Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:1.4.1.2 (glutamate dehydrogenase)
4,380 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Trypanosoma (Schizotrypanum) cruzi epimastigotes (EP stock) grown in complex LIT medium rapidly consume the glucose present but, under aerobic conditions, continue growth in its absence with the concomitant excretion of ammonia, suggesting the utilization of amino acids for energy production. A search for metabolic pathways responsible for amino acid oxidation led to the detection of a NAD+-dependent glutamate dehydrogenase (L-glutamate:NAD+ oxidoreductase, E.C.1.4.1.2) which is different from an NADP+-dependent enzyme previously reported. The enzyme has been partially purified and its kinetic and regulatory properties studied in both directions of the reaction. Km values were 3.6 mM for alpha-ketoglutarate, 0.170 mM for NADH and 16 mM for NH+4, Vmax = 0.67 mumol min-1/mg-1 protein for aminative reduction; Km values were 23.5 mM for L-glutamate and 2.9 mM for NAD+, Vmax = 0.02 mumol min-1 mg-1 protein for deaminative oxidation, Tris buffer, pH 7.6. The enzyme is strongly inhibited by ATP, GTP, ADP and GDP (50% inhibition at 0.75 mM ATP, 3 mM MgCl2). S-Acetyl-CoA is also a potent inhibitor of the enzyme. The results demonstrate the presence of a specific pathway for the oxidation of amino acids, which is tightly regulated by the energy charge and the Krebs cycle activity in T. cruzi epimastigotes.
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PMID:Regulation of energy metabolism in Trypanosoma (Schizotrypanum) cruzi epimastigotes. II. NAD+-dependent glutamate dehydrogenase. 637 48

The effects of ATP and GTP on the activities of ox liver and brain glutamate dehydrogenase were determined in the absence and presence of added Mg2+ ions. Although GTP was an inhibitor of the enzyme reaction assayed in the direction of NAD+ reduction, the magnesium complex of this nucleotide had no effect on the activity. Similarly the magnesium complex of ATP was without effect on the activity of the enzyme although the free nucleotide was an activator. These results suggest that it is important to take account of magnesium complex formation when considering the regulatory actions of these nucleotides.
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PMID:The effects of magnesium ions on the interactions of ox brain and liver glutamate dehydrogenase with ATP and GTP. 646 7

The distance between the catalytic site on bovine liver glutamate dehydrogenase labeled with 4-(iodoacetamido)salicylic acid (ISA) and the adenosine 5'-diphosphate (ADP) activatory site occupied by the analogue 2',3'-O-(2,4,6-trinitrocyclohexadienylidene)adenosine 5'-diphosphate (TNP-ADP) was evaluated by energy transfer. Native enzyme and enzyme containing about 1 mol of acetamidosalicylate/mol of subunit bind about 0.5 mol of TNP-ADP/mol of subunit, and TNP-ADP competes for binding with ADP to native and modified enzyme, indicating that the analogue is a satisfactory probe of the ADP site. From the quenching of acetamidosalicylate donor fluorescence upon addition of TNP-ADP, an average distance of 33 A was determined between the catalytic and ADP sites. The fluorescent nucleotide analogue 5'-[p-(fluorosulfonyl)benzoyl]-2-aza-1,N6-ethenoadenosine (5'-FSBa epsilon A) reacts covalently with glutamate dehydrogenase to about 1 mol/peptide chain. As compared to native enzyme, the SBa epsilon A-enzyme exhibits decreased sensitivity to GTP inhibition but retains its catalytic activity as well as its ability to be activated by ADP and inhibited by high concentrations of NADH. Complete protection against decreased sensitivity to GTP inhibition is provided by GTP in the presence of NADH. It is concluded that 5'-FSBa epsilon A modifies a GTP site on glutamate dehydrogenase. The distance of 23 A between the catalytic site labeled with ISA and a GTP site labeled with 5'-FSBa epsilon A was measured from the quenching of salicylate donor fluorescence in the presence of the SBa epsilon A acceptor on a doubly labeled enzyme. The average distance between the ADP and GTP sites was previously measured as 18 A [Jacobson, M. A., & Colman, R. F. (1983) Biochemistry 22, 4247-4257], indicating that the regulatory sites of glutamate dehydrogenase are closer to each other than to the catalytic site.
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PMID:Distance relationships between the catalytic site labeled with 4-(iodoacetamido)salicylic acid and regulatory sites of glutamate dehydrogenase. 648 74

Bovine liver glutamate dehydrogenase reacts covalently with the new adenosine analogue 6-[(4-bromo-2,3-dioxobutyl)thio]-6-deaminoadenosine 5'-diphosphate with incorporation of about 1 mol of reagent/mol of enzyme subunit. Modified enzyme completely loses its normal ability to be inhibited by high concentrations of reduced diphosphopyridine nucleotide (DPNH) (greater than 100 microM), which binds at a regulatory site distinct from the catalytic site; however, the modified enzyme retains its full activity when assayed at 100 microM DPNH in the absence of allosteric compounds. The enzyme is still activated by ADP, is inhibited by GTP (albeit at higher concentrations), and binds 1.5-2 mol of [14C]GTP/subunit. A plot of initial velocity vs. DPNH concentration for the modified enzyme, in contrast to the native enzyme, followed Michaelis-Menten kinetics. The rate constant (k) for loss of DPNH inhibition (as measured at 0.6 mM DPNH) exhibits a nonlinear dependence on reagent concentration, suggesting a reversible binding of reagent (Kd = 0.19 mM) prior to irreversible modification. At 0.1 mM 6-[(4-bromo-2,3-dioxobutyl)thio]-6-deaminoadenosine 5'-diphosphate, k = 0.036 min-1 and is not affected by alpha-ketoglutarate, 100 microM DPNH, or GTP alone but is decreased to 0.0094 min-1 by 5 mM DPNH and essentially to zero by 5 mM DPNH plus 100 microM GTP. Incorporation after incubation with 0.25 mM 6-[(4-bromo-2,3-dioxobutyl)thio]-6-deaminoadenosine 5'-diphosphate for 2 h at pH 7.1 is 1.14 mol/mol of subunit in the absence but only 0.24 mol/mol of subunit in the presence of DPNH plus GTP.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Affinity labeling of the reduced diphosphopyridine nucleotide inhibitory site of glutamate dehydrogenase by 6-[(4-bromo-2,3-dioxobutyl)thio]-6-deaminoadenosine 5'-diphosphate. 649 69

The fluorescent nucleotide analogue 5'-[p-(fluorosulfonyl)benzoyl]-1,N6-ethenoadenosine (5'-FSB epsilon A) was shown previously to react at a GTP inhibitory site on bovine liver glutamate dehydrogenase. The incorporation was limited to 1.28 mol of reagent/mol of subunit and was attributed to 0.95 mol of modified tyrosine/mol of subunit and 0.33 mol of modified lysine/mol of subunit, quantitatively accounting for the total incorporation prior to acid hydrolysis [Jacobson, M. A., & Colman, R. F. (1983) Biochemistry 22, 4247-4257]. The specific tyrosyl peptide modified by 5'-FSB epsilon A has been isolated from a tryptic and chymotryptic digest of modified enzyme by gel filtration and reverse-phase high-performance liquid chromatography and characterized by amino acid and amino-terminal analysis. A unique residue, tyrosine-262, was identified as an essential amino acid within the GTP binding site. The stacked conformation of the fluorescent analogue when enzyme bound suggests that tyrosine-262 may be located in the region of the GTP site which binds the purine ring.
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PMID:Isolation and identification of a tyrosyl peptide labeled by 5'-[p-(fluorosulfonyl)benzoyl]-1,N6-ethenoadenosine at a GTP site of glutamate dehydrogenase. 652 53

The glutamate dehydrogenase activity found in the serum of patients with Reye's syndrome is shown to be inhibited about 1000-fold more potently by GTP than is the normal human enzyme. 1 mM ADP, which with the normal enzyme effectively reverses GTP inhibition, has no effect in the GTP inhibition of the Reye's syndrome serum activity.
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PMID:Glutamate dehydrogenase in Reye's syndrome. Evidence for the presence of an altered enzyme in serum with increased susceptibility to inhibition by GTP. 663 55

Glutamic dehydrogenase extracted with tris buffer from fresh freeze-thawed rat heart mitochondria was purified by ammonium sulphate fractionation, affinity chromatography on GTP agarose, hydroxyapatite chromatography and concentration using a molecular sieve. The final specific activity is 80 units/mg protein. Thin gel SDS electrophoresis of the purified enzyme preparation after reduction with dithiothreitol shows a major band with a molecular weight of 38 000 Daltons. Two minor bands are also present. Sucrose density gradient centrifugation reveals a molecular weight of 230 000 Daltons for unreduced mitochondrial GDH activity. By gel filtration rat heart mitochondrial glutamic dehydrogenase has a major peak at 230 000 Daltons, a minor peak at 300 000 Daltons and some larger molecular weight species. Rat liver mitochondrial glutamic dehydrogenase has a minor peak at 230 000, a major peak at 300 000 and some larger molecular weight species. The rat liver mitochondrial glutamic dehydrogenase predominance at 300 000 is unchanged by incubation, extraction and purification with rat heart mitochondria. The purified GDH is stable frozen at -10 degrees C in tris-HCl buffer with EDTA. It loses activity at 4 degrees C especially when stored in 0.2 M phosphate buffer. It also loses activity when dialyzed for 24 h. This loss of activity is not completely prevented by adding nucleotides to the buffer (AMP or ADP) but is decreased by their presence.
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PMID:Glutamic dehydrogenase from rat heart mitochondria. I. Purification and physical properties including molecular weight determination. 672 19

Glutamic dehydrogenase purified from rat heart mitochondria has been characterized with regard to its substrate kinetics and the influence of nucleotides and potassium phosphate on its kinetic properties. The enzyme had characteristics similar to liver mitochondrial glutamic dehydrogenase. These included several double reciprocal plots which were biphasic, indicating homotropic interaction; inhibition by GTP, which was overcome by ADP and phosphate; and activity with both NAD(H) and NADP(H). There were a number of significant differences however, in the specific kinetic properties of heart mitochondrial glutamic dehydrogenase. The Vmax of reductive amination was four-fold greater with NADH than with NADPH. The maximum rate of oxidative deamination was ten-fold greater with NAD compared to NADP. The differences also included: saturating levels of NADH and NADPH were stimulatory rather than inhibitory; ammonia was stimulatory at millimolar levels; NADP and alpha-ketoglutarate were both inhibitory at saturating levels; and ADP increased reductive amination 30% at lower levels of NADH but inhibited at higher (stimulatory) levels of NADH.
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PMID:Glutamic dehydrogenase from rat heart mitochondria. II. Kinetic characteristics. 672 20

The binding of 1,N6-etheno-NAD (epsilon NAD) to bovine liver glutamate dehydrogenase (L-glutamate:NAD(P)+ oxidoreductase (deaminating), EC 1.4.1.3) saturated with glutarate has been investigated at pH 7.0, 0.05 M phosphate buffer at 20 degrees C, by fluorescence titrations. epsilon NAD binds to the protein in a simple fashion: one molecule of coenzyme per enzyme polypeptide chain in the range of enzyme concentrations investigated (from above 50 to a few micromoles of enzyme polypeptide chains/liter). The fluorescence enhancement factor, Q, of bound epsilon NAD relative to free epsilon NAD is independent of the saturation degree, as deduced from the constant value of the long fluorescence decay lifetime (about 21 ns), and is about 17, as deduced from Fmax/F0 ratio values obtained after extrapolation from double reciprocal plots of 1/delta F vs. 1/[glutamate dehydrogenase]. This value for the Q factor is also independent of enzyme concentration, as well as of the presence of either GTP or ADP. At low enzyme concentrations (below 20 mumol polypeptide chains/liter), the dissociation constant of epsilon NAD increases progressively from a plateau value of about 50 microM to about 100 microM at infinite dilution. This is interpreted as being due to a minor affinity of glutamate dehydrogenase hexamers, with respect to higher aggregation states of the enzyme, towards epsilon NAD. As expected, GTP and ADP change the affinity of glutamate dehydrogenase towards epsilon NAD in an opposite manner: GTP strongly increases it, whereas ADP strongly decreases it (Kappd around 6 microM with saturating GTP and around 300 microM with saturating ADP). Furthermore, in the case of GTP, both GTP and epsilon NAD bind to glutamate dehydrogenase with positive cooperativity, with a Hill coefficient of approx. 1.8 for both and a Kappd approximately equal to 30 microM for the binding of GTP to glutamate dehydrogenase saturated with epsilon NAD and glutarate. The value of the Q factor remains the same, even in the presence of the effectors (again from lifetime measurements), as well as the number of epsilon NAD binding sites per enzyme polypeptide chain. These results are interpreted in terms of independent active sites, in the case without effectors. With ADP the binding appears to be simple, but no careful investigation has been attempted at low enzyme concentrations because of the low saturation degree achievable, whereas with GTP the cooperativity can be explained as due to a shift towards hexamers from higher aggregation states.
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PMID:The binding of 1,N6-etheno-NAD to bovine liver glutamate dehydrogenase. 674 62

L-Leucine and its nonmetabolized analogue, 2-aminobicyclo-[2,2,1]heptane-2-carboxylic acid (BCH) activate glutamate dehydrogenase in pancreatic islets, whether the reaction velocity is measured in the direction of glutamate synthesis or glutamate deamination. The rate of glutamate oxidative deamination is increased by ADP and inhibited by 2-ketoglutarate, NH4+ and GTP. The islet homogenate catalyzes the transamination between L-glutamate and either 2-ketoisocaproate or pyruvate, and between 2-ketoglutarate and L-leucine, L-aspartate, L-alanine, L-isoleucine, L-valine, L-norvaline or L-norleucine, but not b (+/-) BCH. The glutamate-aspartate transaminase is preferentially located in mitochondria relative to other transaminases. The parallel effects of L-leucine and BCH on glutamate dehydrogenase and their vastly different abilities to act as transamination partners may account for both analogies and discrepancies in the metabolic and functional responses of the islets to these two branched-chain amino acids.
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PMID:The stimulus-secretion coupling of amino acid-induced insulin release. XI. Kinetics of deamination and transamination reactions. 675 75


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