Amino acid oxidation and alanine production in rat hemidiaphragm in vitro. Effects of dichloroacetate.
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ABSTRACT:
SUBMITTER: Palmer TN
PROVIDER: S-EPMC1144271 | biostudies-other | 1984 Oct
REPOSITORIES: biostudies-other
Similar Datasets
Project description:Dichloroacetate (which activates pyruvate dehydrogenase) decreases the release of alanine, pyruvate and lactate in hemidiaphragm incubations with valine. Dichloroacetate interferes with alanine formation by diverting pyruvate into oxidative pathways, which not only limits pyruvate availability for direct transamination to form alanine but also indirectly affects branched-chain amino acid transamination by limiting 2-oxoglutarate regeneration from glutamate.
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Project description:Muscle branched-chain amino acid metabolism is coupled to alanine formation via branched-chain amino acid aminotransferase and alanine aminotransferase, but the subcellular distributions of these and other associated enzymes are uncertain. Recovery of branched-chain aminotransferase in the cytosol fraction after differential centrifugation was shown to be accompanied by leakage of mitochondrial-matrix marker enzymes. By using a differential fractional extraction procedure, most of the branched-chain aminotransferase activity in rat muscle was located in the mitochondrial compartment, whereas alanine aminotransferase was predominantly in the cytosolic compartment. Phosphoenolpyruvate carboxykinase, like aspartate aminotransferase, was approximately equally distributed between these subcellular compartments. This arrangement necessitates a transfer of branched-chain amino nitrogen and carbon from the mitochondria to the cytosol for alanine synthesis de novo to occur. In incubations of hemidiaphragms from 48 h-starved rats with 3mM-valine or 3mM-glutamate, the stimulation of alanine release was inhibited by 69% by 1 mM-aminomethoxybut-3-enoate, a selective inhibitor of aspartate aminotransferase. Leucine-stimulated alanine release was unaffected. These data implicate aspartate aminotransferase in the transfer of amino acid carbon and nitrogen from the mitochondria to the cytosol, and suggest that oxaloacetate, via phosphoenolpyruvate carboxykinase, can serve as an intermediate on the route of pyruvate formation for muscle alanine synthesis.
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Project description:An N-carbamoyl-beta-alanine amidohydrolase of industrial interest from Agrobacterium tumefaciens C58 (beta car(At)) has been characterized. Beta car(At) is most active at 30 degrees C and pH 8.0 with N-carbamoyl-beta-alanine as a substrate. The purified enzyme is completely inactivated by the metal-chelating agent 8-hydroxyquinoline-5-sulfonic acid (HQSA), and activity is restored by the addition of divalent metal ions, such as Mn(2+), Ni(2+), and Co(2+). The native enzyme is a homodimer with a molecular mass of 90 kDa from pH 5.5 to 9.0. The enzyme has a broad substrate spectrum and hydrolyzes nonsubstituted N-carbamoyl-alpha-, -beta-, -gamma-, and -delta-amino acids, with the greatest catalytic efficiency for N-carbamoyl-beta-alanine. Beta car(At) also recognizes substrate analogues substituted with sulfonic and phosphonic acid groups to produce the beta-amino acids taurine and ciliatine, respectively. Beta car(At) is able to produce monosubstituted beta(2)- and beta(3)-amino acids, showing better catalytic efficiency (k(cat)/K(m)) for the production of the former. For both types of monosubstituted substrates, the enzyme hydrolyzes N-carbamoyl-beta-amino acids with a short aliphatic side chain better than those with aromatic rings. These properties make beta car(At) an outstanding candidate for application in the biotechnology industry.
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Project description:Amino acid analyses of mitochondrial membranes are compared with the amino acid composition of whole mitochondria (Alberti, 1964) and found to be very similar except in the cystine content. The composition of the endogenous amino acids found in freshly prepared mitochondria has been established and shown to differ considerably from the amino acid composition of membranes or whole mitochondria. The amino acids produced during anaerobic incubation of mitochondria at pH7.4, on the other hand, resemble the membrane in composition, supporting the view that neutral proteinase activity is responsible for their appearance. Aerobic incubation produces a similar pattern of amino acids except that amino acids such as proline, serine, asparagine, glutamic acid and glutamine, which can be metabolically utilized under aerobic conditions, are present to a smaller extent. The presence of large relative concentrations of endogenous taurine, cysteic acid and oxidized glutathione and the accumulation of taurine during incubation is found. The selective retention of taurine and cysteic acid within the mitochondria is established. It is proposed that the first step in the degeneration of isolated mitochondria results from lipid hydroperoxide accumulation caused by the lack of glutathione reductase in isolated mitochondria.
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Project description:Significant body of evidence suggests that abnormal kynurenic acid (KYNA) level is involved in the pathophysiology of central nervous system disorders. In the brain, KYNA is synthesized from kynurenine (KYN) by kynurenine aminotransferases (KATs), predominantly by KAT II isoenzyme. Blockage of ionotropic glutamate (GLU) receptors is a main cellular effect of KYNA. High KYNA levels have been linked with psychotic symptoms and cognitive dysfunction in animals and humans. As immunological imbalance and impaired glutamatergic neurotransmission are one of the crucial processes in neurological pathologies, we aimed to analyze the effect of anti-inflammatory agents, inhibitors of cyclooxygenase-2 (COX-2): celecoxib, niflumic acid, and parecoxib, on KYNA synthesis and KAT II activity in rat brain in vitro. The influence of COX-2 inhibitors was examined in rat brain cortical slices and on isolated KAT II enzyme. Niflumic acid and parecoxib decreased in a dose-dependent manner KYNA production and KAT II activity in rat brain cortex in vitro, whereas celecoxib was ineffective. Molecular docking results suggested that niflumic acid and parecoxib interact with an active site of KAT II. In conclusion, niflumic acid and parecoxib are dual COX-2 and KAT II inhibitors.
| S-EPMC6313367 | biostudies-literature
Project description:In a sheep model of intrauterine growth restriction (IUGR) produced from placental insufficiency, late gestation fetuses had smaller skeletal muscle mass, myofiber area, and slower muscle protein accretion rates compared with normally growing fetuses. We hypothesized that IUGR fetal muscle develops adaptations that divert amino acids (AAs) from protein accretion and activate pathways that conserve substrates for other organs. We placed hindlimb arterial and venous catheters into late gestation IUGR (n = 10) and control (CON, n = 8) fetal sheep and included an external iliac artery flow probe to measure hindlimb AA uptake rates. Arterial and venous plasma samples and biceps femoris muscle were analyzed by mass spectrometry-based metabolomics. IUGR fetuses had greater abundance of metabolites enriched within the alanine, aspartate, and glutamate metabolism pathway compared with CON. Net uptake rates of branched-chain AA (BCAA) were lower by 42%-73%, and muscle ammoniagenic AAs (alanine, glycine, and glutamine) were lower by 107%-158% in IUGR hindlimbs versus CON. AA uptake rates correlated with hindlimb weight; the smallest hindlimbs showed net release of ammoniagenic AAs. Gene expression levels indicated a decrease in BCAA catabolism in IUGR muscle. Plasma purines were lower and plasma uric acid was higher in IUGR versus CON, possibly a reflection of ATP conservation. We conclude that IUGR skeletal muscle has lower BCAA uptake and develops adaptations that divert AAs away from protein accretion into alternative pathways that sustain global energy production and nitrogen disposal in the form of ammoniagenic AAs for metabolism in other organs.
| S-EPMC6879841 | biostudies-literature
Project description:Protein supplementation during human pregnancy does not improve fetal growth and may increase small-for-gestational-age birth rates and mortality. To define possible mechanisms, sheep with twin pregnancies were infused with amino acids (AA group, n = 7) or saline (C group, n = 4) for 4 days during late gestation. In the AA group, fetal plasma leucine, isoleucine, valine, and lysine concentrations were increased (P < 0.05), and threonine was decreased (P < 0.05). In the AA group, fetal arterial pH (7.365 +/- 0.007 day 0 vs. 7.336 +/- 0.012 day 4, P < 0.005), hemoglobin-oxygen saturation (46.2 +/- 2.6 vs. 37.8 +/- 3.6%, P < 0.005), and total oxygen content (3.17 +/- 0.17 vs. 2.49 +/- 0.20 mmol/l, P < 0.0001) were decreased on day 4 compared with day 0. Fetal leucine disposal did not change (9.22 +/- 0.73 vs. 8.09 +/- 0.63 micromol x min(-1) x kg(-1), AA vs. C), but the rate of leucine oxidation increased 43% in the AA group (2.63 +/- 0.16 vs. 1.84 +/- 0.24 micromol x min(-1) x kg(-1), P < 0.05). Fetal oxygen utilization tended to be increased in the AA group (327 +/- 23 vs. 250 +/- 29 micromol x min(-1) x kg(-1), P = 0.06). Rates of leucine incorporation into fetal protein (5.19 +/- 0.97 vs. 5.47 +/- 0.89 micromol x min(-1) x kg(-1), AA vs. C), release from protein breakdown (4.20 +/- 0.95 vs. 4.62 +/- 0.74 micromol x min(-1) x kg(-1)), and protein accretion (1.00 +/- 0.30 vs. 0.85 +/- 0.25 micromol x min(-1) x kg(-1)) did not change. Consistent with these data, there was no change in the fetal skeletal muscle ubiquitin ligases MaFBx1 or MuRF1 or in the protein synthesis regulators 4E-BP1, eEF2, eIF2alpha, and p70(S6K). Decreased concentrations of certain essential amino acids, increased amino acid oxidation, fetal acidosis, and fetal hypoxia are possible mechanisms to explain fetal toxicity during maternal amino acid supplementation.
| S-EPMC2739698 | biostudies-literature
Project description:A short sequence of 11 amino acids belonging to the cj0669 protein from Campylobacter jejuni NCTC 11168, which was previously identified as potentially immunogenic, was analyzed via alanine scanning to narrow down the significant amino acid residues within the sequence. Twelve peptides, one representing the original sequence and eleven peptides with each residue replaced by alanine in turn, were synthesized on microarrays by JPTs Pepstar Technology. For each microarray, nine replicates for each peptide were spotted. The microarray was separated into three incubation chambers by the ProPlate 3-well module (Grace Biolabs) to allow for incubation with different antibodies in parallel. For specific interactions, rabbit polyclonal IgG to C. jejuni was used, while unspecific binding to the epitope sequence was checked using rabbit polyclonal IgG to Salmonella enterica.
2013-04-06 | E-GEOD-45556 | biostudies-arrayexpress
Project description:A short sequence of 11 amino acids belonging to the cj0669 protein from Campylobacter jejuni NCTC 11168 which was previously identified as potentially immunogenig was analyzed via alanine scanning to narrow down the significant amino acid residues within the sequence. Twelve peptides, one representing the original sequence and eleven peptides with each residue replaced by alanine in turn, were synthesized on microarrays by JPTs Pepstar Technology. For each microarray, nine replicates for each peptide were spotted. The microarray was seperated into three incubation chambers by the ProPlate 3-well module (Grace Biolabs) to allow for incubation with different antibodies in parallel. For specific interaction rabbit polyclonal IgG to C.jejuni was used, while non-specific binding to the epitope sequence was checked using rabbit polyclonal IgG to H. pylori (Abcam ab20459).
2013-05-06 | E-GEOD-46651 | biostudies-arrayexpress
Project description:A short sequence of 11 amino acids belonging to the cj0669 protein from Campylobacter jejuni NCTC 11168 which was previously identified as potentially immunogenig was analyzed via alanine scanning to narrow down the significant amino acid residues within the sequence.
2013-05-06 | GSE46651 | GEO