by phosphorylation/dephosphorylation. both organisms that the intracellular pool size of L-glutamate is dependent on the level of its precursor, citrate, in the medium [12, 131. It is, therefore, reasonable that L-glutamate plays a key role in the regulation of citrate metabolism in these organisms. A high concentration of L-glutamate signals that citrate is available and that it can be cleaved via the fermentation pathway, while at low concentrations citrate is utilized for the biosynthesis of L-glutamate. In R. gelatinosus a futile cycle is avoided by the deacetylation (inactivation) of citrate lyase after the exhaustion of citrate in the medium. The enzyme catalyzing this reaction is citrate lyase deacetylase, whose activity is strongly inhibited by L-glutamate [13].After citrate exhaustion another futile cycle has to be avoided, the one between citrate lyase deacetylase and citrate lyase ligase. The latter enzyme has been shown to be rapidly inactivated in R . gelatinosus [9]. A similar inactivation has been observed in C. sphenoides and it has now been studied in detail in this organism. In this communication we present conclusive evidence that a covalent modification of citrate lyase ligase occurs and that this modification consists of a phosphorylation/dephosphorylation of this enzyme. MATERIALS AND METHODS Organism and growth conditionsClostridium sphenoides strain C 2 (DSM 614) was used for this investigation. The growth medium contained 40 mM trisodium citrate and was prepared as described in [12].Before the total consumption of citrate (17 h) growth was stopped and the harvested cells were stored at -20°C (yield 2.5-3.5 g wet weight/l). In this phase of growth, citrate lyase ligase was present in its active form. For the purification of partially active citrate lyase ligase cells were harv...

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“…The assay of various metabolites was carried out by the following methods: L-malate, Hohorst [19]; citrate, Moellering and Gruber [20]; threo-Dsisocitrate, Siebert [Zl]; protein was measured by the method of Gornall, Bardawill and David [22].…”

Section: Methomentioning

confidence: 99%

The Effects of 2‐Ethylcitrate and Tricarballylate on Citrate Transport in Rat Liver Mitochondria and Fatty Acid Synthesis in Rat White Adipose Tissue

Robinson

Williams

Halperin

et al. 1970

European Journal of Biochemistry

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2-Ethylcitrate and propane 1,2,3 tricarboxylic acid (tricarballylate) were found to be inhibitors of the citrate transporting system as monitored by cis-aconitate and isocitrate oxidation. Kinetic data showed the inhibitions to be competitive with tricarboxylate anion. When the exchange of intramitochondrial [14C]citrate with added extramitochondrial citrate or L-malate was measured at low temperatures, 2-ethylcitrate and 2-propylcitrate were found to be good inhibitors, while tricarballylate did not inhibit and in fact was found to be a good substrate for exchange on the transporting system. Neither 2-ethylcitrate nor tricarballylate a t low concentrations (< 10 mM) were found to inhibit fatty acid synthesis with pyruvate as precursor in rat white adipose tissue but both were potent inhibitors of fatty acid synthesis with glucose as precursor. The implications of these findings are discussed.The rate of egress of citrate from the mitochondrial to the cytoplasmic compartment is of fundamental importance t o two major pathways. Firstly, it has been postulated that cytoplasmic citrate levels control the flow through the glycolytic pathway by influencing the activity of phosphofructokinase in some but not all mammalian tissues [l -41. Secondly the pathway for fatty acid synthesis in rat white adipose tissue and rat liver is thought to involve exit of citrate from the mitochondria to acetyl-CoA when cleaved by citrate-ATP-lyase EXPERIMENTAL PROCEDURE RatsEpididymal fat pads and livers obtained from male Wistar rats (150 g) fed on a purina chow diet. I n all experiments the animals were allowed free access to food before the time of killing (approximately 1O:OO a. m.). Rat liver mitochondria were isolated by methods described previously

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Determination of citrate with citrate lyase

Cited by 423 publications

References 6 publications

5-Aminoimidazole-4-carboxamide-1-β-D-ribofuranoside-induced AMP-activated Protein Kinase Phosphorylation Inhibits Basal and Insulin-stimulated Glucose Uptake, Lipid Synthesis, and Fatty Acid Oxidation in Isolated Rat Adipocytes

5-Aminoimidazole-4-carboxamide-1-β-D-ribofuranoside-induced AMP-activated Protein Kinase Phosphorylation Inhibits Basal and Insulin-stimulated Glucose Uptake, Lipid Synthesis, and Fatty Acid Oxidation in Isolated Rat Adipocytes

Covalent modification of citrate lyase ligase from Clostridium sphenoides by phosphorylation/dephosphorylation

The Effects of 2‐Ethylcitrate and Tricarballylate on Citrate Transport in Rat Liver Mitochondria and Fatty Acid Synthesis in Rat White Adipose Tissue

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