Oxaloacetate decarboxylases of rat liver.
Ontology highlight
ABSTRACT:
SUBMITTER: Dean B
PROVIDER: S-EPMC1165882 | biostudies-other | 1973 Dec
REPOSITORIES: biostudies-other
Similar Datasets
Project description:To compare the powers of the corresponding enzymes as catalysts, the rates of uncatalyzed decarboxylation of several aliphatic acids (oxalate, malonate, acetoacetate, and oxaloacetate) were determined at elevated temperatures and extrapolated to 25 °C. In the extreme case of oxalate, the rate of the uncatalyzed reaction at pH 4.2 was 1.1 × 10(-12) s(-1), implying a 2.5 × 10(13)-fold rate enhancement by oxalate decarboxylase. Whereas the enzymatic decarboxylation of oxalate requires O(2) and Mn(II), the uncatalyzed reaction is unaffected by the presence of these cofactors and appears to proceed by heterolytic elimination of CO(2).
| S-EPMC3077560 | biostudies-literature
Project description:1. The rates of translocation of oxaloacetate and l-malate into rat liver mitochondria were measured by a direct spectrophotometric assay. 2. Penetration obeyed Michaelis-Menten kinetics, and apparent K(m) values were 40mum for oxaloacetate and 0.13mm for l-malate. 3. Arrhenius plots of the temperature-dependence of rates of penetration gave activation energies of +10kcal./mole for oxaloacetate and +8kcal./mole for l-malate. 4. The translocation of both oxaloacetate and l-malate was competitively inhibited by d-malate, succinate, malonate, meso-tartrate, maleate and citraconate. The K(i) values of these inhibitors were similar for the penetration of both oxaloacetate and l-malate. 5. Rates of penetration were stimulated by NNN'N'-tetramethyl-p-phenylenediamine dihydrochloride plus ascorbate under aerobic conditions or by ATP under anaerobic conditions. 6. The energy-dependent stimulation of translocation was abolished by uncouplers of oxidative phosphorylation. Oligomycin A, aurovertin, octyl-guanidine and atractyloside prevented the stimulation by ATP, but did not inhibit the stimulation by NNN'N'-tetramethyl-p-phenylenediamine dihydrochloride plus ascorbate. 7. Mitochondria prepared in the presence of ethylene-dioxybis(ethyleneamino)tetra-acetic acid did not exhibit the energy-dependent translocation, but this could be restored by the addition of 50mum-calcium chloride. 8. Valinomycin or gramicidin plus potassium chloride enhanced the energy-dependent translocation of oxaloacetate and l-malate. 9. Addition of oxaloacetate stimulated the adenosine triphosphatase activity of the mitochondria, and the ratio of ;extra' oxaloacetate translocation to ;extra' adenosine triphosphatase activity was 1.6:1. 10. Possible mechanisms for the energy-dependent entry of oxaloacetate and l-malate into mitochondria are discussed in relation to the above results.
| S-EPMC1187045 | biostudies-other
Project description:Chemical injury is partly due to free radical lipid peroxidation, which can induce oxidative stress and produce a large number of reactive oxygen species (ROS). Oxaloacetic acid is an important intermediary in the tricarboxylic acid cycle (TCA cycle) and participates in metabolism and energy production. In our study, we found that oxaloacetate (OA) effectively alleviated liver injury which was induced by hydrogen peroxide (H₂O₂) in vitro and carbon tetrachloride (CCl₄) in vivo. OA scavenged ROS, prevented oxidative damage and maintained the normal structure of mitochondria. We further confirmed that OA increased adenosine triphosphate (ATP) by promoting the TCA production cycle and oxidative phosphorylation (OXPHOS). Finally, OA inhibited the mitogen-activated protein kinase (MAPK) and apoptotic pathways by suppressing tumor necrosis factor-α (TNF-α). Our findings reveal a mechanism for OA ameliorating chemical liver injury and suggest a possible implementation for preventing the chemical liver injury.
| S-EPMC6032239 | biostudies-literature
Project description:Hepatocyte lipotoxicity is characterized by aberrant mitochondrial metabolism, which predisposes cells to oxidative stress and apoptosis. Previously, we reported that translocation of calcium from the endoplasmic reticulum to mitochondria of palmitate-treated hepatocytes activates anaplerotic flux from glutamine to α-ketoglutarate (αKG), which subsequently enters the citric acid cycle (CAC) for oxidation. We hypothesized that increased glutamine anaplerosis fuels elevations in CAC flux and oxidative stress following palmitate treatment. To test this hypothesis, primary rat hepatocytes or immortalized H4IIEC3 rat hepatoma cells were treated with lipotoxic levels of palmitate while modulating anaplerotic pathways leading to αKG. We found that culture media supplemented with glutamine, glutamate, or dimethyl-αKG increased palmitate lipotoxicity compared with media that lacked these anaplerotic substrates. Knockdown of glutamate-oxaloacetate transaminase activity significantly reduced the lipotoxic effects of palmitate, whereas knockdown of glutamate dehydrogenase (Glud1) had no effect on palmitate lipotoxicity. 13C flux analysis of H4IIEC3 cells co-treated with palmitate and the pan-transaminase inhibitor aminooxyacetic acid confirmed that reductions in lipotoxic markers were associated with decreases in anaplerosis, CAC flux, and oxygen consumption. Taken together, these results demonstrate that lipotoxic palmitate treatments enhance anaplerosis in cultured rat hepatocytes, causing a shift to aberrant transaminase metabolism that fuels CAC dysregulation and oxidative stress.
| S-EPMC6398133 | biostudies-literature
Project description:Blood glutamate scavengers have been shown to effectively reduce blood glutamate concentrations and improve neurological outcome after traumatic brain injury and stroke in rats. This study investigates the efficacy of blood glutamate scavengers oxaloacetate and pyruvate in the treatment of subarachnoid hemorrhage (SAH) in rats. Isotonic saline, 250 mg/kg oxaloacetate, or 125 mg/kg pyruvate was injected intravenously in 60 rats, 60 minutes after induction of SAH at a rate of 0.1 ml/100 g/min for 30 minutes. There were 20 additional rats that were used as a sham-operated group. Blood samples were collected at baseline and 90 minutes after SAH. Neurological performance was assessed at 24 h after SAH. In half of the rats, glutamate concentrations in the cerebrospinal fluid were measured 24 h after SAH. For the remaining half, the blood brain barrier permeability in the frontal and parieto-occipital lobes was measured 48 h after SAH. Blood glutamate levels were reduced in rats treated with oxaloacetate or pyruvate at 90 minutes after SAH (p < 0.001). Cerebrospinal fluid glutamate was reduced in rats treated with pyruvate (p < 0.05). Neurological performance was significantly improved in rats treated with oxaloacetate (p < 0.05) or pyruvate (p < 0.01). The breakdown of the blood brain barrier was reduced in the frontal lobe in rats treated with pyruvate (p < 0.05) and in the parieto-occipital lobes in rats treated with either pyruvate (p < 0.01) or oxaloacetate (p < 0.01). This study demonstrates the effectiveness of blood glutamate scavengers oxaloacetate and pyruvate as a therapeutic neuroprotective strategy in a rat model of SAH.
| S-EPMC3441925 | biostudies-literature
Project description:Blood glutamate scavenging is a novel and attractive protecting strategy to reduce the excitotoxic effect of extracellular glutamate released during ischemic brain injury. Glutamate oxaloacetate transaminase 1 (GOT1) activation by means of oxaloacetate administration has been used to reduce the glutamate concentration in the blood. However, the protective effect of the administration of the recombinant GOT1 (rGOT1) enzyme has not been yet addressed in cerebral ischemia. The aim of this study was to analyze the protective effect of an effective dose of oxaloacetate and the human rGOT1 alone and in combination with a non-effective dose of oxaloacetate in an animal model of ischemic stroke. Sixty rats were subjected to a transient middle cerebral artery occlusion (MCAO). Infarct volumes were assessed by magnetic resonance imaging (MRI) before treatment administration, and 24 h and 7 days after MCAO. Brain glutamate levels were determined by in vivo MR spectroscopy (MRS) during artery occlusion (80 min) and reperfusion (180 min). GOT activity and serum glutamate concentration were analyzed during the occlusion and reperfusion period. Somatosensory test was performed at baseline and 7 days after MCAO. The three treatments tested induced a reduction in serum and brain glutamate levels, resulting in a reduction in infarct volume and sensorimotor deficit. Protective effect of rGOT1 supplemented with oxaloacetate at 7 days persists even when treatment was delayed until at least 2 h after onset of ischemia. In conclusion, our findings indicate that the combination of human rGOT1 with low doses of oxaloacetate seems to be a successful approach for stroke treatment.
| S-EPMC4040715 | biostudies-literature
Project description:The irreversible decarboxylation step, which commits 2-oxo acids to the Ehrlich pathway, was initially attributed to pyruvate decarboxylase. However, the yeast genome was shown to harbour no fewer than 5 genes that show sequence similarity with thiamine-diphosphate dependent decarboxylase genes. Three of these (PDC1, PDC5 and PDC6) encode pyruvate decarboxylases { while ARO10 and THI3 represent alternative candidates for Ehrlich-pathway decarboxylases. Transcriptome analysis and decarboxylase activity measurements on an S. cerevisiae aro10 strain, a double aro10 thi3 deletion strain and a quadruple pdc1,5,6,aro10 mutant strains grown in carbon–limited chemostat with phenylalanine as nitrogen source indicated that: i) PDC5 is strongly upregulated in an aro10 background (Fig. 2) and also encodes a broad-substrate α-keto acid decarboxylase. ii) PDC5 expression depends on the presence of THI3 (Fig. 2), and iii) in contrast to cell extracts from a strain expressing ARO10 only (pdc1,5,6, thi3), cell extract from a strain that only contains THI3 (pdc1,5,6,aro10) did not produce any α-keto acid decarboxylase activity . THI3 has recently been demonstrated to be involved in regulation of thiamine homeostasis in S. cerevisiae, which further suggests that its role in the Ehrlich pathway may be regulatory rather than catalytic. A systematic investigation of the catalytic properties of all five (putative) TPP-dependent decarboxylases (Aro10p, Thi3p, Pdc1p, Pdc5p, Pdc6p) is essential for a final resolution of the substrate specificity of these key enzymes in the Ehrlich pathway. Keywords: Strain comparison
2007-11-15 | GSE9590 | GEO
Project description:Since the catalytic-centre activity of mouse kidney ornithine decarboxylase (ODC) has been assumed to be twice as high as that of rat liver ODC, we compared relative catalytic-centre activity of the two enzymes by titration with antizyme, which inhibits ODC by stoichiometric binding. In either a crude or a purified state, both enzymes were inhibited by rat liver antizyme to the same extent, indicating that they have nearly identical catalytic-centre activities. This conclusion was supported by comparison of affinity labelling of the enzymes with alpha-difluoromethyl[14C]ornithine.
| S-EPMC1148793 | biostudies-other
Project description:Biosynthetic arginine decarboxylase (ADC; also known as SpeA) plays an important role in the biosynthesis of polyamines from arginine in bacteria and plants. SpeA is a pyridoxal-5'-phosphate (PLP)-dependent enzyme and shares weak sequence homology with several other PLP-dependent decarboxylases. Here, the crystal structure of PLP-bound SpeA from Campylobacter jejuni is reported at 3.0?Å resolution and that of Escherichia coli SpeA in complex with a sulfate ion is reported at 3.1?Å resolution. The structure of the SpeA monomer contains two large domains, an N-terminal TIM-barrel domain followed by a ?-sandwich domain, as well as two smaller helical domains. The TIM-barrel and ?-sandwich domains share structural homology with several other PLP-dependent decarboxylases, even though the sequence conservation among these enzymes is less than 25%. A similar tetramer is observed for both C. jejuni and E. coli SpeA, composed of two dimers of tightly associated monomers. The active site of SpeA is located at the interface of this dimer and is formed by residues from the TIM-barrel domain of one monomer and a highly conserved loop in the ?-sandwich domain of the other monomer. The PLP cofactor is recognized by hydrogen-bonding, ?-stacking and van der Waals interactions.
| S-EPMC2998355 | biostudies-literature
Project description:The concentration of metabolically active (i.e. 'free') oxaloacetate in the mitochondrial compartment of isolated liver cells was investigated by two independent approaches. On the basis of mitochondrial aspartate aminotransferase maintaining equilibrium and the direct measurements of mitochondrial aspartate, 2-oxoglutarate and glutamate, the concentration of free oxaloacetate was calculated to be 5 microM after incubation of hepatocytes in the presence of 1.5 mM-lactate and 0.05 mM-oleate. Gradually increasing oleate up to 0.5 mM decreased the free oxaloacetate to 2 microM. Very similar results were obtained when free oxaloacetate concentration was derived from the CO2 production of hepatocytes as a measure of citrate flux through the tricarboxylic acid cycle, and the kinetic data on citrate synthase in situ. The decrease in free oxaloacetate on increasing oleate concentration was associated with lowered rates of cycle-dependent CO2 output and O2 uptake, indicating a decrease in the disposal of acetyl-CoA into the tricarboxylic acid cycle. This decrease could explain 25-30% of the increase in ketone-body production occurring at elevated fatty acid supply. This work documents on a quantitative basis the role of free oxaloacetate in the regulation of ketogenesis.
| S-EPMC1153321 | biostudies-other