scholarly journals Amino acid oxidation and alanine production in rat hemidiaphragm in vitro. Effects of dichloroacetate

1984 ◽  
Vol 223 (1) ◽  
pp. 113-117 ◽  
Author(s):  
T N Palmer ◽  
M A Caldecourt ◽  
M C Sugden
Keyword(s):  
Amino Acids ◽  
Pyruvate Dehydrogenase ◽  
Tricarboxylic Acid Cycle ◽  
Acid Oxidation ◽  
14Co2 Production ◽  
In Vitro Effects ◽  
Tricarboxylic Acid Cycle Intermediates ◽  
Rate Limiting ◽  
Main Carbon

Dichloroacetate (an activator of pyruvate dehydrogenase) stimulates 14CO2 production from [U-14C]glucose, but not from [U-14C]glutamate, [U-14C]aspartate, [U-14C]- and [1-14C]-valine and [U-14C]- and [1-14C]-leucine. It is concluded (1) that pyruvate dehydrogenase is not rate-limiting in the oxidation to CO2 of amino acids that are metabolized to tricarboxylic acid-cycle intermediates, and (2) that carbohydrate (and not amino acids) is the main carbon precursor in alanine formation in muscle.

scholarly journals Adrenergic inhibition of branched-chain 2-oxo acid dehydrogenase in rat diaphragm muscle in vitro

1983 ◽  
Vol 216 (1) ◽  
pp. 63-70 ◽  
Author(s):  
T N Palmer ◽  
M A Caldecourt ◽  
M C Sugden
Keyword(s):  
Cyclic Amp ◽  
Tricarboxylic Acid Cycle ◽  
Diaphragm Muscle ◽  
Dibutyryl Cyclic Amp ◽  
Adrenergic Agents ◽  
Acid Dehydrogenase ◽  
14Co2 Production ◽  
Branched Chain ◽  
Tricarboxylic Acid Cycle Intermediates

In theory, the complete oxidation to CO2 of amino acids that are metabolized by conversion into tricarboxylic acid-cycle intermediates may proceed via their conversion into acetyl-CoA. The possible adrenergic modulation of this oxidative pathway was investigated in isolated hemidiaphragms from 40 h-starved rats. Adrenaline (5.5 microM), phenylephrine (0.49 mM) and dibutyryl cyclic AMP (10 microM) inhibited 14CO2 production from 3 mM-[U-14C]valine by 35%, 28% and 19% respectively. At the same time, these agents stimulated glycogen mobilization (measured as a decrease in glycogen content) and glycolysis (measured as lactate release). Adrenaline, phenylephrine and dibutyryl cyclic AMP did not inhibit 14CO2 production from 3 mM-[U-14C]aspartate or 3 mM-[U-14C]glutamate, although, as in the presence of valine, the agents stimulated glycogen mobilization and glycolysis. The rate of proteolysis (measured as tyrosine release in the presence of cycloheximide) was not changed by adrenaline. The data indicate that the adrenergic inhibition of 14CO2 production from [U-14C]valine was not a consequence of radiolabel dilution. Inhibition was apparently specific for branched-chain amino acid metabolism in that the adrenergic agonists also inhibited 14CO2 production from [1-14C]valine, [1-14C]leucine and [U-14C]isoleucine. Since 14CO2 production from the 1-14C-labelled substrates is a specific measure of decarboxylation in the reaction catalysed by the branched-chain 2-oxo acid dehydrogenase complex, it is at this site that the adrenergic agents are concluded to act.

scholarly journals Glycolytic origin of alanine formed in rat diaphragm muscle in vitro

1985 ◽  
Vol 231 (3) ◽  
pp. 801-804 ◽  
Author(s):  
M A Caldecourt ◽  
D J Cox ◽  
M C Sugden ◽  
T N Palmer
Keyword(s):  
Amino Acids ◽  
De Novo ◽  
Tricarboxylic Acid Cycle ◽  
Tricarboxylic Acid ◽  
Diaphragm Muscle ◽  
Rat Diaphragm ◽  
Acid Cycle ◽  
Tricarboxylic Acid Cycle Intermediates ◽  
Alanine Production

In quarter-diaphragms from 40 h-starved rats the rate of glycogen mobilization is sufficient to account for the rate of lactate+pyruvate+alanine production. It is concluded, therefore, that alanine derives its carbon skeleton predominantly via glycolysis and not via synthesis de novo from tricarboxylic acid-cycle intermediates and related amino acids.

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.

scholarly journals The rate-limiting step of protein synthesis in vivo and in vitro and the distribution of growing peptides between the puromycinlabile and puromycin-non-labile sites on polyribosomes

1971 ◽  
Vol 122 (3) ◽  
pp. 267-276 ◽  
Author(s):  
D. C. N. Earl ◽  
Susan T. Hindley
Keyword(s):  
Amino Acids ◽  
Protein Synthesis ◽  
Donor Site ◽  
Peptide Bond ◽  
Rate Limiting Step ◽  
Limiting Step ◽  
Donor And Acceptor ◽  
Rate Limiting

1. At 3 min after an intravenous injection of radioactive amino acids into the rat, the bulk of radioactivity associated with liver polyribosomes can be interpreted as growing peptides. 2. In an attempt to identify the rate-limiting step of protein synthesis in vivo and in vitro, use was made of the action of puromycin at 0°C, in releasing growing peptides only from the donor site, to study the distribution of growing peptides between the donor and acceptor sites. 3. Evidence is presented that all growing peptides in a population of liver polyribosomes labelled in vivo are similarly distributed between the donor and acceptor sites, and that the proportion released by puromycin is not an artifact of methodology. 4. The proportion released by puromycin is about 50% for both liver and muscle polyribosomes labelled in vivo, suggesting that neither the availability nor binding of aminoacyl-tRNA nor peptide bond synthesis nor translocation can limit the rate of protein synthesis in vivo. Attempts to alter this by starvation, hypophysectomy, growth hormone, alloxan, insulin and partial hepatectomy were unsuccessful. 5. Growing peptides on liver polyribosomes labelled in a cell-free system in vitro or by incubating hemidiaphragms in vitro were largely in the donor site, suggesting that either the availability or binding of aminoacyl-tRNA, or peptide bond synthesis, must be rate limiting in vitro and that the rate-limiting step differs from that in vivo. 6. Neither in vivo nor in the hemidiaphragm system in vitro was a correlation found between the proportion of growing peptides in the donor site and changes in the rate of incorporation of radioactivity into protein. This could indicate that the intracellular concentration of amino acids or aminoacyl-tRNA limits the rate of protein synthesis and that the increased incorporation results from a rise to a higher but still suboptimum concentration.

Tricarboxylic acid cycle activity in perfused rat lungs after O2 exposure

1992 ◽  
Vol 262 (4) ◽  
pp. L495-L501 ◽  
Author(s):  
D. J. Bassett ◽  
S. S. Reichenbaugh
Keyword(s):  
Tricarboxylic Acid Cycle ◽  
Tricarboxylic Acid ◽  
Enzyme Inactivation ◽  
Pyridine Nucleotides ◽  
Acid Cycle ◽  
Metabolic Conditions ◽  
Steady State Levels ◽  
14Co2 Production ◽  
Tricarboxylic Acid Cycle Intermediates

O2-induced impairment of mitochondrial energy generation was examined in intact lungs isolated from rats after 18-30 h exposure to either air or 100% O2 in vivo. Mitochondrial metabolic rates were determined by separate measurements of 14CO2 production from [1-14C]pyruvate and [U-14C]palmitate, perfused under normal and stimulated metabolic conditions brought about by perfusion with the uncoupler of oxidative phosphorylation, 2,4-dinitrophenol (DNP). In the absence of DNP, O2 exposure did not significantly alter 14CO2 productions from either substrate. DNP increased lung pyruvate and palmitate catabolism to CO2 twofold in air-exposed lungs but did not alter 14CO2 production in lungs isolated from O2-exposed rats. These data demonstrated an O2-induced impairment of maximal mitochondrial metabolism of both pyruvate and palmitate that could not be explained by alterations in tissue free coenzyme A or by loss of pyridine nucleotides. However, comparisons of the steady-state levels of tricarboxylic acid cycle intermediates between O2- and air-exposed lungs did identify isocitrate dehydrogenase as a possible site of O2-induced enzyme inactivation.

scholarly journals Branched-chain amino acid metabolism and alanine formation in rat diaphragm muscle in vitro. Effects of dichloroacetate

1984 ◽  
Vol 223 (3) ◽  
pp. 831-835 ◽  
Author(s):  
K Snell ◽  
D A Duff
Keyword(s):  
Amino Acid ◽  
Pyruvate Dehydrogenase ◽  
Amino Acid Metabolism ◽  
Acid Metabolism ◽  
Diaphragm Muscle ◽  
Rat Diaphragm ◽  
Branched Chain Amino Acid ◽  
In Vitro Effects ◽  
Branched Chain

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.

scholarly journals Degradation of Aromatics and Chloroaromatics by Pseudomonas sp. Strain B13: Cloning, Characterization, and Analysis of Sequences Encoding 3-Oxoadipate:Succinyl-Coenzyme A (CoA) Transferase and 3-Oxoadipyl-CoA Thiolase

2002 ◽  
Vol 184 (1) ◽  
pp. 216-223 ◽  
Author(s):  
Markus Göbel ◽  
Kerstin Kassel-Cati ◽  
Eberhard Schmidt ◽  
Walter Reineke
Keyword(s):  
Amino Acids ◽  
Coenzyme A ◽  
Tricarboxylic Acid Cycle ◽  
Tricarboxylic Acid ◽  
Open Reading Frames ◽  
Amino Acid Residues ◽  
Coa Transferase ◽  
Tricarboxylic Acid Cycle Intermediates ◽  
Reading Frames

ABSTRACT 3-Oxoadipate:succinyl-coenzyme A (CoA) transferase and 3-oxoadipyl-CoA thiolase carry out the ultimate steps in the conversion of benzoate and 3-chlorobenzoate to tricarboxylic acid cycle intermediates in bacteria utilizing the 3-oxoadipate pathway. This report describes the characterization of DNA fragments with the overall length of 5.9 kb from Pseudomonas sp. strain B13 that encode these enzymes. DNA sequence analysis revealed five open reading frames (ORFs) plus an incomplete one. ORF1, of unknown function, has a length of 414 bp. ORF2 (catI) encodes a polypeptide of 282 amino acids and starts at nucleotide 813. ORF3 (catJ) encodes a polypeptide of 260 amino acids and begins at nucleotide 1661. CatI and CatJ are the subunits of the 3-oxoadipate:succinyl-CoA transferase, whose activity was demonstrated when both genes were ligated into expression vector pET11a. ORF4, termed catF, codes for a protein of 401 amino acid residues with a predicted mass of 41,678 Da with 3-oxoadipyl-CoA thiolase activity. The last three ORFs seem to form an operon since they are oriented in the same direction and showed an overlapping of 1 bp between catI and catJ and of 4 bp between catJ and catF. Conserved functional groups important for the catalytic activity of CoA transferases and thiolases were identified in CatI, CatJ, and CatF. ORF5 (catD) encodes the 3-oxoadipate enol-lactone hydrolase. An incomplete ORF6 of 1,183 bp downstream of ORF5 and oriented in the opposite direction was found. The protein sequence deduced from ORF6 showed a putative AMP-binding domain signature.

scholarly journals Cysteine and iron accelerate the formation of ribose-5-phosphate, providing insights into the evolutionary origins of the metabolic network structure

PLoS Biology ◽  
2021 ◽  
Vol 19 (12) ◽  
pp. e3001468
Author(s):  
Gabriel Piedrafita ◽  
Sreejith J. Varma ◽  
Cecilia Castro ◽  
Christoph Messner ◽  
Lukasz Szyrwiel ◽  
...  
Keyword(s):  
Amino Acids ◽  
Metabolic Network ◽  
Reaction Network ◽  
High Specificity ◽  
Pentose Phosphate ◽  
Evolutionary Origins ◽  
Metal Catalyzed ◽  
Rate Limiting ◽  
Nonenzymatic Reactions

The structure of the metabolic network is highly conserved, but we know little about its evolutionary origins. Key for explaining the early evolution of metabolism is solving a chicken–egg dilemma, which describes that enzymes are made from the very same molecules they produce. The recent discovery of several nonenzymatic reaction sequences that topologically resemble central metabolism has provided experimental support for a “metabolism first” theory, in which at least part of the extant metabolic network emerged on the basis of nonenzymatic reactions. But how could evolution kick-start on the basis of a metal catalyzed reaction sequence, and how could the structure of nonenzymatic reaction sequences be imprinted on the metabolic network to remain conserved for billions of years? We performed an in vitro screening where we add the simplest components of metabolic enzymes, proteinogenic amino acids, to a nonenzymatic, iron-driven reaction network that resembles glycolysis and the pentose phosphate pathway (PPP). We observe that the presence of the amino acids enhanced several of the nonenzymatic reactions. Particular attention was triggered by a reaction that resembles a rate-limiting step in the oxidative PPP. A prebiotically available, proteinogenic amino acid cysteine accelerated the formation of RNA nucleoside precursor ribose-5-phosphate from 6-phosphogluconate. We report that iron and cysteine interact and have additive effects on the reaction rate so that ribose-5-phosphate forms at high specificity under mild, metabolism typical temperature and environmental conditions. We speculate that accelerating effects of amino acids on rate-limiting nonenzymatic reactions could have facilitated a stepwise enzymatization of nonenzymatic reaction sequences, imprinting their structure on the evolving metabolic network.

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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