Bovine liver glutamate dehydrogenase
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ABSTRACT:
SUBMITTER: Edward T Eng
PROVIDER: EMPIAR-10217 | biostudies-other |
REPOSITORIES: biostudies-other
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Project description:In this work, we re-examine the previously reported phenomenon of the creation of a superactive glutamate dehydrogenase by proteolytic modification by chymotrypsin and explore the various discrepancies that came to light during those studies. We find that superactivation is caused by cleavage at the N terminus of the protein and not the C-terminal allosteric site, as has previously been suggested. N-terminal sequencing reveals that TLCK-treated chymotrypsin cleaves bovine glutamate dehydrogenase at phenylalanine 10. We suggest that trypsin contamination in nontreated chymotrypsin may have led to the production of the larger 4-5 kDa digestion product, previously misinterpreted as having caused the activation. In line with some previous studies, we can confirm that GTP inhibition is attenuated to some extent by the proteolysis, while ADP activation is almost abolished. Utilizing the recently solved structures of bovine glutamate dehydrogenase, we illustrate the cleavage points.
| S-EPMC2492812 | biostudies-literature
Project description:1. Ox liver glutamate dehydrogenase is activated by bovine pancreatic alpha-chymotrypsin, but the extent of activation is dependent on the age of the dehydrogenase preparation. 2. The degree of activation is constant and the pseudo-first-order rate constant of activation is directly proportional to the concentration of proteinase used. 3. Commercial preparations of alpha-chymotrypsin differ in their ability to produce a secondary inactivation phase, and this was shown to be due to low tryptic contamination. The 'superactive' form of glutamate dehydrogenase has an increased sensitivity to tryptic inactivation as compared with the native enzyme. 4. Analysis of the activation by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis revealed that the subunit molecular weight of 'superactive' glutamate dehydrogenase differs by less than 5% from that of the native subunit.
| S-EPMC1158448 | biostudies-other
Project description:1. Initial rates of oxidative deamination of L-glutamate with NAD+ as coenzyme, and of reductive aminiation of 2-oxoglutarate with NADH as coenzyme, catalysed by bovine liver glutamate dehydrogenase were measured in 0.111 M-sodium phosphate buffer, pH 7, at 25 degrees C, in the absence and presence of product inhibitors. All 12 possible combinations of variable substrate and product inhibitor were used. 2. Strict competition was observed between NAD+ and NADH, and between glutamate and 2-oxoglutarate. All other inhibition patterns were clearly non-competitive, except for inhibition by NH4+ with NAD+ as variable substrate. Here the extrapolation did not permit a clear distinction between competitive and non-competitive inhibition. 3. Mutually non-competitive behaviour between glutamate and NH4+ indicates that these substrates can be bound at the active site simultaneously. 4. Primary Lineweaver-Burk plots and derived secondary plots of slopes and intercepts against inhibitor concentration were linear, with one exception: with 2-oxoglutarate as variable substrate, the replot of primary intercepts against inhibitory NAD+ concentration was curved. 5. Separate Ki values were evaluated for the effect of each product inhibitor on the individual terms in the reciprocal initial-rate equations. With this information it is possible to calculate rates for any combination of substrate concentrations within the experimental range with any concentration of a single product inhibitor. 6. The inhibition patterns are consistent with neither a simple compulsory-order mechanism nor a rapid-equilibrium random-order mechanism without modification. They can, however, be reconciled with either type of mechanism by postulating appropirate abortive complexes. Of the two compulsory sequences that have been proposed, one, that in which the order of binding is NADH, NH4+, 2-oxoglutarate, requires an implausible pattern of abortive complex-formation to account for the results. 7. On the basis of a rapid-equilibrium random-order mechanism, dissociation constants can be calculated from the Ki values. Where these can be compared with independent estimates from the kinetics of the uninhibited reaction or from direct measurements of substrate binding, the agreement is reasonable good. On balance, therefore, the results provide further support for the rapid-equilibrium random-order mechanism under these conditions.
| S-EPMC1172361 | biostudies-other
Project description:Bovine liver glutamate dehydrogenase is potently inhibited by Zn2+ ions. At pH 7.0 a kinetic dissociation constant for Zn2+ of 18 microM is obtained. The fluorescent lanthanide Eu3+ competes for the Zn2+-binding site and relieves the Zn2+-induced inhibition, but does not cause inhibition. Studies on the effects of Zn2+ or Eu3+ on the tertiary and quaternary structure of the enzyme by the use of protein fluorescence, heat-stability and re-activation after guanidinium chloride denaturation indicate that, whereas Zn2+ affects both tertiary and quaternary structure, Eu3+ does not affect either, consistent with its lack of effect on enzymic properties. Eu3+ fluorescence had a strong excitation peak at 395 nm with emission at 456 nm. In the presence of glutamate dehydrogenase the fluorescence emission is shifted to 501 nm. Eu3+, with high-affinity binding site and distinctive fluorescence properties after binding, would appear to be an ideal fluorophore for use in conformational studies or resonance-energy-transfer studies.
| S-EPMC1148258 | biostudies-other
Project description:The liver is the most essential organ for the metabolism of ammonia, in where most of ammonia is removed by urea and glutamine synthesis. Regulated by leucine, glutamate dehydrogenase (GDH) catalyzes the reversible inter-conversion of glutamate to ammonia. To determine the mechanism of leucine regulating GDH, pigs weighing 20 ± 1 kg were infused for 80 min with ammonium chloride or alanine in the presence or absence of leucine. Primary pig hepatocytes were incubated with or without leucine. In the in vivo experiments with either ammonium or alanine as the nitrogen source, addition of leucine significantly inhibited ureagenesis and promoted the production of glutamate and glutamine in the perfused pig liver (P < 0.05). Similarly, leucine stimulated GDH activity and inhibited sirtuin4 (SIRT4) gene expression (P < 0.01). Leucine could also activate mammalian target of rapamycin complex 1 (mTORC1) signaling (P < 0.05), as evidenced by the increased phosphorylation levels of ribosomal protein S6 kinase 1 (S6K1) and ribosomal protein S6 (S6). Interestingly, the leucine-induced mTORC1 pathway activation suitably correlated with increased GDH activity and decreased expression of SIRT4. Similar results were observed in primary cultured hepatocytes. Notably, leucine exerted no significant change in GDH activity in SIRT4-deficient hepatocytes (P > 0.05), while mTORC1 signaling was activated. Leucine exerted no significant changes in both GDH activity and SIRT4 gene expression in rapamycin treated hepatocytes (P > 0.05). In conclusion, L-leucine increases GDH activity and stimulates glutamate synthesis from different nitrogen sources by regulating mTORC1/SIRT4 pathway in the liver of pigs.
| S-EPMC6116330 | biostudies-literature
Project description:Glutamate dehydrogenase is an important enzyme in the hepatic regulation of nitrogen and energy metabolism. It catalyzes one of the most relevant anaplerotic reactions. Although its relevance in liver homeostasis has been widely described, its daily pattern and responsiveness to restricted feeding protocols has not been studied. We explored the daily variations of liver glutamate dehydrogenase transcription, protein, activity, and histochemical and subcellular location in a protocol of daytime food synchronization in rats. Restricted feeding involved food access for 2 h each day for three weeks. Control groups included food ad libitum as well as acute fasting (21 h fasting) and refeeding (22 h fasting followed by 2 h of food access). Glutamate dehydrogenase mRNA, protein, activity, and histological location were measured every 3 h by qPCR, Western blot, spectrophotometry, and immunohistochemistry, respectively, to generate 24-h profiles. Restricted feeding promoted higher levels of mitochondrial glutamate dehydrogenase protein and activity, as well as a loss of 24-h rhythmicity, in comparison to ad libitum conditions. The rhythmicity of glutamate dehydrogenase activity detected in serum was changed. The data demonstrated that daytime restricted feeding enhanced glutamate dehydrogenase protein and activity levels in liver mitochondria, changed the rhythmicity of its mRNA and serum activity, but without effect in its expression in hepatocytes surrounding central and portal veins. These results could be related to the adaptation in nitrogen and energy metabolism that occurs in the liver during restricted feeding and the concomitant expression of the food entrainable oscillator. Impact statement For the first time, we are reporting the changes in daily rhythmicity of glutamate dehydrogenase (GDH) mRNA, protein and activity that occur in the liver during the expression of the food entrained oscillator (FEO). These results are part of the metabolic adaptations that modulate the hepatic timing system when the protocol of daytime restricted feeding is applied. As highlight, it was demonstrated higher GDH protein and activity in the mitochondrial fraction. These results contribute to a better understanding of the influence of the FEO in the energy and nitrogen handling in the liver. They could also be significant in the pathophysiology of hepatic diseases related with circadian abnormalities.
| S-EPMC5407590 | biostudies-literature
Project description:1. Kinetic aspects of the reaction between crystalline bovine liver glutamate dehydrogenase and formiminoglutamate were investigated to establish the conditions under which the latter may interfere with the assay of glutamate by using glutamate dehydrogenase and to explain why formiminoglutamate accumulates in vivo after histidine loading, although it can react with glutamate dehydrogenase. The Km and Vmax. values were compared with those of the enzyme reacting with glutamate. At pH 7.4 Km for formiminoglutamate was much higher and Vmax. much lower than the values for glutamate. 2. The equilibrium constant at pH 7.0 was 0.017 micrometer with formiminoglutamate, i.e. about one two-hundredths that with glutamate. 3. In vivo the interaction between glutamate dehydrogenase and formiminoglutamate is minimal even when the concentration of the latter in the liver is greatly raised, as in cobalamine or folate deficiency after histidine loading. 4. At pH 9.3, i.e. under the conditions for the assay of glutamate by glutamate dehydrogenase, formiminoglutamate reacts readily with the enzyme.
| S-EPMC1183950 | biostudies-other
Project description:Initial-rate studies were made of the oxidation of L-glutamate by NAD+ and NADP+ catalysed by highly purified preparations of dogfish liver glutamate dehydrogenase. With NAD+ as coenzyme the kinetics show the same features of coenzyme activation as seen with the bovine liver enzyme [Engel & Dalziel (1969) Biochem. J. 115, 621--631]. With NADP+ as coenzyme, initial rates are much slower than with NAD+, and Lineweaver--Burk plots are linear over extended ranges of substrate and coenzyme concentration. Stopped-flow studies with NADP+ as coenzyme give no evidence for the accumulation of significant concentrations of NADPH-containing complexes with the enzyme in the steady state. Protection studies against inactivation by pyridoxal 5'-phosphate indicate that NAD+ and NADP+ give the same degree of protection in the presence of sodium glutarate. The results are used to deduce information about the mechanism of glutamate oxidation by the enzyme. Initial-rate studies of the reductive amination of 2-oxoglutarate by NADH and NADPH catalysed by dogfish liver glutamate dehydrogenase showed that the kinetic features of the reaction are very similar with both coenzymes, but reactions with NADH are much faster. The data show that a number of possible mechanisms for the reaction may be discarded, including the compulsory mechanism (previously proposed for the enzyme) in which the sequence of binding is NAD(P)H, NH4+ and 2-oxoglutarate. The kinetic data suggest either a rapid-equilibrium random mechanism or the compulsory mechanism with the binding sequence NH4+, NAD(P)H, 2-oxoglutarate. However, binding studies and protection studies indicate that coenzyme and 2-oxoglutarate do bind to the free enzyme.
| S-EPMC1186394 | biostudies-other
Project description:Serum activities of alanine and aspartate aminotransferases (ALT and AST) are used as gold standard biomarkers for the diagnosis of hepatocellular injury. Since ALT and AST lack liver specificity, the diagnosis of the onset of hepatocellular injury in patients with underlying muscle impairments is severely limited. Thus, we evaluated the potential of glutamate dehydrogenase (GLDH) as a liver specific alternative biomarker of hepatocellular injury. In our study, serum GLDH in subjects with Duchene muscular dystrophy (DMD) was equivalent to serum GLDH in age matched healthy subjects, while serum ALT was increased 20-fold in DMD subjects. Furthermore, serum GLDH in 131 subjects with variety of muscle impairments was similar to serum GLDH of healthy subjects while serum ALT corelated with serum creatine kinase, a widely accepted biomarker of muscle impairment. In addition, significant elevations of ALT, AST, and CK were observed in a case of a patient with rhabdomyolysis, while serum GLDH stayed within the normal range until the onset of hypoxia-induced liver injury. In a mouse model of DMD (DMDmdx), serum GLDH but not serum ALT clearly correlated with the degree of acetaminophen-induced liver injury. Taken together, our data support the utility of serum GLDH as a liver-specific biomarker of liver injury that has a potential to improve diagnosis of hepatocellular injury in patients with underlying muscle impairments. In drug development, GLDH may have utility as a biomarker of drug induced liver injury in clinical trials of new therapies to treat muscle diseases such as DMD.
| S-EPMC7224523 | biostudies-literature
Project description:Previous workers have shown that the hexamers of glutamate dehydrogenase are dissociated first into trimers and subsequently into monomers by increasing guanidinium chloride concentrations. In renaturation experiments it is shown that trimers of glutamate dehydrogenase can be reassociated to give the hexamer form of the enzyme, with full regain of activity. Monomeric subunits produced at high guanidinium chloride concentrations cannot be renatured. The trimer form of the enzyme is shown to have no catalytic activity, although the hexamer form in guanidinium chloride has full activity.
| S-EPMC1153214 | biostudies-other