Oxidative Deamination Of Glutamate Research Articles

Effects of hypo-osmotic stress on osmoregulation, antioxidant response, and energy metabolism in sea cucumber Holothuria moebii under desalination environment

With global climate changing, hypo-salinity events are increasing in frequency and duration because of continuous rainfall and freshwater inflow, which causes reduced cytosolic osmolarity and cellular stress responses in aquatic animals. Sea cucumbers are considered stenohaline because they lack osmoregulatory organs and are vulnerable to salinity fluctuations. In this study, we performed multiple biochemical assays, de novo transcriptomics, and widely targeted metabolomics to comprehensively explore the osmoregulatory mechanisms and physiological responses of sea cucumber Holothuria moebii to hypo-osmotic stress, which is a representative specie that is frequently exposed to hypo-saline intertidal zones. Our results found that H. moebii contracted their ambulacral feet and oral tentacles, and the coelomic fluid ion concentrations were reduced to be consistent with the environment. The microvilli of intestines and respiratory trees underwent degeneration, and the cytoplasm exhibited swelling and vacuolation. Moreover, the Na+, K+, and Cl− concentrations and Na+/K+-ATPase activity were significantly reduced under hypo-osmotic stress. The decrease in protein kinase A activity and increase in 5′-AMP level indicated a significant inhibition of the cAMP signaling pathway to regulate ion concentrations. And small intracellular organic molecules (amino acids, nucleotides and their derivatives) also play crucial roles in osmoregulation through oxidative deamination of glutamate, nucleotide catabolism, and nucleic acid synthesis. Moreover, lysosomes and peroxisomes removed oxidative damage, whereas antioxidant metabolites, such as N-acetyl amino acids and glutathione, were increased to resist oxidative stress. With prolonged hypo-osmotic stress, glycerophospholipid metabolism was enhanced to maintain membrane stability. Furthermore, acyl-CoA-binding protein activity was significantly inhibited, and only a small amount of acylcarnitine was significantly accumulated, which indicated a disruption in energy metabolism. PPAR signaling pathway and choline content were up-regulated to promote fatty acid metabolism under hypo-osmotic stress. Overall, our results provide new insights into the osmoregulatory mechanisms and physiological responses of sea cucumbers to hypo-osmotic stress.

The Glutamate Dehydrogenase Pathway and Its Roles in Cell and Tissue Biology in Health and Disease.

Glutamate dehydrogenase (GDH) is a hexameric enzyme that catalyzes the reversible conversion of glutamate to α-ketoglutarate and ammonia while reducing NAD(P)+ to NAD(P)H. It is found in all living organisms serving both catabolic and anabolic reactions. In mammalian tissues, oxidative deamination of glutamate via GDH generates α-ketoglutarate, which is metabolized by the Krebs cycle, leading to the synthesis of ATP. In addition, the GDH pathway is linked to diverse cellular processes, including ammonia metabolism, acid-base equilibrium, redox homeostasis (via formation of fumarate), lipid biosynthesis (via oxidative generation of citrate), and lactate production. While most mammals possess a single GDH1 protein (hGDH1 in the human) that is highly expressed in the liver, humans and other primates have acquired, via duplication, an hGDH2 isoenzyme with distinct functional properties and tissue expression profile. The novel hGDH2 underwent rapid evolutionary adaptation, acquiring unique properties that enable enhanced enzyme function under conditions inhibitory to its ancestor hGDH1. These are thought to provide a biological advantage to humans with hGDH2 evolution occurring concomitantly with human brain development. hGDH2 is co-expressed with hGDH1 in human brain, kidney, testis and steroidogenic organs, but not in the liver. In human cerebral cortex, hGDH1 and hGDH2 are expressed in astrocytes, the cells responsible for removing and metabolizing transmitter glutamate, and for supplying neurons with glutamine and lactate. In human testis, hGDH2 (but not hGDH1) is densely expressed in the Sertoli cells, known to provide the spermatids with lactate and other nutrients. In steroid producing cells, hGDH1/2 is thought to generate reducing equivalents (NADPH) in the mitochondria for the biosynthesis of steroidal hormones. Lastly, up-regulation of hGDH1/2 expression occurs in cancer, permitting neoplastic cells to utilize glutamine/glutamate for their growth. In addition, deregulation of hGDH1/2 is implicated in the pathogenesis of several human disorders.

Molecular cloning and functional analysis of the second gene encoding glutamate dehydrogenase in triticale

Glutamate dehydrogenase (GDH; EC 1.4.1.2) catalyzes the reductive amination of 2-oxoglutarate with ammonium and the oxidative deamination of glutamate. In some plants species, GDH is a hexamer and can be separated into seven isoenzymes that are composed of two distinct subunits: α and β. The large number of isoenzymes is the reason why GDH functions are still being intensively researched and widely studied. Until recently, the β subunit of GDH in triticale, a common Polish cereal, was thought to be encoded by the TsGDH1 gene, which can undergo posttranslational modifications and form a heterohexameric enzyme. Here, we report the cloning and molecular characterization of a second glutamate dehydrogenase gene—TsGDH2 (encoding α subunits). The TsGDH2 cDNA contains a 1236-bp open reading frame encoding a 411-amino-acid polypeptide with a calculated molecular mass of 44.5 kDa. To clarify the role of TsGDH2 in triticale, we used triticale GDH α-subunit cDNA to generate transgenic A. thaliana lines with increased and decreased GDH activity via alternation of α-subunit levels.

Plasmodium falciparum glutamate dehydrogenase a is dispensable and not a drug target during erythrocytic development

BackgroundPlasmodium falciparum contains three genes encoding potential glutamate dehydrogenases. The protein encoded by gdha has previously been biochemically and structurally characterized. It was suggested that it is important for the supply of reducing equivalents during intra-erythrocytic development of Plasmodium and, therefore, a suitable drug target.MethodsThe gene encoding the NADP(H)-dependent GDHa has been disrupted by reverse genetics in P. falciparum and the effect on the antioxidant and metabolic capacities of the resulting mutant parasites was investigated.ResultsNo growth defect under low and elevated oxygen tension, no up- or down-regulation of a number of antioxidant and NADP(H)-generating proteins or mRNAs and no increased levels of GSH were detected in the D10Δgdha parasite lines. Further, the fate of the carbon skeleton of [13C] labelled glutamine was assessed by metabolomic studies, revealing no differences in the labelling of α-ketoglutarate and other TCA pathway intermediates between wild type and mutant parasites.ConclusionsFirst, the data support the conclusion that D10Δgdha parasites are not experiencing enhanced oxidative stress and that GDHa function may not be the provision of NADP(H) for reductive reactions. Second, the results imply that the cytosolic, NADP(H)-dependent GDHa protein is not involved in the oxidative deamination of glutamate but that the protein may play a role in ammonia assimilation as has been described for other NADP(H)-dependent GDH from plants and fungi. The lack of an obvious phenotype in the absence of GDHa may point to a regulatory role of the protein providing glutamate (as nitrogen storage molecule) in situations where the parasites experience a limiting supply of carbon sources and, therefore, under in vitro conditions the enzyme is unlikely to be of significant importance. The data imply that the protein is not a suitable target for future drug development against intra-erythrocytic parasite development.

Branched-chain Amino Acid Metabolon: INTERACTION OF GLUTAMATE DEHYDROGENASE WITH THE MITOCHONDRIAL BRANCHED-CHAIN AMINOTRANSFERASE (BCATm)

The catabolic pathway for branched-chain amino acids includes deamination followed by oxidative decarboxylation of the deaminated product branched-chain alpha-keto acids, catalyzed by the mitochondrial branched-chain aminotransferase (BCATm) and branched-chain alpha-keto acid dehydrogenase enzyme complex (BCKDC). We found that BCATm binds to the E1 decarboxylase of BCKDC, forming a metabolon that allows channeling of branched-chain alpha-keto acids from BCATm to E1. The protein complex also contains glutamate dehydrogenase (GDH1), 4-nitrophenylphosphatase domain and non-neuronal SNAP25-like protein homolog 1, pyruvate carboxylase, and BCKDC kinase. GDH1 binds to the pyridoxamine 5'-phosphate (PMP) form of BCATm (PMP-BCATm) but not to the pyridoxal 5'-phosphate-BCATm and other metabolon proteins. Leucine activates GDH1, and oxidative deamination of glutamate is increased further by addition of PMP-BCATm. Isoleucine and valine are not allosteric activators of GDH1, but in the presence of 5'-phosphate-BCATm, they convert BCATm to PMP-BCATm, stimulating GDH1 activity. Sensitivity to ADP activation of GDH1 was unaffected by PMP-BCATm; however, addition of a 3 or higher molar ratio of PMP-BCATm to GDH1 protected GDH1 from GTP inhibition by 50%. Kinetic results suggest that GDH1 facilitates regeneration of the form of BCATm that binds to E1 decarboxylase of the BCKDC, promotes metabolon formation, branched-chain amino acid oxidation, and cycling of nitrogen through glutamate.

Photochemical Enzyme Co-Factor Regeneration: Towards Continuous Glutamate Monitoring with a Sol-Gel Optical Biosensor

AbstractSol-gel encapsulation has recently surfaced as a successful approach to biomolecule immobilization. Proteins, including enzymes, are trapped in the pores of the sol-gel derived glass while retaining their spectroscopic properties and biological activity. Our current work extends the unique capabilities of biomolecule-doped sol-gel materials to the detection of glutamate, the major excitatory neurotransmitter in the central nervous system. We are developing an in vivo fiber optic biosensor for glutamate along with methods to achieve continuous monitoring. In our research to date we have encapsulated GDH in a silica sol-gel film on the tip of an optical fiber. GDH catalyzes the oxidative deamination of glutamate to α-ketoglutarate and the simultaneous reduction of NAD+ to NADH. To quantify the glutamate concentration, we observe the rate of change of NADH fluorescence as a function of time. An important consideration for continuous in vivo monitoring is the incorporation of a selfsustaining NAD+ source. We have adopted a photochemical means of regenerating NAD+ from NADH, by irradiating thionine (3,7-diaminophenothiazin-5-ium) which we incorporate into the sol-gel sensor material. When excited with visible light (λabc∼ 596 nm), thionine undergoes a reaction with NADH resulting in a non-fluorescent form of thionine and NAD+. We have characterized the kinetics of this reaction in the sol-gel matrix, and have shown that the reaction results in regenerated co-factor that is usable by GDH for the oxidation of glutamate.

Contribution of glutamate dehydrogenase to mitochondrial glutamate metabolism studied by 13C and 31P nuclear magnetic resonance

The relative contribution of glutamate dehydrogenase (GDH) and the aminotransferase activity to mitochondrial glutamate metabolism was investigated in dilute suspensions of purified mitochondria from potato (Solanum tuberosum) tubers. Measurements of glutamate-dependent oxygen consumption by mitochondria in different metabolic states were complemented by novel in situ NMR assays of specific enzymes that metabolize glutamate. First, a new assay for aminotransferase activity, based on the exchange of deuterium between deuterated water and glutamate, provided a method for establishing the effectiveness of the aminotransferase inhibitor amino-oxyacetate in situ, and thus allowed the contribution of the aminotransferase activity to glutamate oxidation to be assessed unambiguously. Secondly, the activity of GDH in the mitochondria was monitored in a coupled assay in which glutamine synthetase was used to trap the ammonium released by the oxidative deamination of glutamate. Thirdly, the reversibility of the GDH reaction was investigated by monitoring the isotopic exchange between glutamate and [(15)N]ammonium. These novel approaches show that the oxidative deamination of glutamate can make a significant contribution to mitochondrial glutamate metabolism and that GDH can support the aminotransferases in funneling carbon from glutamate into the TCA cycle.

Contribution of glutamate dehydrogenase to mitochondrial glutamate metabolism studied by 13 C and 31 P nuclear magnetic resonance

The relative contribution of glutamate dehydrogenase (GDH) and the aminotransferase activity to mitochondrial glutamate metabolism was investigated in dilute suspensions of purified mitochondria from potato ( Solanum tuberosum ) tubers. Measurements of glutamate‐dependent oxygen consumption by mitochondria in different metabolic states were complemented by novel in situ NMR assays of specific enzymes that metabolize glutamate. First, a new assay for aminotransferase activity, based on the exchange of deuterium between deuterated water and glutamate, provided a method for establishing the effectiveness of the aminotransferase inhibitor amino‐oxyacetate in situ , and thus allowed the contribution of the aminotransferase activity to glutamate oxidation to be assessed unambiguously. Secondly, the activity of GDH in the mitochondria was monitored in a coupled assay in which glutamine synthetase was used to trap the ammonium released by the oxidative deamination of glutamate. Thirdly, the reversibility of the GDH reaction was investigated by monitoring the isotopic exchange between glutamate and [ 15 N]ammonium. These novel approaches show that the oxidative deamination of glutamate can make a significant contribution to mitochondrial glutamate metabolism and that GDH can support the aminotransferases in funnelling carbon from glutamate into the TCA cycle.

Sol-Gel Optical Sensors for Glutamate

AbstractThe amino acid glutamate is the major excitatory neurotransmitter used in the nervous system for interneuronal communication. It is used throughout the brain by various neuronal pathways including those involved in learning and memory, locomotion, and sensory perception. Because glutamate is released from neurons on a millisecond time scale into sub-micrometer spaces, the development of a glutamate biosensor with high temporal and spatial resolution is of great interest for the study of neurological function and disease. Here, we demonstrate the feasibility of an optical glutamate sensor based on the sol-gel encapsulation of the enzyme glutamate dehydrogenase (GDH). GDH catalyses the oxidative deamination of glutamate and the reduction of NAD+ to NADH. NADH fluorescence is the basis of the sensor detection. Thermodynamic and kinetic studies show that GDH remains active in the sol-gel matrix and that the reaction rate is correlated to the glutamate concentration.

Glutamate dehydrogenase of Sphagnum

Glutamate dehydrogenase (GDH) of S. fallax was purified 2000-fold to apparent homogeneity. As estimated by gel filtration and SDS-PAGE, a native molecular weight of M r 210 000 and a subunit molecular weight of M r 52 000 were determined, indicating that the enzyme is tetrameric with subunits of identical molecular weights. Isoforms could not be detected. Purified GDH displayed pH-optima of 8.6 with NAD(P)H and 7.7 with NAD + and a high K m for ammonium of 28 and 41 mM with NADH and NADPH as coenzymes, respectively. The enzyme is located in the mitochondria. The occurrence of GDH activity in the cytosolic fraction was addressed to organelle breakage. Enhanced ammonium concentrations and a reduced carbon supply caused a substantial increase in GDH activity. Labelling studies with [ 15N]ammonium and [ 15N]glutamate were consistent with the role of the enzyme in the oxidative deamination of glutamate. © 1997 Elsevier Science Ltd. All rights reserved

Two soluble forms of glutamate dehydrogenase isoproteins from bovine brain.

Two soluble forms of novel glutamate dehydrogenase isoproteins, designated GDH I and GDH II, have been purified from bovine brain. GDH I and GDH II were separated on a hydroxyapatite column and eluted by a step gradient at different phosphate concentrations (30 mM and 50 mM for GDH I and GDH II, respectively). The preparations were homogeneous on SDS/PAGE. GDH I and GDH II showed similarity in their molecular sizes and are composed of six identical subunits having a molecular size of 57,500 Da. Differences between the biochemical properties of GDH I and GDH II, such as N-terminal amino acid sequences of intact and tryptic-digested enzymes, kinetic parameters, optimum pH and heat stability, were extensively examined in both reductive amination of alpha-oxoglutarate and oxidative deamination of glutamate. The different effects of ADP on GDH isoproteins were also studied under various conditions. These results indicate that GDH I and GDH II, isolated from bovine brain, are novel and distinct polypeptides.

Evidence for deamination by glutamate dehydrogenase in higher plants: Commentary

Glutamate dehydrogenase (GDH; EC 1.4.1.2) catalyses the interconversion of 2-oxoglutarate and glutamate. For many years it was assumed that GDH established a net flux in the direction of glutamate synthesis in higher plants and that this ubiquitous enzyme was responsible for the assimilation of ammonium (Davies 1968). However, this view changed with the discovery of glutamate synthase (GOGAT; EC 1.4.1.13), and it is now generally accepted that it is the glutamate synthase cycle, involving the successive operation of glutamine synthetase (GS; EC 6.3.1.2) and GOGAT, that is the major pathway for the assimilation of ammonium by higher plants (Lea and Miflin 1974; Stewart et al. 1980; Lea et al. 1990; Oaks 1994). Although it has been argued that there may be circumstances in which GDH could still provide a route for ammonium assimilation (Cammaerts and Jacobs 1985; Srivastava and Singh 1987; Loyola-Vargas et al. 1988; Magalhaes et al. 1990), recent attention has focused on the oxidative deamination of glutamate by GDH and the hypothesis that GDH performs an anaplerotic function in plants (Robinson et al. 1991, 1992). We used a combination of in vivo and in vitro methods to investigate the function of GDH in carrot (Daucus carota L.) cell suspension cultures, and the results were entirely consistent with a role for the enzyme in the deamination of glutamate. Firstly, enzyme assays of cell extracts showed that GDH activity was lowest in cells in the exponential phase of growth and reached a maximum in the stationary phase (Robinson et al. 1991). This suggests that GDH is unlikely to be important in ammonium assimilation, since the exponential phase coincides with a period of rapid nitrogen assimila-

Evidence That Glutamate Dehydrogenase Plays a Role in the Oxidative Deamination of Glutamate in Seedlings of Zea mays

In order to investigate the role of glutamate dehydrogenase we have compared the metabolism of [15N]glutamate in young seedlings of wild-type and a glutamate dehydrogenase-null mutant of Zea mays. The principal labelled products in roots of wild-type seedlings are the amide nitrogen of glutamine, glutamine-amino nitrogen and ammonium. The incorporation of label into glutamine-amide is markedly inhibited by methionine sulfoximine. In contrast, little or no labelling of glutamine-amide or ammonium occurs in roots of the GDH1-null mutant, the major labelled product is the amino group of asparagine. In shoots of the wild type, 15N is recovered in the amide of glutamine, ammonium, the amino group of asparagine and other amino acids. In mutant shoots, over 75% of the label is recovered in the asparagine-amino group and there is little labelling of glutamine-amide or ammonium. These major differences in glutamate metabolism of wild-type and mutant seedlings are consistent with glutamate dehydrogenase functioning in the direction of oxidative deamination and having a role in protein catabolism of germinating seeds.

Inhibition of glutamate dehydrogenase in brain mitochondria and synaptosomes by Mg2+ and polyamines: a possible cause for its low in vivo activity.

Magnesium and the polyamines putrescine, spermidine, and spermine inhibited the activity of glutamate dehydrogenase in permeabilized rat brain mitochondria in a concentration-dependent manner. The inhibitory effect was observed on both the reductive amination of 2-oxoglutarate and oxidative deamination of glutamate, as well as in the presence and absence of ADP and leucine, the allosteric activators of the enzyme. Kinetic studies at various concentrations of substrates showed that inhibition by magnesium and spermine was very pronounced at 2-oxoglutarate concentrations less than 0.5 mM and NADH levels less than 0.08 mM. The presence of the former compounds also accentuated the inhibitory effect of high concentrations of 2-oxoglutarate (> 2.0 mM) and NADH (> 0.32 mM). Addition of magnesium and spermine to suspensions of synaptosomes decreased the amount of ammonia produced from glutamate. It is suggested that polyamines and magnesium, normal constituents of mammalian brain, are responsible, at least in part, for the low glutamate dehydrogenase activity in vivo.

Transport and metabolism of glutamate by rat cerebellar mitochondria during ammonia toxicity

Pathophysiological concentrations of ammonia, both in vivo and in vitro, suppressed the oxidation of glutamate by rat cerebellar mitochondria. The transport of glutamate into mitochondria was either unaltered or enhanced during hyperammonemic states. Activities of mitochondrial enzymes, aspartate aminotransferase, alanine aminotransferase, glutamate dehydrogenase, glutaminase, and GABA-transaminase were suppressed during hyperammonemic states. Suppression of 14CO2 production with (aminooxy)acetic acid but not with glutamic acid diethyl ester indicated that transamination but not oxidative deamination of glutamate plays a major role in glutamate oxidation during normal and hyperammonemic states.

Activation of Glutamate Dehydrogenase by Leucine and Its Nonmetabolizable Analogue in Rat Brain Synaptosomes

Leucine and beta-(+/-)-2-aminobicyclo[2.2.1]heptane-2-carboxylic acid (BCH) stimulated, in a dose-dependent manner, reductive amination of 2-oxoglutarate in rat brain synaptosomes treated with Triton X-100. The concentration dependence curves were sigmoid, with 10-15-fold stimulations at 15 mM leucine (or BCH); oxidative deamination of glutamate also was enhanced, albeit less. In intact synaptosomes, leucine and BCH elevated oxygen uptake and increased ammonia formation, consistent with stimulation of glutamate dehydrogenase (GDH). Enhancement of oxidative deamination was seen with endogenous as well as exogenous glutamate and with glutamate generated inside synaptosomes from added glutamine. With endogenous glutamate, the stimulation of oxidative deamination was accompanied by a decrease in aspartate formation, which suggests a concomitant reduction in flux through aspartate aminotransferase. Activation of reductive amination of 2-oxoglutarate by BCH or leucine could not be demonstrated even in synaptosomes depleted of internal glutamate. It is suggested that GDH in synaptosomes functions in the direction of glutamate oxidation, and that leucine may act as an endogenous activator of GDH in brain in vivo.

Metabolic pathways of ammoniogenesis in the shrimp Crangon crangon L.: Possible role of glutamate dehydrogenase

1. 1. The oxidative deamination of glutamate by glutamate dehydrogenase (GDH) was determined in crude homogenates of the shrimp Crangon crangon. 2. 2. The GDH activity of whole shrimps (1.192 ± 0.164 UI/g wet wt and 0.032 ± 0.004 UI mg protein) (± SD) is probably sufficient to account for all the ammonia excretion of this species. 3. 3. Starvation markedly influenced GDH activity. A 50% decrease of GDH activity was observed following 7 days of fasting but subsequently no further decrease in GDH activity was noticed during starvation up to a maximum of 17 days.

Effect of pH on glutamate efflux from rat kidney mitochondria.

To provide further insight into the relationship between glutamate transport and metabolism in rat kidney mitochondria, the effect of pH on [U-14C]-glutamate efflux from [U-14C]glutamate-loaded mitochondria was tested. Glutamate efflux, measured in rotenone-inhibited energized mitochondria at 28 degrees C, was first order with respect to matrix glutamate. Glutamate efflux was tested at different medium pH values ranging from 6.4 to 7.9. Efflux was fastest at pH 7.9 with a rate constant of 0.988 min-1 and decreased progressively at lower pH values such that the rate constant at pH 6.4 was 0.359 min-1. A highly significant correlation between pH and the rate constant of glutamate efflux was observed. When medium pH was held constant at 7.0, matrix pH (range pH 7.2-8.4) did not affect the rate of glutamate efflux. The reduction in glutamate efflux at low pH provides an explanation for the increased concentration of glutamate observed in the mitochondrial matrix space of isolated rat kidney mitochondria incubated at acid medium pH. The elevated glutamate concentration in turn contributes to the accelerated oxidative deamination of glutamate by glutamate dehydrogenase at acid pH. Moreover, these findings suggest that the reduction in renal cortex glutamate levels observed in acute acidosis in vivo may be explained by a decreased rate of glutamate transport from mitochondria to cytosol coupled with an augmented rate of glutamate oxidation in the matrix.

The stimulus-secretion coupling of amino acid-induced insulin release metabolic interaction of L-glutamine and 2-ketoisocaproate in pancreatic islets

1. The metabolic situation found in pancreatic islets exposed to both L-glutamine and 2-ketoisocaproate was investigated in order to assess its relevance to the synergistic effects of these nutrients upon insulin release. 2. In islet homogenates, several 2-keto acids could be used as partners for the transamination of L-glutamate to 2-ketoglutarate. The rate of transamination did not correlate positively with the capacity of each 2-keto acid to stimulate insulin release in the presence of L-glutamine. 3. L-Glutamine enhanced the production of L-leucine from 2-ketoisocaproate and inhibited the conversion of the 2-keto acid to acetoacetate and CO 2. L-Glutamine also inhibited the oxidation of pyruvate. 4. In the presence of 2-ketoisocaproate, the rate of generation of 2-ketoglutarate from exogenous L-glutamine was increased, but the oxidative deamination of glutamate was suppressed. 5. L-Valine antagonized the effect of 2-ketoisocaproate to augment 14Co 2 output from islets prelabelled with L-[U- 14C]glutamine. 6. L-Glutamine did not increase the islet content of reduced pyridine nucleotides beyond the high level reached in the sole presence of 2-ketoisocaproate. 7. If allowance was made for the influence of exogenous nutrients upon the oxidation of endogenous nutrients, the insulin output evoked by L-glutamine and/or 2-keto acids tightly depended on the increment in oxidation rate attributable to these nutrients. 8. The metabolic and secretory responses to L-glutamine and 2-ketoisocaproate were best explained by a stimulation of transamination reactions between 2-ketoisocaproate and glutamate derived from exogenous glutamine.

Proline metabolism in isolated rat liver cells

The metabolism of proline was studied in liver cells isolated from starved rats. The following observations were made. 1. Consumption of proline could be largely accounted for by production of glucose, urea, glutamate and glutamine. 2. At least 50% of the total consumption of oxygen was used for proline catabolism. 3. Ureogenesis and gluconeogenesis from proline could be stimulated by partial uncoupling of oxidative phosphorylation. 4. Addition of ethanol had little effect on either proline uptake or oxygen consumption, but strongly inhibited the production of both urea and glucose and caused further accumulation of glutamate and lactate. Accumulation of glutamine was not affected by ethanol. 5. The effects of ethanol could be overcome by partial uncoupling of oxidative phosphorylation. 6. The apparent K(m) values of argininosuccinate synthetase (EC 6.3.4.5) for aspartate and citrulline in the intact hepatocyte are higher than those reported for the isolated enzyme. 7. 3-Mercaptopicolinate, an inhibitor of phosphoenolpyruvate carboxykinase (EC 4.1.1.32), greatly enhanced cytosolic aspartate accumulation during proline metabolism, but inhibited urea synthesis. 8. It is concluded that when proline is provided as a source of nitrogen to liver cells, production of ammonia by oxidative deamination of glutamate is inhibited by the highly reduced state of the nicotinamide nucleotides within the mitochondria. 9. Conversion of proline into glucose and urea is a net-energy-yielding process, and the high state of reduction of the nicotinamide nucleotides is presumably maintained by a high phosphorylation potential. Thus when proline is present as sole substrate, the further oxidation of glutamate by glutamate dehydrogenase (EC 1.4.1.3) is limited by the rate of energy expenditure of the cell.