Fordham University Masthead Logo DigitalResearch@Fordham Chemistry Faculty Publications Chemistry 1997 Nerve tissue-specific umh an glutamate dehydrogenase that is thermolabile and highly regulated by adp / P. Shashidharan, Donald D. Clarke, Naveed Ahmed, Nicholas Moschonas, and Andreas Plaitakis Department of Neurology, Mount Sinai School of Medicine, New York; Department of Chemistry, Fordham University, Bronx New York, USA; and Department of Biology and School of Health Sciences, University of Crete, Crete, Greece P. Shashidharan Mount Sinai School of Medicine. Department of Neurology, [email protected] Donald Dudley Clarke PhD Fordham University, [email protected] Recommended Citation Shashidharan, P.; Clarke, Donald Dudley PhD; Ahmed, Naveed; and Moschonas, Nicholas, "Nerve tissue-specific umh an glutamate dehydrogenase that is thermolabile and highly regulated by adp / P. Shashidharan, Donald D. Clarke, Naveed Ahmed, Nicholas Moschonas, and Andreas Plaitakis Department of Neurology, Mount Sinai School of Medicine, New York; Department of Chemistry, Fordham University, Bronx New York, USA; and Department of Biology and School of Health Sciences, University of Crete, Crete, Greece" (1997). Chemistry Faculty Publications. 13. https://fordham.bepress.com/chem_facultypubs/13 This Article is brought to you for free and open access by the Chemistry at DigitalResearch@Fordham. It has been accepted for inclusion in Chemistry Faculty Publications by an authorized administrator of DigitalResearch@Fordham. For more information, please contact [email protected]. Naveed Ahmed Mount Sinai School of Medicine. Department of Neurology Nicholas Moschonas University of Crete. Department of Biology Follow this and additional works at: https://fordham.bepress.com/chem_facultypubs Part of the Biochemistry Commons Journal of Neurochemistry Lippincott-Raven Publishers, Philadelphia © 1997 International Society for Neurochemistry Nerve Tissue-Specific Human Glutamate Dehydrogenase that Is Thermolabile and Highly Regulated by ADP , I *P. Shashidharan, tDonald D. Clarke, *Naveed Ahmed, I l tNicholas Moschonas, and *§Andreas Plaitakis f *Department of Neurology, Mount Sinai School of Medicine, New York; t Department of Chemistry, Fordham University, Bronx, New York, U.S.A.; and *Department of Biology and §School of Health Sciences, University of Crete, Crete, Greece Abstract: Glutamate dehydrogenase (GDH), an enzyme mate to a-ketoglutarate, is expressed at high levels in that is central to the metabolism of glutamate, is present mammalian brain (Smith et al., 1975). Although the at high levels in the mammalian brain. Studies on human exact function of GDH in brain is not fully understood, leukocytes and rat brain suggested the presence of two the enzyme is enriched in glutamatergic receptive re­ GDH activities differing in thermal stability and allosteric gions (Aoki et al., 1987), where it is highly concen­ regulation, but molecular biological investigations led to the cloning of two human GDH-specific genes encoding trated in astrocytes ( 10 mg of GDH protein/ml of mito­ highly homologous polypeptides. The first gene, desig­ chondrial matrix) (Rothe et al., 1994). These observa­ nated GLUD1, is expressed in all tissues (housekeeping tions are consistent with a suggested role for this GDH), whereas the second gene, designated GLUD2, is enzyme in the metabolism of synaptic glutamate expressed specifically in neural and testicular tissues. In (Plaitakis et al., 1984; Colon et al., 1986). this study, we obtained both GDH isoenzymes in pure At present, the functional significance of GDH in form by expressing a GLUD1 eDNA and a GLUD2 eDNA nerve tissue remains uncertain. Although thermody­ in Sf9 cells and studied their properties. The enzymes namically the GDH reaction favors glutamate synthe­ generated showed comparable catalytic properties when sis, aspartate aminotransferase may be more important fully activated by 1 mM ADP. However, in the absence than GDH in forming glutamate from a-ketoglutarate of ADP, the nerve tissue-specific GDH showed only 5% of its maximal activity, compared with ,....,40% showed by in vivo (Palaiologos et al., 1988; Christensen et al., 1991). Cooper et al. ( 1979) previously showed that the housekeeping enzyme. Low physiological levels of 13 ADP (0.05-0.25 mM) induced a concentration-depen­ NH3 ammonia given systemically to rats failed to dent enhancement of enzyme activity that was propor­ incorporate into brain glutamate, thus suggesting that tionally greater for the nerve tissue GDH (by 550- GDH is not functioning toward the synthesis of this 1 ,300%) than of the housekeeping enzyme {by 120- amino acid in nerve tissue. On the other hand, Yu et 150%). Magnesium chloride (1-2 mM) inhibited the al. ( 1982) suggested that GDH may play a role in nonactivated housekeeping GDH (by 45-64%); this inhi­ glutamate oxidation in astrocytes. Also, Erecinska and bition was reversed almost completely by ADP. In con­ 2 Nelson ( 1990) found that activation of GDH activity trast, Mg + did not affect the nonstimulated nerve tissue­ increased the rate of glutamate oxidation by rat brain specific GDH, although the cation prevented much of the allosteric activation of the enzyme at low ADP levels synaptosomes. Other studies (Kuo et al., 1994) have (0.05-0.25 mM). Heat-inactivation experiments revealed also suggested that, in spite of the high GDH concen­ that the half-life of the housekeeping and nerve tissue­ tration in brain, the metabolic flux through this path­ • specific GDH was 3.5 and 0.5 h, respectively. Hence, the way is quite low in vivo, thus inferring that enzyme nerve tissue-specific GDH is relatively thermolabile and activity is regulated. Kuo et al. ( 1994) further sug­ has evolved into a highly regulated enzyme. These allo­ gested that, in rat brain, this is due to the inhibitory steric properties may be of importance for regulating • effect of magnesium and polyamines on GDH activity . brain glutamate fluxes in vivo under changing energy de­ Previous studies have shown that GDH activity is mands. Key Words: Glutamate dehydrogenase-Human reduced in leukocytes and ~broblasts of patients with brain-Glutamate metabolism-ADP enzyme regula­ tion-Enzyme thermolability. J. Neurochem. 68, 1804-1811 (1997). Received September 25, 1996; revised manuscript received Janu­ ary 6, 1997; accepted January 6, 1997. Address correspondence and reprint requests to Dr. A. Plaitakis at Department of Neurology, Mount Sinai School of Medicine, One Gustave L. Levy Place, New York, NY 10029, U.S.A. Glutamate dehydrogenase ( GDH), the enzyme that Abbreviations used: GDH, glutamate dehydrogenase; Sf9, catalyzes the reversible oxidative deamination of gluta- Spodoptera frugiperda. 1804 NERVE TISSUE-SPECIFIC HUMAN GDH 1805 retically distinct isoproteins. More recent studies have shown that two different GDH isoproteins are also present in bovine brain ( Cho et al., 1995). UNCUT Molecular biological studies (Mavrothalassitis et al., 1988; Shashidharan et al., 1994) revealed that multiple GDH-specific genes are present in the human. At least M---- two of these genes are functional. The first, designated Gl G2 Gl G2 GLUD1, is an intron-containing gene mapping to hu­ • FIG. 1. Expression of GLUD1 (housekeeping) and GLUD2 man chromosome 10; the mRNA of this gene is ex­ (nerve tissue-specific) eDNA in Sf9 cells. A: PCR amplification pressed in all tissues (housekeeping) (Hanauer et al., of a 580-bp DNA segment using DNA isolated from Sf9 cell 1987; Mavrothalassitis et al., 1988; Anagnou et al., cultures infected with recombinant baculovirus containing the 1993; Michaelidis et al., 1993) and is the only GDH­ human GLUD1 eDNA (G1; lane 1) or GLUD2 eDNA (G2; lane 1 ). Cloned human GLUD1 eDNA (G1; lane 2) and GLUD2 eDNA specific message found in human liver ( Shashidharan (G2; lane 2) were used as controls. B: Restriction digestion of et al., 1994). The second, designated GLUD2, is en­ amplified DNA with Sa/1. Amplified DNA from human GLUD2 coded by an X-linked intronless gene, the mRNA of eDNA ( G2) generates two smaller fragments (cleaved), whereas which is specifically expressed in neural and testicular that from human GLUD2 eDNA (G1) remains uncleaved (uncut). tissues ( Shashidharan et al., 1994) . The polypeptides predicted by the two genes share a 96% amino acid sequence homology ( Shashidharan multisystemic neurological disorders that are clinically et al., 1994), with this similarity in structure probably and pathologically heterogeneous (Plaitakis et al., accounting for the inability to separate them in a clean 1980, 1982; Duvoisin et al., 1983, 1988; Aubby et al., form from human tissues. The availability of recombi­ 1988; Abe et al., 1992). Our work with neurologic nant DNA technology has permitted us now to obtain patients led to the finding that GDH is present in human and study each of these human GDH isoenzymes in tissues in "heat-labile" and "heat-stable" forms and pure (unmixed), catalytically active forms by express­ that, in these patients, reduction in GDH activity was ing the corresponding cDN As in the insect Spodoptera largely limited to the heat-labile component (Plaitakis frugiperda ( Sf9) cells. Results revealed that the nerve et al., 1984) . Similar results have been obtained by tissue-specific isoform has evolved into a highly regu­ other (Konagaya et al., 1986; Kajiyama et al., 1988; lated enzyme that is markedly activated by ADP at lwattsuji et al., 1989; Abe et al., 1992) but not all concentrations that can occur when