Glutamate dehydrogenase: A study of its physico-chemical properties from the liver of mice of two different age groups

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J. Wahlang1* & D. Syiem2

1Department of Biochemistry, St. Edmund’s College, Shillong, India
2Department of Biochemistry,North Eastern Hills University,Shillong, India.
*Corresponding author: James Wahlang, email:


Glutamate dehydrogenase (GDH) an enzyme which catalyze the reversible formation of glutamate from \alpha-ketoglutarate, occupies a central position in mammalian nitrogen metabolism since the reaction which it catalyze provides the major pathway by which ammonia become bound to the \alpha-carbon atom of an \alpha-ketoacid to generate glutamate. The activity of GDH is influenced by a number of physical and chemical parameters that contributes to its stability as well as its activity. Physico-chemical studies of partially purified GDH from two age groups (10- and 90-day old), from the mice liver tissue are reported.

Keywords GDH, physico-chemical, mice


GDH is a ubiquitous enzyme present in practically all organisms. In plants GDH catalyzes the reversible reductive amination of \alpha-ketoglutarate to form glutamate in the presence of the co-factor NAD(P)H. In humans two isoforms of GDH exists; hGDH1 a house keeping isozyme; and hGDH2 a nerve specific isozyme. These isoforms have different intracellular locations and cofactor affinities, but share similar pH optima, relative molecular masses, and specificities for \alpha-ketoglutarate and glutamate. Although this enzyme does not exhibit allosteric regulation in plants, bacteria or fungi, its activity is tightly controlled by a number of compounds in mammals (Yip et al., 1995; Knapp et al., 1997; Zaganas and Plaitakis, 2002; Yang et al.,2004; Masterodemos et al., 2005; Kanavouras et al., 2007).

The role of GDH assumed significance about a decade ago with the discovery that hypoglycemia in infants and young children was associated with activating mutations of GDH (Fang et al., 2002, MacMullen et al., 2001; Stanley et al., 1998).

This enzyme is widely distributed in mammals where it is located in the mitochondrial matrix and microsomes and is abundant in the liver, heart and the kidneys (Kawajiri et al., 1977). The mammalian forms of GDH constitute a more or less discrete class in sharing the ability to use either NAD(H) or NADP(H) (transhydrogenase) as coenzymes. GDH occupies a central position in mammalian nitrogen metabolism since the reaction which it catalyze provides the major pathway by which ammonia become bound to the \alpha-carbon atom of an \alpha-ketoacid to generate glutamate (Harvey, 1985). However, recent evidences have suggested that the GDH reactions as observed in patients with hyperinsulinism/hyperammonemia syndrome, operates essentially in the direction of glutamate oxidation (Stanley, 2009). Gain-of-function mutations in GDH have been identified as the cause of this congenital disorder. The consequent increase in GDH activity in the \beta-cells of the pancreas results in increased entry of carbon as \alpha-ketoglutarate into the Krebs cycle, which maintains a high cellular ATP/ADP ratio, triggering the insulin-secretion cascade (MacMullen et al., 2001, Smith et al., 2002, Stanley, 2000). Hyperammonemia, which is brought about as a result of systemic activation of GDH is due to compromised hepatic ammonia handling (Treberg et al., 2010).

The mammalian enzyme is a homohexamer, consisting of six identical subunits and possess the ability to use either NAD(H) or NADP(H) as co-enzymes. GDH is strongly influenced by the positive modulator ADP and the negative modulator GTP. Further, the activity and stability of this enzyme is known to be influenced by a number of factors which includes the pH, ionic strength, types of substrates, inhibitors and their concentration, and reducing agents (Neumann et al., 1976; Gafni & Yuh, 1989; Smith et al., 1970).

This study sought to compare the enzyme activity and/or stability of GDH from liver mitochondria of immature (10-day) and matured (90-day) mice, using dialysed preparations. Dialysis ensures removal of endogenous substrates and co-factors/ metal ions which might otherwise interfere with the physico-chemical assay.

Materials and methods

Preparation of GDH extract: Male Swiss-albino mice of the various post natal ages were killed by cervical dislocation and the organs were quickly removed and washed in 0.9% saline solution. A 20% (w/v) homogenate of the minced tissue in 25 mM Sucrose Tris-HCl, pH 7.4 (isolation medium; IM), was prepared by homogenizing in a borosil homogenizing tube with a motor driven pestle in cold. The homogenates were then centrifuged at 800-1000 x g for 15 min at 4ºC. The supernatant obtained was further fractionated twice at 14,000 x g for 20 min at 2ºC. The mitochondrial pellets obtained were then suspended in a 25 mM Tris-HCl, pH 7.4 containing Triton X-100 (0.05% final concentration) and used for enzyme assay.

Enzyme Assay: GDH activity was assayed according  to the procedure of Wrzeszczynski & Colman (1994) with slight modifications. The change in absorbance per minute (\DeltaE) was monitored spectrophotometrically in a Cary 50 Bio UV-visible Spectrophotometer (Varian, USA), in the direction of reductive amination of \alpha-ketoglutarate in a medium containing the following final concentrations; 84.7 mM immidazole buffer (2.5 ml) pH 7.9, 217 mM ammonium acetate (50 µl), 0.12 mM NADH (30 µl), 0.9 mM EDTA (100 µl), and 1.7 mM ADP (50 µl). The reaction was initiated by the addition of 200 µl of  \alpha-ketoglutarate to a final concentration of 13.6 mM or as specified otherwise. A unit of GDH is defined as the amount required to oxidise 1µmol of NADH/min at 25ºC. Protein concentrations were determined by the dye binding method of Bradford (1976) using bovine serum albumin as reference standard.

Physico-chemical characterization of the enzyme was carried out using dialysed sample preparations and or the purified enzyme. Crude GDH extracted from the liver of 10- and 90-day old mice was made free of endogenous substrate and metal ions, which might otherwise interfere during analysis, by dialyzing against the isolation medium containing 25 mM sucrose-Tris HCl buffer, pH 7.4 added with 1 mM 2-mercaptoethanol and 1mM phenylmethyl sulphonyl fluoride (PMSF) maintained at 4ºC. Dialysed sample was then used to assess the effects of various effects of substrates, metal ions and other inhibitors.

Determination of optimum ionic strength: Varying ionic strength (10-200 mM) of immidazole buffer was used for determining the optimal buffer ionic strength for GDH from the liver 10- and 90-day old. The other conditions of assay remained as described under method section.

Determination of pH optima: The pH optima was determined using 25 mM sucrose tris HCl and or immidazole buffer pH (7.0-9.0) with buffering capacities in the alkaline range of same ionic strength and sodium acetate buffer of the same ionic strength, covering the lower pH range. The other conditions of assay remained as described earlier. The pH optima for GDH of the two age groups were determined.

Reducing effects of Dithiothreitol and 2-mercaptoethanol: The enzyme preparations from the two age groups (10-and 90-day) were separately incubated in assay buffer containing varying concentrations of DTT (0-10 mM) and 2-mercaptoethanol (0-10 mM) for one hour in the cold followed by normal enzyme assay. The results are expressed in per cent activity.

Inactivation studies: Using various concentrations (0-5M) of guanidine hydrochloride prepared in assay buffer, the enzyme preparations from the two age groups (10- and 90-day) were incubated in separated test tubes for one hour in the cold followed by normal enzyme assay. The results are expressed in terms of percentage activity.

Results and Discussions

Effect of ionic strength on GDH: In both the ages the enzyme activity, expressed as percentage activity from the  maximum, showed identical molar requirement with the maximum activity at 175 mM of immidazole (Figure 1). Using buffer of varying ionic strength, it was observed that GDH from the 10- and 90-day old mice exhibited a similar molar optima, indicating that there was no alteration in the conformation of the enzyme at these ages.

pH stability of GDH: The pH profile of GDH from both the ages (10- and 90-day) was apparently similar with optimum enzyme activity observed at pH 8.0, beyond which a decrease in enzyme activity was observed (Figure 2). Neumann et al., (1976) have reported that the conformational transition of GDH from Candida utilis is pH- and temperature-dependent. The observed loss of activity at higher pH values may reflect deprotonation of cationic groups with pK values of 9 or higher. Such cationic groups might be those expected to interact with the carboxylic groups of the substrates, glutamate or \alpha-ketoglutarate, or with the phosphate groups of pyridine nucleotide co-enzymes (Piszkiewicz & Smith, 1971).

Effect of temperature on the stability of GDH: As shown in Figure 3, the activity of GDH from both the ages showed a typical temperature-activity profile with optimal activity expressed as % activity from the maximum observed at 25ºC and decreases to about 4% and 3% in 10 and 90- day respectively at 65ºC. The effect of temperature on the stability of GDH from the two ages was also found to be similar. The enzyme activity was observed to be highest at 25ºC for both the ages and looses more than 80% of enzyme activity above 55ºC. In contrast, age related changes with respect to heat stability have been reported for many enzymes for example phosphoglycerate kinase, a glycolytic enzyme exhibited a marked increase in the protein’s heat stability as a result of aging-related modification (Gafni & Yuh, 1989).

Effects of reducing agents: The effects of thiol-containing reducing agents, 2-mercaptoethanol and dithiothreitol (DTT) on the enzyme from either age exhibited similar pattern of activity (Figure 4A & B). Although the pattern of influence on the activity was similar in both the ages, DTT confer higher stability on liver GDH from both 10- and 90-day old mice. This signifies the role of the sulphydryl groups of the reagent in contributing to the stability of the enzyme, and the property did not change with age.

Effect of guanidine hydrochloride on GDH: The dialyzed enzyme from both age groups showed similar susceptibility to Gdn.HCl. More than 50% inactivation of enzyme activity by Gdn.HCl at 0.1M was observed and become almost completely inactivated at 1.25 M with activity falling below 10% from the control (Figure 5). Dialysed GDH preparation from the liver of the immature and matured mice exhibited a similar response to guanidinium hydrochloride (Gdn.HCl) inactivation, with more than 50% decrease in enzyme activity observed at 0.1M for both ages. Sensitivity to this denaturant may provide an indication to the conformation of this enzyme. It was earlier suggested that, at concentrations of Gdn.HCl insufficient to cause denaturation, the hexamer dissociated into inactive trimers without any gross structural changes that could be detected by fluorescence or circular dichroism techniques (West & Price, 1988). Conformational changes including non-covalent modifications in proteins during aging have also been reported (Rothstein, 1979; 1985). Other studies shown that enolase, a glycolytic enzyme isolated from young nematodes, when subjected to denaturation by guanidine hydrochloride and allowed to refold, the folded protein showed a resemblance to the isomeric form with different catalytic property, present in the old nematode, signifying that the presence of young age enolase in tissues of young animals attests to the presence in these tissues, of conditions that favour folding into this species (Sharma & Rothstein, 1978).

Earlier biochemical works led to the finding that multiple forms of GDH are present in mammalian system and that the activities of these isotypes differ in their relative resistance to thermal inactivation, detergent extractability and allosteric regulation characteristics (Plaitakis et al., 1984, 2000; Cho et al., 1995; Shashidharan et al., 1997). These forms have been designated as soluble and particulate GDH (Plaitakis et al., 1984; Colon et al., 1986; Hussain et al., 1989). The GDH isoproteins are differentially distributed in the two catalytically active isoforms of the enzyme (Colon et al., 1986; Plaitakis et al., 1993). The four different forms of GDH isoproteins were detected from the human cerebellum of normal subjects and patients with neurodegenerative disorders (Duvoisin et al., 1983; Plaitakis et al., 1984 Hussain et al., 1989). Our studies however indicate that the liver enzyme conformation remains unchanged with age.




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