Skip to main content
SpringerLink
Log in

The effect of pH and ADP on ammonia affinity for human glutamate dehydrogenases

  • Published:
Metabolic Brain Disease Aims and scope Submit manuscript

Abstract

Glutamate dehydrogenase (GDH) uses ammonia to reversibly convert α-ketoglutarate to glutamate using NADP(H) and NAD(H) as cofactors. While GDH in most mammals is encoded by a single GLUD1 gene, humans and other primates have acquired a GLUD2 gene with distinct tissue expression profile. The two human isoenzymes (hGDH1 and hGDH2), though highly homologous, differ markedly in their regulatory properties. Here we obtained hGDH1 and hGDH2 in recombinant form and studied their Km for ammonia in the presence of 1.0 mM ADP. The analyses showed that lowering the pH of the buffer (from 8.0 to 7.0) increased the Km for ammonia substantially (hGDH1: from 12.8 ± 1.4 mM to 57.5 ± 1.6 mM; hGDH2: from 14.7 ± 1.6 mM to 62.2 ± 1.7 mM), thus essentially precluding reductive amination. Moreover, lowering the ADP concentration to 0.1 mM not only increased the K0.5 [NH4 +] of hGDH2, but also introduced a positive cooperative binding phenomenon in this isoenzyme. Hence, intra-mitochondrial acidification, as occurring in astrocytes during glutamatergic transmission should favor the oxidative deamination of glutamate. Similar considerations apply to the handling of glutamate by the proximal convoluted tubules of the kidney during systemic acidosis. The reverse could apply for conditions of local or systemic hyperammonemia or alkalosis.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Fig. 1
Fig. 2

Similar content being viewed by others

References

  • Adeva MM, Souto G, Blanco N, Donapetry C (2012) Ammonium metabolism in humans. Metabolism 61:1495–1511

    Article  PubMed  CAS  Google Scholar 

  • Azarias G, Perreten H, Lengacher S, Poburko D, Demaurex N, Magistretti PJ, Chatton JY (2011) Glutamate transport decreases mitochondrial pH and modulates oxidative metabolism in astrocytes. J Neurosci 31:3550–3559

    Article  PubMed  CAS  Google Scholar 

  • Bailey J, Bell ET, Bell JE (1982) Regulation of bovine glutamate dehydrogenase. The effects of pH and ADP. J Biol Chem 257:5579–5583

    PubMed  CAS  Google Scholar 

  • Balut C, vande Ven M, Despa S, Lambrichts I, Ameloot M, Steels P, Smets I (2008) Measurement of cytosolic and mitochondrial pH in living cells during reversible metabolic inhibition. Kidney Int 73:226–232

    Article  PubMed  CAS  Google Scholar 

  • Cohen NS, Kyan FS, Kyan SS, Cheung CW, Raijman L (1985) The apparent Km of ammonia for carbamoyl phosphate synthetase (ammonia) in situ. Biochem J 229:205–211

    PubMed  CAS  Google Scholar 

  • Cooper A (2012) The role of glutamine synthetase and glutamate dehydrogenase in cerebral ammonia homeostasis. Neurochem Res 37:2439–2455

    Article  PubMed  CAS  Google Scholar 

  • Engel P (1981) More than one substrate. In: Engel P (ed) Enzyme kinetics, the steady state approach. Chapmann Hall, London

    Google Scholar 

  • Faff-Michalak L, Albrecht J (1993) Hyperammonemia and hepatic encephalopathy stimulate rat cerebral synaptic mitochondrial glutamate dehydrogenase activity specifically in the direction of glutamate oxidation. Brain Res 618:299–302

    Article  PubMed  CAS  Google Scholar 

  • Hudson R, Daniel R (1993) L-glutamate dehydrogenases: distribution, properties and mechanism. Comp Biochem Physiol B 106:767–792

    PubMed  CAS  Google Scholar 

  • Hutson SM, Islam MM, Zaganas I (2011) Interaction between glutamate dehydrogenase (GDH) and l-leucine catabolic enzymes: Intersecting metabolic pathways. Neurochem Int 59:518–524

    Article  PubMed  CAS  Google Scholar 

  • Kimelberg H, Vitarella D, Aschner M (1996) Effects of toxins on glia in brain edema. In: Aschner M, Kimelberg H (eds) The role of glia in neurotoxicity. CRC Press, Boca Raton, pp 311–334

    Google Scholar 

  • Leke R, Bak L, Anker M, Melo TM, Sørensen M, Keiding S, Vilstrup H, Ott P, Portela LV, Sonnewald U, Schousboe A, Waagepetersen HS (2011) Detoxification of ammonia in mouse cortical GABAergic cell cultures increases neuronal oxidative metabolism and reveals an emerging role for release of glucose-derived alanine. Neurotox Res 19:496–510

    Article  PubMed  CAS  Google Scholar 

  • Llopis J, McCaffery JM, Miyawaki A, Farquhar MG, Tsien RY (1998) Measurement of cytosolic, mitochondrial, and Golgi pH in single living cells with green fluorescent proteins. Proc Natl Acad Sci 95:6803–6808

    Article  PubMed  CAS  Google Scholar 

  • Marcaggi P, Coles JA (2001) Ammonium in nervous tissue: transport across cell membranes, fluxes from neurons to glial cells, and role in signalling. Progr Neurobiol 64:157–183

    Article  CAS  Google Scholar 

  • McKenna MC, Sonnewald U, Huang X, Stevenson J, Zielke HR (1996) Exogenous glutamate concentration regulates the metabolic fate of glutamate in astrocytes. J Neurochem 66:386–393

    Article  PubMed  CAS  Google Scholar 

  • Metelkin E, Demin O, Kovacs Z, Chinopoulos C (2009) Modeling of ATP-ADP steady-state exchange rate mediated by the adenine nucleotide translocase in isolated mitochondria. FEBS J 276:6942–6955

    Article  PubMed  CAS  Google Scholar 

  • Murthy MS, Pande SV (1985) Microcompartmentation of transported carnitine, acetylcarnitine and ADP occurs in the mitochondrial matrix. Implications for transport measurements and metabolism. Biochem J 230:657–663

    PubMed  CAS  Google Scholar 

  • Plaitakis A, Zaganas I (2001) Regulation of human glutamate dehydrogenases: implications for glutamate, ammonia and energy metabolism in brain. J Neurosci Res 66:899–908

    Article  PubMed  CAS  Google Scholar 

  • Rama Rao KV, Jayakumar AR, Norenberg DM (2003) Ammonia neurotoxicity: role of the mitochondrial permeability transition. Metab Brain Dis 18:113–127

    Article  PubMed  CAS  Google Scholar 

  • Schoolwerth AC, LaNoue KF, Hoover WJ (1984) Effect of pH on glutamate efflux from rat kidney mitochondria. Am J Physiol - Ren Physiol 246:F266–F271

    CAS  Google Scholar 

  • Shashidharan P, Clarke DD, Ahmed N, Moschonas N, Plaitakis A (1997) Nerve tissue-specific human glutamate dehydrogenase that is thermolabile and highly regulated by ADP. J Neurochem 68:1804–1811

    Article  PubMed  CAS  Google Scholar 

  • Sonnewald U, Westergaard N, Schousboe A (1997) Glutamate transport and metabolism in astrocytes. Glia 21:56–63

    Article  PubMed  CAS  Google Scholar 

  • Swain M, Butterworth R, Blei A (1992) Ammonia and related amino acids in the pathogenesis of brain edema in acute ischemic liver failure in rats. Hepatology 15:449–453

    Article  PubMed  CAS  Google Scholar 

  • Tsacopoulos M, Poitry-Yamate C, Poitry S (1997) Ammonium and glutamate released by neurons are signals regulating the nutritive function of a glial cell. J Neurosci 17:2383–2390

    PubMed  CAS  Google Scholar 

  • Vetterli L, Carobbio S, Pournourmohammadi S, Martin-Del-Rio R, Skytt DM, Waagepetersen HS, Tamarit-Rodriguez J, Maechler P (2012) Delineation of glutamate pathways and secretory responses in pancreatic islets with beta-cell specific abrogation of the glutamate dehydrogenase. Mol Biol Cell 23:3851–3862

    Article  PubMed  CAS  Google Scholar 

  • Waagepetersen HS, Sonnewald U, Larsson OM, Schousboe A (2000) A possible role of alanine for ammonia transfer between astrocytes and glutamatergic neurons. J Neurochem 75:471–479

    Article  PubMed  CAS  Google Scholar 

  • Wiederkehr A, Park K-S, Dupont O, Demaurex N, Pozzan T, Cline GW, Wollheim CB (2009) Matrix alkalinization: a novel mitochondrial signal for sustained pancreatic β-cell activation. EMBO J 28:417–428

    Article  PubMed  CAS  Google Scholar 

  • Williamson DH, Lund P, Krebs HA (1967) The redox state of free nicotinamide-adenine dinucleotide in the cytoplasm and mitochondria of rat liver. Biochem J 103:514–527

    PubMed  CAS  Google Scholar 

  • Yu AC, Schousboe A, Hertz L (1982) Metabolic fate of 14C-labeled glutamate in astrocytes in primary culture. J Neurochem 39:954–960

    Article  PubMed  CAS  Google Scholar 

  • Zaganas I, Plaitakis A (2002) Single amino acid substitution (G456A) in the vicinity of the GTP binding domain of human housekeeping glutamate dehydrogenase markedly attenuates GTP inhibition and abolishes the cooperative behavior of the enzyme. J Biol Chem 277:26422–26428

    Article  PubMed  CAS  Google Scholar 

  • Zaganas I, Spanaki C, Plaitakis A (2012) Expression of human GLUD2 glutamate dehydrogenase in human tissues: functional implications. Neurochem Int 61:455–462

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgments

This research has been co-financed by the European Union (European Social Fund – ESF) and Greek national funds through the Operational Program “Education and Lifelong Learning” of the National Strategic Reference Framework (NSRF) - Research Funding Program: THALIS –UOC, Title “Mitochondrial dysfunction in neurodegenerative diseases” (MDND grant, University of Crete Code 3578). Also, support was obtained from the “Association for research and treatment of neurologic diseases of Crete-EY ZHN”, the Lundbeck Foundation and The Danish Medical Research Council 09-066319.

Author information

Authors and Affiliations

  1. Neurology Laboratory, Medical School, University of Crete, Heraklion, Crete, Greece

    Ioannis Zaganas, Kamilla Pajęcka, Camilla Wendel Nielsen & Andreas Plaitakis

  2. Department of Drug Design and Pharmacology, Faculty of Health and Medical Sciences, University of Copenhagen, 2100, Copenhagen, Denmark

    Kamilla Pajęcka, Camilla Wendel Nielsen, Arne Schousboe & Helle S. Waagepetersen

Authors
  1. Ioannis Zaganas
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Kamilla Pajęcka
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Camilla Wendel Nielsen
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Arne Schousboe
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Helle S. Waagepetersen
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Andreas Plaitakis
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Ioannis Zaganas.

Additional information

Ioannis Zaganas and Kamilla Pajęcka contributed equally to this work.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Zaganas, I., Pajęcka, K., Wendel Nielsen, C. et al. The effect of pH and ADP on ammonia affinity for human glutamate dehydrogenases. Metab Brain Dis 28, 127–131 (2013). https://doi.org/10.1007/s11011-013-9382-6

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s11011-013-9382-6

Keywords

Search

Navigation