The role of phosphoenolpyruvate carboxykinase in muscle alanine synthesis

3-Mercaptopicolinic acid (3-MPA) is reportedly a specific inhibitor of phosphoenolpyruvate (PEP) carboxykinase and has hitherto been used accordingly to elucidate the metabolic role of PEP carboxykinase in vitro and in vivo. We show that 3-MPA has multiple effects on intermediary metabolism in hemidiaphragms from 40 h-starved rats. It decreases the release of lactate + pyruvate and alanine in hemidiaphragms provided with no added substrate or with valine, leucine or isoleucine. Moreover, irrespective of the substrate provided (none, valine, leucine, isoleucine, glucose, acetate, oleate), 3-MPA decreases the [lactate]/[pyruvate] ratio. 3-MPA is without effect on 14CO2 production from [U-14C]valine, [1-14C]valine, [1-14C]leucine, [U-14C]isoleucine or [1-14C]oleate, but stimulates 14CO2 production from [U-14C]glucose and [1-14C]pyruvate and inhibits 14CO2 production from [1-14C]acetate. Glycolytic flux (measured as 3H2O formation from [5-3H]glucose) is stimulated by 3-MPA. It is concluded that 3-MPA has site(s) of actions other than PEP carboxykinase and that the putative role of PEP carboxykinase in alanine synthesis de novo in skeletal muscle from tricarboxylic acid-cycle intermediates and related amino acids requires reappraisal.

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Stimulation of [1-14C]oleate oxidation to 14CO2 in isolated rat hepatocytes by the catecholamines, vasopressin and angiotensin. A possible mechanism of action

Biochemical Journal ◽

10.1042/bj2120085 ◽

1983 ◽

Vol 212 (1) ◽

pp. 85-91 ◽

Cited By ~ 18

Author(s):

M C Sugden ◽

D I Watts

Keyword(s):

Phosphoenolpyruvate Carboxykinase ◽

Tricarboxylic Acid Cycle ◽

Rat Hepatocytes ◽

Tricarboxylic Acid ◽

Isolated Rat Hepatocytes ◽

Acid Cycle ◽

14Co2 Production ◽

Oxoglutarate Dehydrogenase ◽

Stimulation Of

Adrenaline, noradrenaline, vasopressin and angiotensin increased 14CO2 production from [1-14C]oleate by hepatocytes from fed rats but not by hepatocytes from starved rats. The hormones did not increase 14CO2 production when hepatocytes from fed rats were depleted of glycogen in vitro. Increased 14CO2 production from]1-14C]oleate in response to the hormones was observed when hepatocytes from starved rats were incubated with 3-mercaptopicolinate, an inhibitor of phosphoenolpyruvate carboxykinase. 3-Mercaptopicolinate inhibited uptake and esterification of [1-14C]oleate, slightly increased 14CO2 production from [1-14C]oleate and greatly increased the [3-hydroxybutyrate]/[acetoacetate] ratio. In the presence of 3-mercaptopicolinate 14CO2 production in response to the catecholamines was blocked by the α-antagonist phentolamine and required extracellular Ca2+. The effects of vasopressin and angiotensin were also Ca2+-dependent. The actions of the hormones of 14CO2 production from [I-14C]oleate by hepatocytes from starved rats in the presence of 3-mercaptopicolinate thus have the characteristics of the response to the hormones found with hepatocytes from fed rats incubated without 3-mercaptopicolinate. The stimulatory effects of the hormones on 14CO2 production from [1-14C]oleate were not the result of decreased esterification (as the hormones increased esterification) or increased β-oxidation. It is suggested that the effect of the hormones to increase 14CO2 production from [1-14C]oleate are mediated by CA2+-activation of NAD+-linked isocitrate dehydrogenase, the 2-oxoglutarate dehydrogenase complex, and/or electron transport. The results also demonstrate that when the supply of oxaloacetate is limited it is utilized for gluconeogenesis rather than to maintain tricarboxylic acid-cycle flux.

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Regulation of l-Alanine Dehydrogenase in Rhizobium leguminosarum bv. viciae and Its Role in Pea Nodules

Journal of Bacteriology ◽

10.1128/jb.186.3.842-849.2004 ◽

2004 ◽

Vol 186 (3) ◽

pp. 842-849 ◽

Cited By ~ 26

Author(s):

Emma Lodwig ◽

Shalini Kumar ◽

David Allaway ◽

Alex Bourdes ◽

Jürgen Prell ◽

...

Keyword(s):

Rhizobium Leguminosarum ◽

De Novo ◽

Dry Weight ◽

Northern Blotting ◽

Alanine Dehydrogenase ◽

Living Culture ◽

Optimal Functioning ◽

Alanine Synthesis ◽

Sole Product

ABSTRACT Alanine dehydrogenase (AldA) is the principal enzyme with which pea bacteroids synthesize alanine de novo. In free-living culture, AldA activity is induced by carboxylic acids (succinate, malate, and pyruvate), although the best inducer is alanine. Measurement of the intracellular concentration of alanine showed that AldA contributes to net alanine synthesis in laboratory cultures. Divergently transcribed from aldA is an AsnC type regulator, aldR. Mutation of aldR prevents induction of AldA activity. Plasmid-borne gusA fusions showed that aldR is required for transcription of both aldA and aldR; hence, AldR is autoregulatory. However, plasmid fusions containing the aldA-aldR intergenic region could apparently titrate out AldR, sometimes resulting in a complete loss of AldA enzyme activity. Therefore, integrated aldR::gusA and aldA::gusA fusions, as well as Northern blotting, were used to confirm the induction of aldA activity. Both aldA and aldR were expressed in the II/III interzone and zone III of pea nodules. Overexpression of aldA in bacteroids did not alter the ability of pea plants to fix nitrogen, as measured by acetylene reduction, but caused a large reduction in the size and dry weight of plants. This suggests that overexpression of aldA impairs the ability of bacteroids to donate fixed nitrogen that the plant can productively assimilate. We propose that the role of AldA may be to balance the alanine level for optimal functioning of bacteroid metabolism rather than to synthesize alanine as the sole product of N2 reduction.

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Identification of In Vivo Enzyme Activities in the Cometabolism of Glucose and Acetate by Saccharomyces cerevisiae by Using 13C-Labeled Substrates

Eukaryotic Cell ◽

10.1128/ec.2.3.599-608.2003 ◽

2003 ◽

Vol 2 (3) ◽

pp. 599-608 ◽

Cited By ~ 44

Author(s):

Margarida Moreira dos Santos ◽

Andreas Karoly Gombert ◽

Bjarke Christensen ◽

Lisbeth Olsson ◽

Jens Nielsen

Keyword(s):

Saccharomyces Cerevisiae ◽

Enzyme Activities ◽

Malic Enzyme ◽

Phosphoenolpyruvate Carboxykinase ◽

Tricarboxylic Acid Cycle ◽

Central Carbon Metabolism ◽

Isopropylmalate Synthase ◽

Very High

ABSTRACT A detailed characterization of the central metabolic network of Saccharomyces cerevisiae CEN.PK 113-7D was carried out during cometabolism of different mixtures of glucose and acetate, using aerobic C-limited chemostats in which one of these two substrates was labeled with 13C. To confirm the role of malic enzyme, an isogenic strain with the corresponding gene deleted was grown under the same conditions. The labeling patterns of proteinogenic amino acids were analyzed and used to estimate metabolic fluxes and/or make inferences about the in vivo activities of enzymes of the central carbon metabolism and amino acid biosynthesis. Malic enzyme flux increased linearly with increasing acetate fraction. During growth on a very-high-acetate fraction, the activity of malic enzyme satisfied the biosynthetic needs of pyruvate in the mitochondria, while in the cytosol pyruvate was supplied via pyruvate kinase. In several cases enzyme activities were unexpectedly detected, e.g., the glyoxylate shunt for a very-low-acetate fraction, phosphoenolpyruvate carboxykinase for an acetate fraction of 0.46 C-mol of acetate/C-mol of substrate, and glucose catabolism to CO2 via the tricarboxylic acid cycle for a very-high-acetate fraction. Cytoplasmic alanine aminotransferase activity was detected, and evidence was found that α-isopropylmalate synthase has two active forms in vivo, one mitochondrial and the other a short cytoplasmic form.

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Citrate Synthesis by Lymphocytes

Clinical Chemistry ◽

10.1093/clinchem/21.7.825 ◽

1975 ◽

Vol 21 (7) ◽

pp. 825-828 ◽

Cited By ~ 1

Author(s):

Padmakar K Dixit ◽

Ruth Cadwell

Keyword(s):

De Novo ◽

Tricarboxylic Acid Cycle ◽

Tricarboxylic Acid ◽

Radiometric Method ◽

Chronic Lymphatic Leukemia ◽

Acetyl Coa ◽

Acid Cycle ◽

Lymphatic Leukemia ◽

Cellmediated Immunity

Abstract The important role of lymphocytes in humoral and cellmediated immunity indicates that they need a readily available intracellular source of energy. Here, we demonstrate that these cells contain enzymes involved in citrate formation. The citrate is oxidized through the tricarboxylic acid cycle to furnish energy. A newly devised, simple radiometric method was used to determine the condensation of labeled acetate or acetyl CoA with oxaloacetate to form citrate. The de novo synthesized citrate was oxidized to pentabromoacetone, and radioactivity of the resulting CO2 was directly related to the amount of citrate synthesized. Both lymphocytes from chronic lymphatic leukemia patients and cultured lymphoblasts possess this active citrate-synthesizing apparatus, although the lymphoblasts appeared to have a considerably higher activity, perhaps because they are larger.

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γδ-Dioxovalerate as a substrate for the glyoxalase enzyme system

Biochemical Journal ◽

10.1042/bj1350713 ◽

1973 ◽

Vol 135 (4) ◽

pp. 713-719 ◽

Cited By ~ 29

Author(s):

Tadeusz Jerzykowski ◽

Romana Winter ◽

Wojciech Matuszewski

Keyword(s):

Enzyme System ◽

Tricarboxylic Acid Cycle ◽

Tricarboxylic Acid ◽

Glyoxalase I ◽

Glyoxalase System ◽

Thiol Ester ◽

Glyoxalase Ii ◽

Metabolic Functions ◽

Metabolic Role

1. Crude γδ-dioxovalerate was synthesized from laevulinate by two different methods and was purified by Sephadex chromatography. Some analytical reactions of the compound are described. 2. γδ-Dioxovalerate is a substrate for glyoxalase I and the GSH derivative formed by this enzyme is hydrolysed by glyoxalase II to form d-α-hydroxyglutarate. The Km of glyoxalase I for γδ-dioxovalerate is 1.0×10−3m at pH5.8.3. The u.v.-absorption spectrum of thiol ester, synthesized enzymically from γδ-dioxovalerate and GSH by glyoxalase I, is almost identical with that for S-lactoylglutathione. Some optical properties of this thiol ester were measured. 4. Attempts to show reversibility of the glyoxalase system reactions with d-α-hydroxyglutarate as substrate were unsuccessful. 5. The possible metabolic role of the γδ-dioxovalerate reaction is discussed. It is suggested that one of the metabolic functions of the glyoxalase system may be to provide a mechanism for the entry of this compound into the tricarboxylic acid cycle.

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Effects of hyperthyroidism on stimulation of [1-14C]oleate oxidation to 14CO2 in isolated hepatocytes from fed rats by the catecholamines, vasopressin, and angiotensin II

Bioscience Reports ◽

10.1007/bf01120987 ◽

1983 ◽

Vol 3 (8) ◽

pp. 757-765 ◽

Cited By ~ 1

Author(s):

Mary C. Sugden ◽

Amal El-Saadi ◽

Anthony W. Goode ◽

J. Stewart Orr

Keyword(s):

Angiotensin Ii ◽

Concentration Ratio ◽

Redox State ◽

Phosphoenolpyruvate Carboxykinase ◽

Isolated Hepatocytes ◽

Glycolytic Flux ◽

Impaired Ability ◽

Mitochondrial Redox State ◽

14Co2 Production ◽

Stimulation Of

Possible effects of adrenaline, noradrenaline, vasopressin, and angiotensin II to increase 14CO2 production from [1-14C]oleate were examined in hepatocytes from fed L-triiodothyronine(T3)-treated or control rats. Rates of 14CO2 production were decreased and rates of ketogenesis increased in hepatocytes from T3-treated rats. These changes were accompanied by a marked shift of the 3-hydroxybutyrate:acetoacetate concentration ratio towards acetoacetate. Rates of glucose and lactate release were decreased. Whereas the Ca2+-mobilizing hormones increased 14CO2 production from [1-14C]oleate by 64–84% with hepatocytes from control rats, they increased 14CO2 production from [1-14C]oleate by only 24–32% with hepatocytes from T3-treated rats. The magnitude of the response to the Ca2+-mobilizing hormones in hepatocytes from T3-treated rats was increased by the addition of 3-mercaptopicolinate, an inhibitor of phosphoenolpyruvate carboxykinase, to the incubation medium (increases of 52–88%). In the presence of 3-mercaptopicolinate, the 3-hydroxybutyrate:acetoacetate concentration ratio in hepatocytes from fed, T3-treated rats was similar to that in hepatocytes from control rats in the absence of 3-mercaptopicolinate. The results demonstrate that hyperthyroidism per se does not lead to a loss of sensitivity, in terms of oleate oxidation, either to the catecholamines or to vasopressin and angiotensin II. The impaired ability of hepatocytes from T3-treated rats to respond to these hormones is a consequence of decreased net glycolytic flux or a more oxidized mitochondrial redox state.

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What Is the Metabolic Role of Phosphoenolpyruvate Carboxykinase?

Journal of Biological Chemistry ◽

10.1074/jbc.r109.040543 ◽

2009 ◽

Vol 284 (40) ◽

pp. 27025-27029 ◽

Cited By ~ 152

Author(s):

Jianqi Yang ◽

Satish C. Kalhan ◽

Richard W. Hanson

Keyword(s):

Phosphoenolpyruvate Carboxykinase ◽

Metabolic Role

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Posttransplant CMV infection and the role of immunosuppression (Sponsor: Novartis Pharma GmbH, Nürnberg)

Therapieforum Transplant ◽

10.1055/a-0711-9047 ◽

2016 ◽

Vol 04 (01) ◽

pp. 4-10

Keyword(s):

Lower Incidence ◽

Paradigm Shift ◽

Mtor Inhibitor ◽

De Novo ◽

Expert Panel ◽

Risk Of Infection ◽

Cmv Infection ◽

Expert Meeting ◽

Selection Of

AbstractImmunosuppression permits graft survival after transplantation and consequently a longer and better life. On the other hand, it increases the risk of infection, for instance with cytomegalovirus (CMV). However, the various available immunosuppressive therapies differ in this regard. One of the first clinical trials using de novo everolimus after kidney transplantation [1] already revealed a considerably lower incidence of CMV infection in the everolimus arms than in the mycophenolate mofetil (MMF) arm. This result was repeatedly confirmed in later studies [2–4]. Everolimus is now considered a substance with antiviral properties. This article is based on the expert meeting “Posttransplant CMV infection and the role of immunosuppression”. The expert panel called for a paradigm shift: In a CMV prevention strategy the targeted selection of the immunosuppressive therapy is also a key element. For patients with elevated risk of CMV, mTOR inhibitor-based immunosuppression is advantageous as it is associated with a significantly lower incidence of CMV events.

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