scholarly journals Pyruvate Carboxylase Is an Essential Protein in the Assembly of Yeast Peroxisomal Oligomeric Alcohol Oxidase

2003 ◽  
Vol 14 (2) ◽  
pp. 786-797 ◽  
Author(s):  
Paulina Ozimek ◽  
Ralf van Dijk ◽  
Kantcho Latchev ◽  
Carlos Gancedo ◽  
Dong Yuan Wang ◽  
...  
Keyword(s):  
Pyruvate Carboxylase ◽  
Tricarboxylic Acid Cycle ◽  
Hansenula Polymorpha ◽  
Alcohol Oxidase ◽  
Methylotrophic Yeast ◽  
Tricarboxylic Acid ◽  
Matrix Proteins ◽  
Growth Media ◽  
Fad Binding

Hansenula polymorpha ass3 mutants are characterized by the accumulation of inactive alcohol oxidase (AO) monomers in the cytosol, whereas other peroxisomal matrix proteins are normally activated and sorted to peroxisomes. These mutants also have a glutamate or aspartate requirement on minimal media. Cloning of the corresponding gene resulted in the isolation of the H. polymorpha PYC gene that encodes pyruvate carboxylase (HpPyc1p). HpPyc1p is a cytosolic, anapleurotic enzyme that replenishes the tricarboxylic acid cycle with oxaloacetate. The absence of this enzyme can be compensated by addition of aspartate or glutamate to the growth media. We show that HpPyc1p protein but not the enzyme activity is essential for import and assembly of AO. Similar results were obtained in the related yeast Pichia pastoris. In vitro studies revealed that HpPyc1p has affinity for FAD and is capable to physically interact with AO protein. These data suggest that in methylotrophic yeast pyruvate carboxylase plays a dual role in that, besides its well-characterized metabolic function as anapleurotic enzyme, the protein fulfils a specific role in the AO sorting and assembly process, possibly by mediating FAD-binding to AO monomers.

Glucose utilization by sheep embryos derived in vivo and in vitro

1991 ◽  
Vol 3 (5) ◽  
pp. 571 ◽  
Author(s):  
JG Thompson ◽  
AC Simpson ◽  
PA Pugh ◽  
RW Wright ◽  
HR Tervit
Keyword(s):  
Glucose Oxidation ◽  
Glucose Utilization ◽  
Tricarboxylic Acid Cycle ◽  
Tricarboxylic Acid ◽  
Cell Stage ◽  
Glycolytic Activity ◽  
Total Utilization ◽  
Oxidation Of Glucose

Embryos were collected from superovulated donors at various intervals from onset of oestrus, ranging from Day 1.5 to Day 6. In addition, blastocysts obtained from the culture of 1-cell embryos collected in vivo or of oocytes matured and fertilized in vitro were used to assess the effects of in vitro manipulation and culture on glucose utilization. Glycolytic activity was determined by the conversion of [5-3H]glucose to 3H2O, and oxidation of glucose was determined by the conversion of [U-14C]glucose to 14CO2. Glucose utilization increases significantly from the 8-cell stage and during compaction and blastulation. Glucose oxidation was at a relatively low level (5-12% of total utilization) compared with glycolysis. No difference was observed between the glycolytic activity of blastocysts derived from in vivo or in vitro sources. However, glucose oxidation was lower (P less than 0.05) in blastocysts derived from the culture of 1-cell embryos or from oocytes matured and fertilized in vitro. Exogenous tricarboxylic acid cycle substrates (i.e. pyruvate and lactate supplied in the medium) affected the level of glucose oxidation.

scholarly journals Metabolic phases during the development of granulation tissue

1967 ◽  
Vol 105 (1) ◽  
pp. 333-341 ◽  
Author(s):  
Kirsti Lampiaho ◽  
E. Kulonen
Keyword(s):  
Granulation Tissue ◽  
Collagen Synthesis ◽  
Tricarboxylic Acid Cycle ◽  
Tricarboxylic Acid ◽  
Pentose Phosphate ◽  
Acid Cycle ◽  
Short Period ◽  
Tricarboxylic Acid Cycle Intermediates ◽  
Observation Period

1. The metabolism of incubated slices of sponge-induced granulation tissue, harvested 4–90 days after the implantation, was studied with special reference to the capacity of collagen synthesis and to the energy metabolism. Data are also given on the nucleic acid contents during the observation period. Three metabolic phases were evident. 2. The viability of the slices for the synthesis of collagen was studied in various conditions. Freezing and homogenization destroyed the capacity of the tissue to incorporate proline into collagen. 3. Consumption of oxygen reached the maximum at 30–40 days. There was evidence that the pentose phosphate cycle was important, especially during the phases of the proliferation and the involution. The formation of lactic acid was maximal at about 20 days. 4. The capacity to incorporate proline into collagen hydroxyproline in vitro was limited to a relatively short period at 10–30 days. 5. The synthesis of collagen was dependent on the supply of oxygen and glucose, which latter could be replaced in the incubation medium by other monosaccharides but not by the metabolites of glucose or tricarboxylic acid-cycle intermediates.

Determination of gluconeogenesis in vivo with 14C-labeled substrates

1985 ◽  
Vol 248 (4) ◽  
pp. R391-R399 ◽  
Author(s):  
J. Katz
Keyword(s):  
Pyruvate Carboxylase ◽  
Tricarboxylic Acid Cycle ◽  
Specific Radioactivity ◽  
Tricarboxylic Acid ◽  
Pyruvate Metabolism ◽  
Acetyl Coenzyme A ◽  
Glucose Carbon ◽  
Labeling Pattern

A mitochondrial model of gluconeogenesis and the tricarboxylic acid cycle, where pyruvate is metabolized via pyruvate carboxylase and pyruvate dehydrogenase, and pyruvate kinase is examined. The effect of the rate of tricarboxylic acid flux and the rates of the three reactions of pyruvate metabolism on the labeling patterns from [14C]pyruvate and [24C]acetate are analyzed. Expressions describing the specific radioactivities and 14C distribution in glucose as a function of these rates are derived. Specific radioactivities and isotopic patterns depend markedly on the ratio of the rates of pyruvate carboxylation and decarboxylation to the rate of citrate synthesis, but the effect of phosphoenolpyruvate hydrolysis is minor. The effects of these rates on 1) specific radioactivity of phosphoenolpyruvate, 2) labeling pattern in glucose, and 3) contribution of pyruvate, acetyl-coenzyme A, and CO2 to glucose carbon are illustrated. To determine the contribution of lactate or alanine to gluconeogenesis, experiments with two compounds labeled in different carbons are required. Methods in current use to correct for the dilution of 14C in gluconeogenesis from [14C]pyruvate are shown to be erroneous. The experimental design and techniques to determine gluconeogenesis from 14C-labeled precursors are presented and illustrated with numerical examples.

Tricarboxylic acid cycle dehydrogenases inhibition by naringenin: experimental and molecular modelling evidence

2020 ◽  
Vol 123 (10) ◽  
pp. 1117-1126
Author(s):  
Pauline Maciel August ◽  
Mateus Grings ◽  
Marcelo Sartori Grunwald ◽  
Geancarlo Zanatta ◽  
Vinícius Stone ◽  
...  
Keyword(s):  
Citrate Synthase ◽  
Tricarboxylic Acid Cycle ◽  
Cell Metabolism ◽  
Tricarboxylic Acid ◽  
Metabolic Effects ◽  
Similar Reduction ◽  
Acid Cycle ◽  
Docking Simulations ◽  
Female Wistar Rats

AbstractThe study of polyphenols’ effects on health has been gaining attention lately. In addition to reacting with important enzymes, altering the cell metabolism, these substances can present either positive or negative metabolic alterations depending on their consumption levels. Naringenin, a citrus flavonoid, already presents diverse metabolic effects. The objective of this work was to evaluate the effect of maternal naringenin supplementation during pregnancy on the tricarboxylic acid cycle activity in offspring’s cerebellum. Adult female Wistar rats were divided into two groups: (1) vehicle (1 ml/kg by oral administration (p.o.)) or (2) naringenin (50 mg/kg p.o.). The offspring were euthanised at 7th day of life, and the cerebellum was dissected to analyse citrate synthase, isocitrate dehydrogenase (IDH), α-ketoglutarate dehydrogenase (α-KGDH) and malate dehydrogenase (MDH) activities. Molecular docking used SwissDock web server and FORECASTER Suite, and the proposed binding pose image was created on UCSF Chimera. Data were analysed by Student’s t test. Naringenin supplementation during pregnancy significantly inhibited IDH, α-KGDH and MDH activities in offspring’s cerebellum. A similar reduction was observed in vitro, using purified α-KGDH and MDH, subjected to pre-incubation with naringenin. Docking simulations demonstrated that naringenin possibly interacts with dehydrogenases in the substrate and cofactor binding sites, inhibiting their function. Naringenin administration during pregnancy may affect cerebellar development and must be evaluated with caution by pregnant women and their physicians.

scholarly journals Up-regulation of glucose metabolism in Kupffer cells following infusion of tumour necrosis factor

1991 ◽  
Vol 278 (2) ◽  
pp. 515-519 ◽  
Author(s):  
Z Spolarics ◽  
G J Bagby ◽  
C H Lang ◽  
J J Spitzer
Keyword(s):  
Endothelial Cells ◽  
Kupffer Cells ◽  
Tumour Necrosis ◽  
Glucose Oxidation ◽  
Tricarboxylic Acid Cycle ◽  
Tricarboxylic Acid ◽  
Acid Cycle ◽  
Necrosis Factor

Alterations of glucose metabolism and the oxidation of glutamine and palmitate were studied, by using specifically labelled substrates, in freshly isolated Kupffer cells and hepatic endothelial cells after infusion in vivo of human recombinant tumour necrosis factor-alpha (TNF; 7.5 x 10(5) IU/30 min per kg body wt., intravenously). Cells were incubated in a medium containing 5 mM-glucose, 0.4 mM-palmitate, 1 mM-lactate and 0.5 mM-glutamine. Administration of TNF in vivo increased glucose use in Kupffer cells by 70%. Glucose oxidation in the tricarboxylic acid cycle and flux in the Embden-Meyerhof (EM) pathway were elevated by 40 and 80% respectively. Treatment in vitro with 1 microM-phorbol 12-myristate 13-acetate (PMA) resulted in a similar percentage increase in glucose use by Kupffer cells prepared from either saline- or TNF-treated rats. However, PMA increased the activity of the hexose monophosphate shunt (HMS) by 3- and 10-fold in cells isolated from saline- or TNF-infused animals respectively. A phagocyte stimulus in vitro, opsonized zymosan, increased glucose use by 30% and doubled the flux through the HMS in Kupffer cells from saline-infused animals. The activity of the HMS in response to zymosan was increased by 400% after TNF treatment. In endothelial cells, basal glucose utilization was not altered by TNF treatment. PMA increased HMS activity in endothelial cells to a similar degree after saline or TNF infusion. Zymosan, however, increased HMS activity only in endothelial cells from TNF-treated rats. Oxidation of palmitate or glutamine was not affected by TNF treatment either under basal conditions or after challenge in vitro. Our data indicate that, after phagocytosis in vitro or protein kinase C activation, glucose use and flux through the HMS increase in Kupffer cells. This is accompanied by increased glycolytic flux, with no changes in glucose oxidation in the tricarboxylic acid cycle. After TNF exposure, followed by a secondary stimulus, the enhanced glucose use by Kupffer cells is primarily channelled through the HMS pathway. These data suggest that the increased glucose use in vivo by Kupffer cells found after immune-stimulated conditions may subserve primarily the increased need for NADPH and HMS intermediates.

scholarly journals IN VITRO EFFECTS OF FLUORIDE ON TRICARBOXYLIC ACID CYCLE DEHYDROGENASES AND OXIDATIVE PHOSPHORYLATION: PART I

1967 ◽  
Vol 15 (4) ◽  
pp. 195-201 ◽  
Author(s):  
C. JAMES LOVELACE ◽  
GENE W. MILLER
Keyword(s):  
Oxidative Phosphorylation ◽  
Competitive Inhibition ◽  
Tricarboxylic Acid Cycle ◽  
Succinic Dehydrogenase ◽  
Tricarboxylic Acid ◽  
Initial Increase ◽  
Acid Cycle ◽  
Oxidase System ◽  
Vitro Effect

Studies were conducted on the in vitro effect of fluoride on the succinic oxidase system utilizing mitochondria obtained from cauliflower. Preincubation of mitochondria with fluoride did not increase inhibition of succinic oxidase. Various other tricarboxylic acid cycle substrates were used to determine their sensitivity to fluoride; only succinate oxidation was affected. A series of succinate concentrations in the presence and in the absence of fluoride showed increased activity of succinic dehydrogenase, which indicated competitive inhibition. Various concentrations of phosphate in the absence of fluoride showed that phosphate had only slight effects on the succinic 2,6-dichlorophenolindophenol reductase component of the succinic oxidase system. In the absence of phosphate, various concentrations of fluoride showed an initial increase in activity followed by a decrease in activity of succinic 2,6-dichlorophenolindophenol reductase. In the presence of phosphate, fluoride caused marked inhibition of succinic 2,6-dichlorophenolindophenol reductase. It is believed that this inhibition results from an enzyme-fluorophosphate complex which has a lower dissociation constant than that of the enzyme-substrate complex. An oxidative phosphorylation study indicated that both respiration and phosphorylation were inhibited.

scholarly journals Routing of Hansenula polymorpha Alcohol Oxidase: An Alternative Peroxisomal Protein-sorting Machinery

2004 ◽  
Vol 15 (3) ◽  
pp. 1347-1355 ◽  
Author(s):  
Katja Gunkel ◽  
Ralf van Dijk ◽  
Marten Veenhuis ◽  
Ida J. van der Klei
Keyword(s):  
Amino Acids ◽  
Protein Translocation ◽  
Hansenula Polymorpha ◽  
Alcohol Oxidase ◽  
N Terminus ◽  
Dependent Protein ◽  
Peptides Synthesis ◽  
Translocation Pathway ◽  
Tpr Domain ◽  
Fad Binding

Import of Hansenula polymorpha alcohol oxidase (AO) into peroxisomes is dependent on the PTS1 receptor, HpPex5p. The PTS1 of AO (-LARF) is sufficient to direct reporter proteins to peroxisomes. To study AO sorting in more detail, strains producing mutant AO proteins were constructed. AO containing a mutation in the FAD binding fold was mislocalized to the cytosol. This indicates that the PTS1 of AO is not sufficient for import of AO. AO protein in which the PTS1 was destroyed (-LARA) was normally sorted to peroxisomes. Moreover, C-terminal deletions of up to 16 amino acids did not significantly affect AO import, indicating that the PTS1 was not necessary for targeting. Consistent with these observations we found that AO import occurred independent from the C-terminal TPR-domain of HpPex5p, known to bind PTS1 peptides. Synthesis of the N-terminal domain (amino acids 1-272) of HpPex5p in pex5 cells restored AO import, whereas other PTS1 proteins were mislocalized to the cytosol. These data indicate that AO is imported via a novel HpPex5p-dependent protein translocation pathway, which does not require the PTS1 of AO and the C-terminal TPR domains of HpPex5p, but involves FAD binding and the N-terminus of HpPex5p.

scholarly journals Transamination of glutamate to tricarboxylic acid-cycle intermediates in cultured neurons correlates with the ability of oxo acids to support neuronal survival in vitro

1986 ◽  
Vol 234 (3) ◽  
pp. 605-610 ◽  
Author(s):  
L Facci ◽  
S D Skaper ◽  
S Varon
Keyword(s):  
Amino Acid ◽  
Neuronal Survival ◽  
Tricarboxylic Acid Cycle ◽  
Tricarboxylic Acid ◽  
Neuronal Cultures ◽  
Acid Cycle ◽  
Ciliary Ganglion Neurons ◽  
Amino Acid Precursors ◽  
Ganglion Neurons

Cultures of central-nervous-system neurons at low densities require for their survival exogenous pyruvate, alpha-oxoglutarate or oxaloacetate, even in the presence of high glucose concentrations. Most other alpha-oxo acids support cell survival only in the presence of alpha-amino acids which transaminate to alpha-oxoglutarate, oxaloacetate or pyruvate. The alpha-oxo acids therefore operate as acceptors of amino groups from appropriate donors to generate tricarboxylic acid-cycle-relevant substrates, and these alpha-oxo acids provide for neuronal support only insofar as they make it possible for exogenously supplied alpha-amino acid precursors to generate intracellularly one of the three critical metabolites. To examine more closely the relationship between transamination activity and neuronal survival, we measured 14CO2 production from [14C]glutamate in the presence of appropriate alpha-oxo acid partners by using 8-day-embryonic chick forebrain, dorsal-root-ganglion and ciliary-ganglion neurons. Neuronal survival was measured concurrently in monolayer neuronal cultures maintained with the corresponding amino acid/oxo acid pairs. Forebrain and ganglionic cell suspensions both produced 14CO2 from [14C]glutamate, which accurately correlated with 24 h neuronal survival. Concentrations of glutamate or alpha-oxo acid which provide for maximal neuronal survival also produced maximal amounts of 14CO2. The same ability to generate CO2 from glutamate (in the presence of the appropriate alpha-oxo acids) can ensure neuronal survival in 24 h cultures and therefore must meet energy or other metabolic needs of those neurons which glucose itself is unable to satisfy.

scholarly journals Studies on the Metabolism of Locust Fat Body

1959 ◽  
Vol 36 (4) ◽  
pp. 665-675
Author(s):  
A. N. CLEMENTS
Keyword(s):  
Amino Acids ◽  
Sodium Acetate ◽  
Flight Muscle ◽  
Fat Body ◽  
Enzyme System ◽  
Tricarboxylic Acid Cycle ◽  
Succinic Dehydrogenase ◽  
Tricarboxylic Acid ◽  
Acid Cycle

1. The incorporation of glycine-14C (G), leucine-14C (G), sodium acetate-2-14C and glucose-14C (G) into Schistocerca fat body was studied under in vitro conditions, and the distribution of radioactivity in the various fat body fractions and the labelling of compounds within the fractions is described. 2. The overall picture was of high incorporation into fat and protein and of very low incorporation into glycogen. 3. Incubation with glycine-14C led to radioactivity appearing in the glycine and serine of the protein and of the amino acid pool. Incubation with sodium acetate-2-14C led to radioactivity appearing in glutamate, proline, aspartate and alanine, showing that the intermediates of the tricarboxylic acid cycle provide the carbon skeletons of certain amino acids. Glucose-14C was largely converted to trehalose. 4. Succinic dehydrogenase and the condensing enzyme system were shown to be present in fat body, contrary to previous reports. The succinic oxidase system was highly labile on homogenizing the tissue. 5. Fat body, unlike flight muscle, used glycine-14C and leucine-14C as respiratory substrates, and it is suggested that fat body acts like the vertebrate liver by transdeaminating amino acids and making them available for further metabolism by other tissues.

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