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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 cellular energy (Aubby et al., 1988; Duvoisin et al., 1988) investiga­ charge declines. tors. Additional studies in our laboratory (Colon et al., 1986) showed the presence of two GDH activities in rat brain differing in their relative resistance to thermal MATERIALS AND METHODS inactivation, detergent extractability, and allosteric Materials regulation characteristics. Further investigations at the Sf9 cells and the baculovirus expression vectors were ob­ protein level (Hussain et al., 1989) revealed that GDH tained from Invitrogen (San Diego; CA; U.S.A.). Insect cell purified from human brain consists of four electropho- media and fetal calf serum were obtained from Life Technol-

Met Arg Ala Gly Tyr ~Ser Arg TyrLeulfij:>.t::::\ Leu~ Pro FIG. 2. N-terminal amino acid se­ ~\@JPAia Leu Ala quence of human recombinant @fP}GIYLeu L Ala GLUD1 and GLU02-derived GDHs. Arg eu AlaAia ~t::;:::.. Leu Gly Ser Gly The expressed proteins were blotted Ser~~@Aia ~ Gin onto polyvinylidene difluoride mem­ Leu Ala Ala Arg Arg His ~ • branes, and their N-terminus was Pro Gly Leu Ala Ty rSer Glu Ja!Val Ala Asp Arg Glu Asp ...... Ala ~ Pro sequenced (amino acids shown in Ala Ala 'C!YG/n GLUD1 boldface). The leader peptide se­ quence is shown in italics. The pre­ • dicted amino acids that are different in the two cDNAs are circled. The cleavage site of the leader peptide AlaAia9Gn / . is indicated by an arrow. In the N­ Ala ProGI ~ Y Leu Ala L HisTyrSerGlu~ValAlaAspArgGluAspt ...... terminus of the mature (processed) ~o OOA~A~A~A~ protein, an alanine to leucine transi­ AI Ser@Q@ Ala Ser tion was detected at the third amino ~;y Ala a Gly acid position; this is predicted by the A Leu rgt:::::\~GI Leu Leu corresponding cDNAs (Shashidha­ ®~ Y tr:;).. Ala ran et al., 1994). Arg Tyr Leu@~AiaLeu Leu~ Ala Ty r v:::!.Jiser Met A rg A Ia Gly Pro GLUD2

J. Neurochem., Vol. 68, No. 5, 1997 1806 P. SHASHIDHARAN ET AL.

Enzyme assay 100 GDH was assayed spectrophotometrically in the direction \ of reductive amination of a-ketoglutarate. For this, the reac­ \ tion mixture of 1 ml contained 50 mM triethanolamine buffer ~ 80 \ (pH 8.0, except as indicated), 100 mM ammonium acetate, ;:> caCJ 150 J.LM NADPH, and 2.6 mM EDTA. ADP was also added Ci) to 1 mM unless otherwise specified. The reaction was started c 60 1"" ·--Ci) with addition of a-ketoglutarate to 8 mM (Colon et al., 0ca ~ 1986). .a GLUD2-derlved GDH ...c 40 Ci) "" CJ ~ Allosteric regulation of human recombinant Ci) ""-._ a. GDHs 20 "'"' - ----_, Stimulation of GDH activity by ADP was carried out by adding increasing amounts of ADP (final concentration, 0 0.05-1.0 mM) to the above assay mixture. Similar ADP 0 20 40 60 80 100 stimulation studies were also carried out at different pH Incubation time (min) at 45°C values using 0.1 M phosphate buffer (pH 6.5, 7 .0, 7 .5, or 2 8.0). The effects of spermine and Mg + on GDH activity FIG. 3. Thermal stability of recombinant human GDHs. were determined by adding either compound to the reac­ Housekeeping (GLUD1-derived GDH; e) and nerve tissue-spe­ tion mixture (final concentration, 1-2 mM) with or with­ cific (GLUD2-derived GDH; •) GDHs were obtained by expres­ out ADP. sion of the corresponding cDNAs in Sf9 cells as described in Materials and Methods. The enzyme extracts were mixed with 1:1 (vol/vol) bovine serum albumin (8 mg/ml) in 100 mM (pH Heat inactivation of recombinant GDHs 7.4) phosphate buffer. The enzyme mixture was incubated at Sf9 cell extracts containing expressed GLUD1- or 45.5°C in Eppendorf tubes. Aliquots were removed at specified GLUD2-derived protein were mixed ( 1:1) with 100 mM intervals and assayed at pH 8.0 in the presence of 1 .0 m M ADP sodium phosphate buffer (pH 7.4) containing 8 mg/ml bo­ as described in Materials and Methods. Data are mean + SEM vine serum albumin. The samples were incubated at 45.0°C (bars) values of three determinations. Baseline activity refers to in a shaking water bath; aliquots were removed at specific enzyme activity at 0 min of incubation. intervals and assayed immediately as described above with the addition of 1.0 mM ADP (final concentration) to the reaction mixture. ogies (Bethesda, MD, U.S.A.). Modified baculovirus (Bacu­ loGold) was from Pharmingen (San Diego, CA, U.S.A.). Purification and N-terminal amino acid NADH, NADPH, a-ketoglutaric acid, ADP, GTP (lithium sequencing of recombinant GDHs salt), and bovine liver GDH were from Boehringer Mann­ Both recombinant human GDHs were partially purified heim Biochemicals (Indianapolis, IN, U.S.A.) . Spermine from Sf9 cell extracts according to a previously described and MgCh were from Sigma Chemical (St. Louis, MO, method (Hussain et al., 1989), and the amino acid sequence U.S.A.). Materials for polyacrylamide gel electrophoresis of their N-terminus was determined. In brief, the purification were from Bio-Rad (Richmond, CA, U.S.A.), and polyvinyl­ method involves ammonium sulfate fractionation and hy­ idene difluoride membranes were from Millipore (Bedford, drophobic interaction chromatography. A 30-65% ammo­ MA, U.S.A.). nium sulfate cut of a high-speed ( 100,000 g for 1 h) superna­ tant of these extracts was obtained and dissolved in Tris­ Expression of GLUDJ and GLUD2 eDNA in HCI buffer (0.05 M, pH 6.0) containing ammonium sulfate to 15% saturation. It was then loaded onto a column ( 1.5 Sf9 Cells X 30 em) packed with pheny1-Sepharose-4B and equili­ A GLUDJ eDNA and a GLUD2 eDNA derived from hu­ brated with the same buffer. The column was washed with man liver and retina mRNA, respectively, were expressed 200 ml of equilibration buffer (to remove any nonspecifi­ in the baculovirus expression system. The coding region of cally bound proteins) and eluted with a total of 250 ml of each of these cDNAs was PCR-amplified (Shashidharan et a double linear gradient of 0-90% ethylene glycol and 15- al., 1994). The products obtained were subcloned, ligated 0 o/o ammonium sulfate. Fractions containing GDH activity to the baculovirus transfer vector (pVL 1392; Invitrogen), were pooled, dialyzed against deionized distilled water, and and used to transform Escherichia coli (INValphaF'). The loaded on a discontinuous 5-15% linear gradient polyacryl­ proper orientation of the insert was verified by DNA se­ amide gel containing 0.1 % sodium dodecyl sulfate as pre­ quencing. Sf9 cells were cotransfected with the plasmid viously described (Hussain et al., 1989). The proteins were DNA and modified baculovirus DNA (BaculoGold; Phar­ transferred to a polyvinylidene difluoride membrane ac­ mingen) and were incubated at 27°C for 4 days. The recom­ cording to the method of Matsudaira ( 1987) using 250 mA binant virus was amplified with two additional rounds of constant current for 4 h in a Bio-Rad electroblotting appara­ infection. Sf9 cells were harvested and homogenized in a tus. The electroblotted polyvinylidene difluoride membrane buffer containing 0.05 M Tris-HCl (pH 7.4), 1% Triton X- was washed in deionized distilled water for 5 min, stained 100, 0.1 mM phenylmethylsulfonyl fluoride, and 0.5 M with 0.1 % Coomassie Brilliant Blue R250 in 50% methanol N aCl. The extracts obtained were used for enzyme assays, for 30 s, and then destained for 3- 5 min in 50% methanol/ kinetic analyses, thermal inactivation, and immunoblotting, 10% acetic acid. It was finally rinsed in deionized distilled as described above. The protein concentrations were deter­ water and air-dried. The GDH band was cut out and used mined as previously described (Lowry et al., 1951). for N -terminal amino acid sequencing ( Matsudaira, 1987).

J. Neurochem. , Vol. 68, No. 5, 1997 NERVE TISSUE-SPECIFIC HUMAN GDH 1807

800 GLUD1-derived GOH -c ·-E 700 -c ·-.! 600 ec.. C) 500 E en -.!! 400 0 E .5. 300 FIG. 4. Allosteric regulation of re­ '>~ combinant human GDHs by ADP. =u 200 Enzyme assays of housekeeping < (GLUD1-derived GDH) and nerve tis­ .~ 100 !: sue-specific ( GLU02-derived GDH) u c.C1) GDH were performed using trietha­ en 0 nolamine buffer (pH 8.0) in the pres­ 0 10 25 50 250 1000 ence of increasing concentrations of AOP btM) ADP (0.01-1.0 mM). Baseline activ­ ity was determined in the absence of ADP ( 100%). Data are mean + SEM (bars) values from three determina­ 7000 7000 tions. GDH activity was measured in 6000 the direction of reductive amination 6000 of a-ketoglutarate as described in 5000 ~ 5000 Materials and Methods. ~ > >; ; u u 4( 4000 4( 4000 4) 4) .5 .5 'i 3000 : 3000 en ftJ mftJ m 2000 0 0 - 2000 fl. -~0 1000 1000

0 0 0 10 25 50 250 1000 0 10 25 50 100 500 1000

AOP (~M) AOP (~M)

Statistical analysis density of which increased between day 2 to 5 postin­ 2 The significance of the effect of Mg + on recombinant fection (data not shown) . human GDH activities was analyzed by an unpaired two­ Sequencing of the N-terminus of the recombinant tailed t test. The statistical analysis was carried out using proteins revealed that both the GLUD1- and GLUD2- Microsoft Excel version 4.0 software. The Hill plot coeffi­ generated polypeptides had been processed within the cient and the cooperativity index were calculated using Enz­ host cells in a manner similar to that occurring for fitter software (Elsevier-BIOSOFf, Cambridge, U.K.). the mature mammalian GDH, which involves removal of the leader peptide (Fig. 2) . Moreover, these se­ RESULTS quencing data confirmed the presence of leucine and alanine at position 3 for the nerve tissue-specific Production of recombinant human GDHs ( GLUD2-derived) and the housekeeping ( GLUDJ­ The specific multiplication of recombinant baculovi­ derived) GDH, respectively, as predicted by the cor­ rus containing either the GLUD 1 or the GLUD2 eDNA responding cDNAs (Fig. 2), thus verifying the spe­ in the Sf9 cell cultures infected by the recombinant cific expression of the cDNAs in the corresponding viruses was verified by Sall digestion of a PCR-ampli­ cell lines. fied DNA segment containing a Sall restriction site unique to the GLUD2 eDNA ( Shashidharan et al., Thermal stability characteristics of GLUD1- and 1994) . As shown in Fig. 1, this treatment resulted in GLUD2-generated GDH complete cleavage of the GLUD2 eDNA-derived PCR Heat-inactivation studies revealed that the two re­ product, but it left uncut the GLUDJ eDNA-derived combinant proteins differed in their relative resistance DNA segment. Immunoblots of Sf9 cell extracts using to thermal denaturation. At 45°C (pH 7.4), heat inacti­ anti-GDH antiserum (Shashidharan et al., 1994) re­ vation proceeded faster for the nerve tissue-specific vealed that cells infected with both recombinant bacu­ GDH (half-life = 0.5 h) than for the housekeeping loviruses showed GDH-positive immunoreactivity, the enzyme (half-life = 3.5 h), as shown in Fig. 3.

J. Neurochem., Vol. 68, No. 5, 1997 1808 P. SHASHIDHARAN ET AL.

250 .------, 700 .------, c c=J GLUD1-GDH pH 6.5 e ~ GLUD2-GDH c::::J GLUD1-GDH 600 ~ GLUD2-GDH ,200 G ~ ~ 500 c > -~150 1; .?! < 400 ~ .. ()• .5 !E "i : 300 t100 Ill ,• 1: i 3.... 200 :;, .. E 50 D. FIG. 5. Effect of pH on the activation of recombinant lc 100 0 GDHs by ADP. Enzyme assays were performed at z various pH values in the absence or the presence of B) o increasing concentrations of ADP. Phosphate buffer A) 6.5 7.0 7.5 8.0 0 0.05 0.10 0.20 0.50 pH ADP (mM) (0.1 M) was used instead of triethanolamine buffer for this set of experiments. Data are mean + SEM (bars) values from three determinations. GDH activity 800 -.------., 1400 .------:--:------, pH7.0 pH7.5 was measured in the direction of reductive amination 700- c::J GLU01-GOH c=J GLUD1-GDH of a-ketoglutarate as described in Materials and ~ GLUD2-GDH 1200 ~ GLU02-GDH Methods. The percentage of activation by ADP at pH 600- 8.0 (data not shown) was similar to that presented ~ f1000 in Fig. 4. > :;: ~ 500- ~ .. .. 800 .5 400- .5 .,.. "i .. 600 Ill 300- Ill= c c -..() - -.. E 400 i 200- G D. D.

100- r- 200

0 -'----'- - '- -- _.._ _.._ '---- 0 -'----'- C) o o.os 0.10 0.20 0.50 D) o o.05 0.10 0.20 o.so ~p~~ ~p~~

Regulation of recombinant GDH activities nerve tissue-specific GDH to '""2,000% of baseline ac­ by ADP tivity while inducing only a 2.5-fold activation of the Enzyme assays of Sf9 cell extracts revealed that housekeeping enzyme (Fig. 4). expression of the GLUD1 or GLUD2 in these cells generated proteins capable of catalyzing the reversible Effect of pH on ADP activation of recombinant interconversion of glutamate to a-ketoglutarate in the human GDHs presence of either NAD(H) or NADP(H). Because Enzyme assays, performed at various pHs, revealed the insect GDH shows an absolute specificity for that the nonstimulated activity (baseline) of the NAD(H) (Shashidharan et al., 1994) and the human GLUDJ-derived GDH (housekeeping) increased by GDH is capable of using both cofactors (Plaitakis et about fourfold as the pH of the reaction buffer in­ al., 1980), measurement of enzyme activity in the pres­ creased from 6.5 to 8.0 (Fig. 5); however, the ADP ence of NADP(H) provided a simple way of eliminat­ activation of the GLUDJ isoenzyme remained propor­ ing all background activity. tionally the same (200-250% of baseline activity; Fig. The specific activities of the two recombinant human 5). In contrast, whereas the baseline activity of the GDHs were similar when analyzed in the presence GLUD2-derived GDH (nerve tissue-specific) was un­ of 1 mM ADP. However, the two enzymes differed affected by varying the pH of the reaction buffer, the markedly in their ability to interconvert glutamate to a­ ADP activation of this isoenzyme was enhanced with ketoglutarate when assayed in the absence of allosteric increasing pH; addition of0.5 mM ADP (final concen­ effectors; the nerve tissue-specific GDH was largely tration) at pH 6.5, 7.0, 7.5, and 8.0 increased the activ­ inactive, whereas the housekeeping enzyme showed ity of the GLUD2-derived GDH to 600, 700, 1,238, about half of its maximal specific activity (Fig. 4). and 2,100% of baseline activity, respectively (Fig. 5). ADP at 0.05-0.25 mM induced a proportionally Hence, increasing the pH of the reaction buffer be­ greater degree of stimulation for the nerve tissue-spe­ tween 6.5 and 8.0 enhances the nonstimulated (base­ cific than for the housekeeping enzyme. This differen­ line) activity of the housekeeping GDH, whereas it tial effect increased at higher nucleotide concentrations renders the nerve tissue-specific isoenzyme increas­ (0.5-1.0 mM), with 1.0 mM ADP stimulating the ingly sensitive to allosteric activation by ADP.

J. Neurochem., Vol. 68, No. 5, 1997 NERVE TISSUE-SPECIFIC HUMAN GDH 1809

Effect of magnesium chloride on recombinant 2400 human GDHs In the absence of ADP, magnesium chloride ( 1.0 2000 and 2.0 mM) inhibited the housekeeping GDH (by 45 / and 64%, respectively), but it had no effect on the l:' 1600 ·s:: J____ ----t/ nonstimulated nerve tissue-specific enzyme (Fig. 6). ~ In the presence of low physiological concentrations of Cl) 1200 c I ADP (0.05-0.25 mM), however, this inhibitory effect Q) I 1/) I of magnesium chloride was completely abolished (Fig. mns 800 7). Magnesium had little effect on the baseline activity ';!. I of the nerve tissue-specific GDH, but it modified its 400 / GLUD1-GDH .,a':...-~-~-o--=---=Q:c--~---c ,. allosteric regulation by preventing most of the acti va­ '? tion of this enzyme at 0.05-0.25 but not at 1.0 mM 0 concentrations of ADP. As a result, the hyperbolic ADP activation curve became sigmoidal in the pres­ 0 250 500 750 1000 ence of magnesium chloride (Fig. 6). This change was ADP (~M) significant by Hill plot analysis, which showed that the FIG. 7. Effect of magnesium chloride on ADP activation of re­ Hill plot coefficient for ADP stimulation was 1.24 + combinant human GDHs. GLU02-derived GDH was assayed in 2 0.02 in the absence of magnesium chloride and 1.81 the absence (0 ) or in the presence of 2.0 mM Mg + (D ); GLU01- derived GDH was also assayed in the absence (6 ) or in the + 0.04 (p < 0.001) in the presence of the cation. presence of 2.0 mM Mg 2 + (\7). Allosteric activation of the recom­ Spermine, shown to be inhibitory to rat brain GDH binant human isoenzymes was studied by varying ADP levels (Kuo et al., 1994), had no effect on either human from 0 to 1,000 p,M while keeping the concentration of the other GDH isoenzyme (data not shown). substrates constant. Data are mean + SEM (bars) values of three determinations. DISCUSSION

In this study, we evaluated the thermal stability and the nerve tissue-specific GDH in these patients. This allosteric regulation characteristics of recombinant hu­ remains to be tested in future studies. man GDH isoproteins generated by expression of Evaluation of the regulatory properties of the two GLUDJ (housekeeping) and GLUD2 (nerve tissue­ expressed GDH isoproteins revealed that the nerve tis­ specific) cDNAs in Sf9 cells. Results revealed that the sue-specific GDH has evolved into a highly regulated housekeeping GDH is relatively heat-stable, whereas enzyme showing little catalytic activity when assayed the nerve tissue-specific enzyme is relatively heat-la­ in the absence of allosteric effectors but being mark­ bile. Because activity of the heat-labile GDH is selec­ edly activated (up to 1,300%) by relatively low physi­ tively reduced in some patients with multisystem de­ ological concentrations of ADP ( 0.02- 0.25 mM). In generation, these results suggest a selective defect in contrast, the housekeeping GD H depends much less on ADP for its activation, maintaining ---50% of its maximal activity in the absence of this nucleotide. 250 Varying the pH of the reaction buffer had a differen­ 1 mM MgCI 2mMMgCI 2 2 tial effect on the allosteric regulation of the two isoen­ -c zymes by ADP. When the pH increased between 6.5 E 200 c and 8.0, the nonstimulated (baseline) activity of the -i 0 housekeeping GDH was enhanced, although the pro­ '- Q. C) 150 portional activation of this isoenzyme by ADP was not E Cii altered. In contrast, increasing the pH between 6.5 and Cl) * • 0 8.0 markedly enhanced the ADP activation of the nerve cE 100 tissue-specific GDH without affecting its baseline spe­ -~ ** cific activity. Bailey et al. ( 1982) previously reported that ADP is inhibitory to bovine liver GDH when the 50 ~u I;:: reaction is carried at pH 6.5 and below. In the present 0 Cl) Q. studies, we found that ADP had no inhibitory effect U) 0 on either human GDH isoenzyme at pH 6.5. Similar GLUD1 GLUD2 GLUD1 GLUD2 results have been reported by Cho et al. ( 1995) using FIG. 6. Effect of magnesium chloride on nonactivated recombi­ bovine brain GDHs. nant human GDHs. Enzyme assays were performed using re­ In addition, regulation of enzyme activity by magne­ combinant human housekeeping ( GLUD1) or nerve tissue-spe­ sium was distinct for the two isoenzymes. The cific (GLUD2) GDH isoenzymes in either the absence (open col­ umns) or presence (hatched columns) of 1.0 or 2.0 mM housekeeping GDH was inhibited substantially by 1.0 magnesium chloride. Data are average + SEM (bars) values of or 2.0 mM magnesium chloride when assayed in the three determinations. *p < 0.02, **p < 0.001. absence of allosteric effectors, but this inhibition was

J. Neurochem., Vol. 68, No. 5, 1997 1810 P. SHASHIDHARAN ET AL. largely reversed by low physiological concentrations Acknowledgment: This work was supported by the Na­ (0.05-0.25 mM) of ADP. Whereas magnesium had tional Institutes of Health grant NS16871. We thank Betsy little effect on the baseline activity of the nerve tissue­ Chalfin for editorial assistance. specific GDH, it modified its allosteric regulation by preventing most of the activation of this enzyme at REFERENCES 0.05-0.25 but not at 1.0 mM ADP. Magnesium levels in the mitochondrial matrix are AbeT., Ishiguro S. 1., Saito H., Kiyosawa M., and Tarnai M. ( 1992) Partially deficient glutamate dehydrogenase activity and attenu­ known to be in the range of 1.0 mM (Taylor et al., ated oscillatory potentials in patients with spinocerebellar de­ 1991 ), but ADP levels can vary from 0.05 to> 1.0 mM generation. Invest. Ophthalmol. Vis. Sci. 33, 447-452. (depending on the rate of oxidative phosphorylation) Anagnou N. P., Seuanez H., Modi W., O'Brien S. J., Papamatheakis (Gabriel et al., 1986). Hence, results of this study J., and Moschonas N. K. ( 1993) Chromosomal mapping of two members of the human glutamate dehydrogenase ( GLUD) gene indicate that physiological levels of ADP and magne­ family to chromosomes 10q22.3-q23 and Xq22-q23. Hum. sium are capable of allosterically regulating human Hered. 43, 351-356. nerve tissue-specific GDH. In particular, magnesium Aoki C., Milner T. S., RexSheu K.-F. R., Blass J.P., and Pickel is capable of shifting the relatively hyperbolic ADP V. M. ( 1987) Regional distribution of astrocytes with intense stimulation curve to one that is clearly sigmoidal. As immunoreactivity for glutamate dehydrogenase in rat brain: im­ plications for neuron-glia interactions in glutamate transmis­ such, magnesium may play a role in keeping the nerve sions. J. Neurosci. 7, 2214-2231. tissue-specific GDH idle at low physiological concen­ Aubby D., Saggu H. K. , Jenner P., Quinn N. 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