Kinetics Of Glutamate Dehydrogenase Research Articles

Effect of pH Dependent Substrate Inhibition on Glutamate Dehydrogenase Kinetics

Glutamate dehydrogenase (GDH) serves as the main regulated enzyme linking protein and carbohydrate metabolism. This enzyme converts the amino acid glutamate to the TCA intermediate, α‐ketoglutarate. While dysregulation of GDH is linked to hyperinsulinism and other diseases, the kinetics this enzyme of have only been analyzed under dilution conditions. However, cells are more crowded than these buffer solutions frequently used to collect enzyme kinetics data because their cytoplasm contains high concentrations of proteins, carbohydrates, and other macromolecules. Evidence has shown that this macromolecular crowding slow diffusion rates and changes protein conformations, which alters the kinetics of enzymes. Scientists often mimic the crowded environment of a cell using large, synthetic polymers, such as dextran, a glucose polymer, which excludes volume to other biological molecules in the same solution. While several studies have characterized these excluded volume effects on enzyme kinetics, this is the first study to analyze the effects of crowding on enzyme inhibition. Investigating how inhibitors like GTP and activators like leucine effect the kinetics of GDH will provide a deeper understanding of the regulation of this enzyme inside out bodies. Furthermore, saturating amounts of glutamate results in substrate inhibition of GDH by forming a “dead end complex” comprised of glutamate, NADH, and GDH at low pH values. To understand the effects of crowding on substrate inhibition, GDH Michaelis Menten kinetics were analyzed in the presence and absence of dextran at four pH values. These assays were repeated with glucose, the monomer of dextran as a control to separate chemical effects from excluded volume effects caused by the large size of dextran. The presence of dextran resulted in more substrate inhibition than in the presence of glucose and this inhibition that was observed at higher pH values than in buffer alone. Leucine, a known activator of GDH, operates by destabilizing the dead‐end complex. While the presence of leucine decreases substrate inhibition in dilute conditions, this compound was unable to active GDH in the presence of dextran. Overall, these results suggest that crowding promotes substrate inhibition by stabilizing the dead‐end complex of GDH which is a stronger interaction than leucine dissociation of the dead‐end complex. Therefore, macromolecular crowding in a cell could provide an additional level of regulation on GDH inside cells.

Building a model of ammonium excretion in two species of marine zooplankton based on glutamate dehydrogenase kinetics

MEPS Marine Ecology Progress Series Contact the journal Facebook Twitter RSS Mailing List Subscribe to our mailing list via Mailchimp HomeLatest VolumeAbout the JournalEditorsTheme Sections MEPS 550:83-99 (2016) - DOI: https://doi.org/10.3354/meps11700 Building a model of ammonium excretion in two species of marine zooplankton based on glutamate dehydrogenase kinetics I. Fernández-Urruzola1,*, N. Osma1, M. Gómez1, S. Montesdeoca-Esponda2, T. T. Packard1 1Marine Ecophysiology Group (EOMAR), Universidad de Las Palmas de Gran Canaria, 35017, Spain 2Environmental Chemical Analysis Group, Universidad de Las Palmas de Gran Canaria, 35017, Spain *Corresponding author: ifernandez@becarios.ulpgc.es ABSTRACT: NH4+ production in 2 species of marine zooplankton was predicted from glutamate dehydrogenase (GDH) activity using general bisubstrate enzyme kinetics during a period of starvation. In addition to starvation, we studied the effect of food quality on both NH4+ excretion rates (RNH4+) and the biochemical composition of zooplankton. The mathematical function used here relies on Michaelis-Menten principles and assumes that the synthesis of NH4+ is controlled mainly by the maximum velocity of GDH, the intracellular pool of substrates and the kinetic constants. This simple model described reasonably well the shifts in RNH4+ that occurred within the time course of starvation in both groups of zooplankton. The calculated RNH4+, however, underestimated the actual RNH4+. Furthermore, we used single substrate kinetics to address the magnitude of either the allosteric inhibition or the activation by purine nucleotides. We found that allosterism is more important in healthy physiological conditions. Overall, the use of first-principles kinetics seems to provide better estimates of RNH4+ than applying a general GDH/RNH4+ ratio on the measurements of GDH; therefore, its application may be of potential interest when studying zooplankton RNH4+ in heterogeneous marine ecosystems. KEY WORDS: Glutamate dehydrogenase · NH4+ excretion · Biochemical kinetics · Brachionus plicatilis · Leptomysis lingvura Full text in pdf format PreviousNextCite this article as: Fernández-Urruzola I, Osma N, Gómez M, Montesdeoca-Esponda S, Packard TT (2016) Building a model of ammonium excretion in two species of marine zooplankton based on glutamate dehydrogenase kinetics. Mar Ecol Prog Ser 550:83-99. https://doi.org/10.3354/meps11700 Export citation RSS - Facebook - Tweet - linkedIn Cited by Published in MEPS Vol. 550. Online publication date: May 25, 2016 Print ISSN: 0171-8630; Online ISSN: 1616-1599 Copyright © 2016 Inter-Research.

Robust regulation of hepatic pericentral amination by glutamate dehydrogenase kinetics.

Impaired glutamate dehydrogenase (GDH) sensitivity to its inhibitors causes excessive insulin secretion by pancreatic beta-cells and defective ammonia metabolism in the liver. These symptoms are commonly associated with hyperinsulinism/hyperammonemia syndrome (HI/HA), which causes recurrent hypoglycaemia in early infancy. Hepatic localization of GDH amination and deamination activities linked with the urea cycle is known to be involved in ammonia metabolism and detoxification. Although deamination activities of hepatic GDH in the periportal zones of liver lobules and its connection to the urea cycle have been exhaustively investigated, physiological roles of GDH amination activity observed at pericentral zones have often been overlooked. Using kinetic modelling approaches, here we report a new role for hepatic GDH amination kinetics in maintaining ammonia homeostasis under an excess intrahepatocyte input of ammonium. We have shown that α-ketoglutarate substrate inhibition kinetics of GDH, which include both random and obligatory ordered association/dissociation reactions, robustly control the ratio between glutamate and ammonium under a wide range of intracellular substrate variation. Dysregulation of this activity under pericentral nitrogen insufficiency contributes to the breaking down of ammonia homeostasis and thereby can significantly affect HI/HA syndrome.

Glutamate Dehydrogenase as a Marker of Alcohol Dependence

The aim of this study was to examine glutamate dehydrogenase (GLDH) in the diagnostic combinations as a result of new findings. GLDH, gama-glutamyltransferase (GGT), aspartate-aminotranferase (AST), alanine-aminotransferase (ALT) and erythrocyte mean cell volume (MCV) were assessed three times in 238 alcoholics admitted to hospital: on admission, after 24 h and after 7 days. All the values were significantly higher than those in healthy persons. The fastest activity decrease was seen in GLDH. The kinetics of GLDH and AST were more applicable than GGT kinetics after a week, but GLDH kinetics were most reliable. GLDH was the most specific laboratory marker with almost 90% specificity. The sensitivity of combination MCV and GLDH kinetics after 1 week of abstinence was pathognomonic by 97.2%. This decision tree gave us a model with 84.5% accuracy. GLDH is an equally accurate marker of alcoholism in comparison to others, if its significantly faster decrease is taken into consideration. We strongly believe that watching changes in the activity of laboratory markers of alcoholism is an effective yet overlooked aid.

Unregulated Insulin Secretion by Pancreatic Beta Cells in Hyperinsulinism/Hyperammonemia Syndrome: Role of Glutamate Dehydrogenase, ATP-Sensitive Potassium Channel, and Nonselective Cation Channel

The hyperinsulinism/hyperammonemia (HI/HA) syndrome is caused by "gain of function" of glutamate dehydrogenase (GDH). Several missense mutations have been found; however, cell behaviors triggered by the excessive GDH activity have not been fully demonstrated. This study was aimed to clarify electrophysiological mechanisms underlying the dysregulated insulin secretion in pancreatic beta cells with GDH mutations. GDH kinetics and insulin secretion were measured in MIN6 cells overexpressing the G446D and L413V. Membrane potentials and channel activity were recorded under the perforated-patch configuration that preserved intracellular environments. In mutant MIN6 cells, sensitivity of GDH to guanosine triphosphate (GTP) was reduced and insulin secretion at low glucose concentrations was enhanced. The basal GDH activity was elevated in L413V bearing a mutation in the antenna-like structure. The L413V cells were depolarized without glucose, often accompanying by repetitive Ca2+ firings. The depolarization was maintained in the presence of adenosine triphosphate (ATP) and disappeared by depleting ATP, suggesting that the depolarization depended on intracellular ATP. In L413V cells, the ATP-sensitive potassium channel (K(ATP) channel) was suppressed and the nonselective cation channel (NSCC) was potentiated, while sensitivity of the channels to their specific blockers or agonists was not impaired. These data suggest that the L413V cells increase the intracellular ATP/adenosine diphosphate (ADP) ratio, which in turn causes sustained depolarization not only by closure of the K(ATP) channel, but also by opening of the NSCC. The resultant activation of the voltage-gated Ca2+ channel appears to induce hyperinsulinism. The present study provides evidence that multiple channels cooperate in unregulated insulin secretion in pancreatic beta cells of the HI/HA syndrome.

Electrochemical study of the effect of ADP and AMP on the kinetics of glutamate dehydrogenase

A chronoamperometric method based on the ‘diffusion’ layer concept of the convective system was used to assay the glutamate dehydrogenase (GLDH) activity. Once the reaction was initiated by adding the enzyme GLDH into a well-stirred nicotinamide adenine dinucleotide (NADH, coenzyme) solution, the steady-state oxidation limiting current of NADH would decrease linearly in a short time. The major advantage of this method is that it directly indicates the continuous in-situ change of the coenzyme concentration, thus, the real initial reaction rate of enzyme-catalyzed reaction, V 0, can be determined. Using this method, the effect of adenosine-5′-monophosphate (AMP) and adenosine-5′-diphosphate (ADP) on the GLDH activity has been monitored. The results showed that ADP and AMP could increase the activity of GLDH. This activation mechanism was proposed by the voltammetric study.

Study of the effect of lanthanide ions on the kinetics of glutamate dehydrogenase by a chronoamperometric method

A chronoamperometric method, based on the oxidation of reduced nicotinamide adenine dinucleotide (NADH) on a glassy carbon electrode, for assaying glutamate dehydrogenase activity is described. Once the reaction was initiated by addition of enzyme into a well stirred assay mixture containing NADH, the concentration of NADH and the steady-state limiting current of NADH oxidation on the electrode decreased linearly in a short time range from the start of the reaction. The rate of the enzyme-catalysed reaction can be calculated from the decrease in the current. This method is reliable for the accurate determination of enzyme activity in purified extracts from liver and crude extracts from cucumber root. The major advantage of this method is the real-time and continuous measurement of the change in coenzyme concentration, so the real initial reaction rate, ν0, can be determined. This method has been used to detect the effect of La3+, Eu3+, Yb3+ and Zn2+ ions on the enzyme activity. The results suggest that lanthanide ions can increase the activity of the enzyme in glutamate synthesis, and Zn2+ acts as a potent inhibitor of the enzyme.