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

---

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

The carboxyl-terminal catalytic domain of the human poly(ADP-ribose) polymerase (PARP) exhibits sequence homology with the NAD(P)(+)-dependent leucine and glutamate dehydrogenases. To clarify the role played by some conserved residues between PARP and NAD(P)(+)-dependent dehydrogenases, point mutations were introduced into the whole enzyme context. Non-conservative mutations of Lys-893 (K893I) and Asp-993 (D993A) completely inactivate human PARP, whereas conservative and nonconservative mutations of Asp-914 (D914E and D914A, respectively) and Lys-953 (K953R and K953I, respectively) partially alter PARP activity. The consequences of conservative substitution of Lys-893 and Asp-993 on the kinetic properties of human poly(ADP-ribose) polymerase enzyme and the polymer it synthesizes suggest that these 2 amino acids are directly involved in the covalent attachment of the first ADP-ribosyl residue from NAD+ onto the acceptor amino acid. In addition, the recent resolution of the three-dimensional structure of the NAD(+)-linked glutamate dehydrogenase from Clostridium symbiosum (Baker, P.J., Britton, K.L., Engel, P.C., Farrants, G.W., Lilley, K.S., Rice, D.W., and Stillman, T.J. (1992) Proteins 12, 75-86) strongly supports our alignment with leucine and glutamate dehydrogenases and provides an interesting structural framework for the analysis of our results of site-directed mutagenesis.
...
PMID:Identification of potential active-site residues in the human poly(ADP-ribose) polymerase. 847 97

Nicotinamide-adenine-dinucleotide-specific glutamate dehydrogenase (NAD-GDH; EC 1.4.1.3) from Bacillus cereus DSM 31 was enriched 260-fold. The molecular mass was determined by gel filtration to be 270 kDa (+/- 25 kDa). The enzyme was highly specific for the coenzyme NAD(H) and catalysed both the formation and the oxidation of glutamate. Apparent Km values of 7.7 mM for glutamate and 0.56 mM for NAD+ during oxidative deamination were measured. Both in crude cell-free extracts and in enriched preparations the enzyme was extremely unstable, especially at low temperatures. The loss of activity in the cold was found to be due to the dissociation of the holoenzyme into catalytically inactive subunits of molecular mass 48 kDa (+/- 5 kDa), indicating that the native enzyme has a hexameric structure. The activity was restored under certain conditions, and no instability of the enzyme in the cold was observed in undisrupted cells.
...
PMID:Properties of the cold-labile NAD(+)-specific glutamate dehydrogenase from Bacillus cereus DSM 31. 851 35

Mitochondrial NAD+, NADH, NADP+ and NADPH were measured in dispersed pancreatic islet cells incubated in the absence or presence of D-glucose and then exposed for 20 s to 0.5 mg/ml digitonin. The latter treatment resulted in the full release of lactate dehydrogenase without any detectable loss of glutamate dehydrogenase. The permeabilized cells were separated from the incubation medium by centrifugation through an oil layer and their content in pyridine nucleotides measured by a radioisotopic procedure coupled to the classical cycling technique. Relative to basal value, D-glucose, in concentrations of 2.8 and 16.7 mM, caused a concentration-related increase in both the NADH/NAD+ and NADPH/NADP+ ratio. These findings provide the first direct evidence for the induction of a more reduced mitochondrial redox state in glucose-stimulated pancreatic islets.
...
PMID:The coupling of metabolic to secretory events in pancreatic islets. Glucose-induced changes in mitochondrial redox state. 861 61

Photoaffinity labeling with [alpha-32P]-8-azidoadenosine 5'-diphosphate (8N3ADP) and [beta-32P]-2-azidoadenosine 5'-diphosphate (2N3ADP) was used to identify overlapping tryptic and chymotryptic generated peptides within the adenine binding domain of the regulatory ADP site of bovine liver glutamate dehydrogenase (GDH). In the absence of UV irradiation, 8N3ADP was able to activate the reverse reaction catalyzed by GDH as well as ADP. Photoinsertion of both [alpha 32P] 8N3ADP and [beta 32P]2N3ADP was reduced best by ADP in comparison to other nucleotides. Photolabeling of GDH with [alpha 32P]8N3-ADP appeared to be biphasic, with saturation occurring near 80 and 130 microM, whereas [beta 32P]2N3ADP showed saturation near 50 microM. When 60 microM [alpha 32P]8N3ADP (below the first saturation value) was used to identify peptides within the ADP binding domain, peptides corresponding to residues G156-K200 and E175-K200 (tryptic) and I158-Y183 (chymotryptic) were photolabeled. However, when 160 microM [alpha 32P]8N3ADP (above the second saturation value) was used, the peptide D403-R418 was also photolabeled. Digestion with both trypsin and chymotrypsin resulted in isolation of peptides E175-Y183 and A184-I192. [beta 32P]2N3ADP at 90 microM also photolabeled tryptic peptides G156-K200 and C270-K289. C270-K289 was shown earlier to be within the NAD+ binding site [Kim, H., and Haley, B. (1991) Bioconjugate Chem. 2, 142-147]. These results are consistent with the residues E175-[192 being within the adenine binding domain of the ADP regulatory site.
...
PMID:Identification of adenine binding domain peptides of the ADP regulatory site within glutamate dehydrogenase. 881 52

By using site-directed mutagenesis, Phe-187, one of the amino-acid residues involved in hydrophobic interaction between the three identical dimers comprising the hexamer of Clostridium symbiosum glutamate dehydrogenase (GDH), has been replaced by an aspartic acid residue. Over-expression in Escherichia coli led to production of large amounts of a soluble protein which, though devoid of GDH activity, showed the expected subunit M(r) on SDS-PAGE, and cross-reacted with an anti-GDH antibody preparation in Western blots. The antibody was used to monitor purification of the inactive protein. F187D GDH showed altered mobility on non-denaturing electrophoresis, consistent with changed size and/or surface charge. Gel filtration on a calibrated column indicated an M(r) of 87000 +/- 3000. The mutant enzyme did not bind to the dye column routinely used in preparing wild-type GDH. Nevertheless suspicions of major misfolding were allayed by the results of chemical modification studies: as with wild-type GDH, NAD+ completely protected one-SH group against modification by DTNB, implying normal coenzyme binding. A significant difference, however, is that in the mutant enzyme both cysteine groups were modified by DTNB, rather than C320 only. The CD spectrum in the far-UV region indicated no major change in secondary structure in the mutant protein. The near-UV CD spectrum, however, was less intense and showed a pronounced Phe contribution, possibly reflecting the changed environment of Phe-199, which would be buried in the hexamer. Sedimentation velocity experiments gave corrected coefficients S20,W of 11.08 S and 5.29 S for the wild-type and mutant proteins. Sedimentation equilibrium gave weight average molar masses M(r,app) of 280000 +/- 5000 g/mol. consistent with the hexameric structure for the wild-type protein and 135000 +/- 3000 g/mol for F187D. The value for the mutant is intermediate between the values expected for a dimer (98000) and a trimer (147000). To investigate the basis of this, sedimentation equilibrium experiments were performed over a range of protein concentrations. M(r,app) showed a linear dependence on concentration and a value of 108 118 g/mol at infinite dilution. This indicates a rapid equilibrium between dimeric and hexameric forms of the mutant protein with an equilibrium constant of 0.13 l/g. An independent analysis of the radial absorption scans with Microcal Origin software indicated a threefold association constant of 0.11 l/g. Introduction of the F187D mutation thus appears to have been successful in producing a dimeric GDH species. Since this protein is inactive it is possible that activity requires subunit interaction around the 3-fold symmetry axis. On the other hand this mutation may disrupt the structure in a way that cannot be extrapolated to other dimers. This issue can only be resolved by making alternative dimeric mutants.
...
PMID:Construction of a dimeric form of glutamate dehydrogenase from Clostridium symbiosum by site-directed mutagenesis. 891 16

Previous studies have capitalized on ordered kinetic mechanisms in the design of biospecific affinity chromatographic methods for highly efficient purifications and mechanistic studies of enzymes. The most direct tactic has been the use of immobilised analogues of the following, usually enzyme-specific substrates, e.g., lactate/pyruvate in the case of lactate dehydrogenase for which NAD+ is the leading substrate. Such immobilised specific substrates are, however, often difficult or impossible to synthesise. The locking-on strategy reverses the tactic by using the more accessible immobilised leading substrate, immobilised NAD+, as adsorbent with soluble analogues of the enzyme-specific ligands (e.g., lactate in the case of lactate dehydrogenase) providing a substantial reinforcement of biospecific adsorption sufficient to effect adsorptive selection of an enzyme from a group of enzymes such as the NAD(+)-specific enzymes. The value of this approach is demonstrated using model studies with lactate dehydrogenase (LDH, EC 1.1.1.27), alcohol dehydrogenase (ADH, EC 1.1.1.1), glutamate dehydrogenase (GDH, EC 1.4.1.3) and malate dehydrogenase (MDH, EC 1.1.1.37). Purification of bovine liver GDH in high yield from crude extracts is described using the tactic.
...
PMID:Further studies on the bioaffinity chromatography of NAD(+)-dependent dehydrogenases using the locking-on effect. 891 27

Visualization of the release of an excitatory neurotransmitter, glutamate (Glu), from a slice preparation of the brain and spinal cord may be of great advantage in studying the release of Glu from a small population of neurons. When capsaicin (10 mu M) was applied to a slice of the rat spinal cord immersed in a medium containing glutamate dehydrogenase (GDH), an oxidized form of nicotinamide adenine dinucleotide (NAD+), and tetrodotoxin, we observed an apparent increase of fluorescence in superficial laminae and lamina X using a confocal laser scanning microscope. Such an increase was not observed in the absence of either NAD+ or GDH, was inhibited by removal of extracellular Ca2+, and was terminated by capsazepine (100 mu M). In contrast to capsaicin, Glu release evoked by high K+ was observed in all laminae throughout the grey matter. The present results suggest that this system enables us to see the site of the release of Glu as an image and that capsaicin releases this amino acid mainly in superficial laminae and lamina X in the spinal cord.
...
PMID:Visualization of glutamate release from rat spinal cord with a confocal laser scanning microscope. 892 25

The mechanism of the binding of reduced coenzyme (NAD+) to clostridial glutamate dehydrogenase (GDH) was determined by transient kinetics. The fluorescent 1,N6-ethenoadenine analogue of NAD+ (epsilonNAD+) was used as a probe of nucleotide binary and ternary complex formation because the binding of NAD+ is optically silent. The kinetics of epsilonNAD+ binding were consistent with a 3-step binding process. The enzyme was found to oscillate between two conformational forms, termed E1 and E2, in the presence and absence of L-glutamate. However, L-glutamate shifted the equilibrium from 96.8% to 99% of the enzyme in the E1 form. The rapid-equilibrium binding of epsilonNAD+ to the E2 form was rate limited by a slow isomerisation of the ternary complex as the binary complex became saturated with epsilonNAD+. The L-glutamate binary complex had a greater affinity for the coenzyme (Kd = 11 microM) than the free enzyme (Km = 39 microM), indicative of a positive interaction of the substrate and coenzyme binding sites. Steady-state studies were also indicative of a positive interaction in the formation of the catalytic complex, with this complex having a Kd for epsilonNAD+ of 6.8 microM. Consequently, there is stabilization of successive complexes on the reaction pathway.
...
PMID:Kinetic studies on the binding of 1,N6-etheno-NAD+ to glutamate dehydrogenase from Clostridium symbiosum. 921 15

In a study of the re-activation of urea-denatured clostridial glutamate dehydrogenase (GDH) the maximum re-activation achieved without any added ligands was about 6%, but with NAD+ and 2-oxoglutarate in combination about 70%. NAD+ alone was also effective but 2-oxoglutarate was not, in striking contrast with the opposite pattern for protection of this enzyme against unfolding in urea [Aghajanian, Martin and Engel (1995) Biochem. J. 311,905-910]. The extent of re-activation was not increased by raising the incubation temperature to 37 degrees C and was independent of the time of enzyme denaturation. CD and fluorimetric studies showed that dilution of denatured enzyme into potassium phosphate buffer led to rapid (half-time <3-5 s)formation of 'structured' intermediates with secondary structure similar to that of native enzyme. These intermediate molecules were inactive, behaved as monomers on a size-exclusion column, and were unable to associate to give the native hexameric structure. Addition of NAD+ facilitated isomerization of these 'structured' monomers into a form(s) capable of re-activation. A side effect in the refolding process was non-specific aggregation, depending on final enzyme concentration. The hexamer fraction from re-activated samples, however, showed the same specific activity as native enzyme. The portion of the enzyme that is not lost through aggregation thus appears to regain the native structure fully. Detailed time-course studies showed that re-activation follows second-order kinetics, suggesting that formation of a dimer may be the rate-limiting step. The possible mechanism for the unfolding and refolding processes of clostridial GDH and effects of coenzyme and substrate on these are discussed in relation to the known crystal structure.
...
PMID:Re-activation of Clostridium symbiosum glutamate dehydrogenase from subunits denatured by urea. 930 12

The triple mutant K89L/A163G/S380A (inactive with glutamate but active with L-Nle and L-Met) and C320S (fully active with glutamate, entirely inactive with L-Nle and L-Met, and also lacking reactive cysteine) mutant of glutamate dehydrogenase (EC 1.4.1.2) of Clostridium symbiosum could be completely denatured by urea with the loss of structure and activity. The mutants denatured by urea could be reassociated to give stable hexamers with recovery of activity of approximately 67% by dilution in 0.1 M potassium phosphate buffer (pH 7.0) containing 2 mM NAD+. The native, urea-denatured, and renatured states of mutant enzymes were characterized by size exclusion chromatography on FPLC and native PAGE. Intersubunit hybrid hexamers containing five subunits of triple mutant and one subunit of C320S mutant were constructed by in vitro subunit hybridization followed by affinity chromatography. Kinetic analysis showed that a 5:1 hybrid hexamer, with only one C320S subunit able to bind NAD+ after DTNB modification, shows classical Michaelis-Menten kinetics with regard to NAD+. This contrasts with the apparent negative co-operativity shown by pure C320S hexamers and suggests that the interaction in NAD+ binding among subunits is eliminated in the hybrid. After removal of thionitrobenzoate, however, all of the subunits in the hybrid are able to bind NAD+. In this state the hybrid enzyme showed slight deviation from classical behavior with regard to NAD+, indicating reintroduction of some level of allosteric interaction. The hybrid hexamer also showed much reduced co-operativity with glutamate at pH 8.8, with a Hill coefficient of 3 for DTNB-treated hybrid (as compared to 5.2 for the pure C320S mutant) and 2.2 for the untreated hybrid. The fact that co-operativity in glutamate binding is not entirely eliminated correlates with evidence that the triple mutant subunits, though inactive toward glutamate, can nevertheless still bind this amino acid.
...
PMID:Intersubunit communication in hybrid hexamers of K89L/A163G/S380A and C320S mutants of glutamate dehydrogenase from Clostridium symbiosum. 939 25


<< Previous 1 2 3 4 5 6 7 8 9 10

---