- Email: [email protected]
Diversity of glutamate dehydrogenase in human brain
Progress in Neuro-Psychopharmacology & Biological Psychiatry 26 (2002) 427 – 435
Article
Diversity of glutamate dehydrogenase in human brain Gulnur Sh. Burbaeva*, Marina S. Turishcheva, Elena A. Vorobyeva, Olga K. Savushkina, Elena B. Tereshkina, Irina S. Boksha Laboratory of Neurochemistry, Mental Health Research Center RAMS, 113152 Zagorodnoje shosse 2-2, Moscow, Russia
Abstract Three forms of glutamate dehydrogenase (GDH, EC 1.4.1.3) are purified from human brain tissue. Two of them, named GDH I (consisting of 58 ± 1-kDa subunit) and GDH II (consisting of 56 ± 1-kDa subunit), are readily solubilized and the third one, GDH III (consisting of 56 ± 1-kDa subunit), is a membrane-associated (particulate bound) isoform. Kinetic constants were determined for GDH III. These GDH forms were found to differ in hydrophobicity as indicated by different affinity to Phenyl-Sepharose. All three GDH forms showed microheterogeneity on two-dimensional (2-D) gel electrophoresis. Specific polyclonal antibodies, which enable to determine the levels of immunoreactivities of all the GDH forms in human brain extracts by enzyme-chemiluminescent amplified (ECL)-Western immunoblotting, were obtained. D 2001 Elsevier Science Inc. All rights reserved. Keywords: Antiglutamate dehydrogenase polyclonal antibodies; Glutamate dehydrogenase isoenzymes; Human brain; Western immunoblotting
1. Introduction Glutamate is the major excitatory neurotransmitter in mammalian brain, and glutamate dehydrogenase (GDH, EC 1.4.1.3) is one of key enzymes of glutamate metabolism. GDH catalyses reversible oxidative deamination of L-glutamate to a-oxoglutarate using NAD/NADP as coenzymes. There is some discrepancy in respect of the role of GDH in glutamate biosynthesis/degradation in vivo (Yudkoff et al., 1991; Kugler, 1993; Kanamori and Ross, 1995; Schmitt and Kugler, 1999), and there are also controversies in results of investigations of GDH cellular localization in central nervous system. According to most biochemical studies, GDH is ubiquitously detected in neurons and glia (Aree et al., 1990; Subbalakshmi and Murthy, 1985). In contrast, immunohistochemical studies have shown that GDH is enriched or exclusively localized in astrocytes (Aoki et al., 1987; Kaneko et al., 1987; Rothe et al., 1990), whereas a significant expression of GDH mRNA in neurons was observed (Schmitt and Kugler, 1999). The contradictory data obtained in brain GDH studies may be due to heterogeneity of this enzyme. Actually, the Abbreviations: GDH, glutamate dehydrogenase; PAG, polyacrylamide gel. * Corresponding author. Tel.: +7-95-952-9129; fax: +7-95-952-8940. E-mail address: [email protected] (G.S. Burbaeva).
existence of soluble and particulate isoproteins of brain GDH has been discovered (Colon et al., 1986; Plaitakis et al., 1993; Rajas and Rouset, 1993). Cho et al. (1995) has detected two different GDH isoproteins (GDH I and GDH II) in bovine brain that were readily solubilized, and no detergents were required for their extraction. Soluble and particulate isoforms of GDH, and also GDH I and GDH II, differ in relative resistance to thermal inactivation and allosteric regulation. The described GDH isoforms may represent the products of different genes. Alternatively, they may be translated from different GDH mRNA species generated by alternative splicing or may arise due to posttranslation modifications. Multiple GDH genes were shown to exist in human (Michaelidis et al., 1993). At least two of them are functional (Mavrothalassitis et al., 1988; Shashidharan et al., 1994). The mRNA encoded by the GLUD1 gene (Michaelidis et al., 1993) is expressed widely (housekeeping), whereas mRNA of the second one, encoded by the GLUD2 gene, is specifically expressed in neural and testicular tissues (Shashidharan et al., 1994). GLUD1 and GLUD2 isoforms of GDH are regulated by distinct allosteric mechanisms (Plaitakis et al., 2000). Besides GDH enzymatic activity, other roles of GDHs have been reported: a particulate GDH form possesses a microtubule-binding activity and acts as a molecule by means of which an of attachment of lysosomes to microtubules
0278-5846/01/$ – see front matter D 2001 Elsevier Science Inc. All rights reserved. PII: S 0 2 7 8 - 5 8 4 6 ( 0 1 ) 0 0 2 7 3 - 1
428
G.S. Burbaeva et al. / Progress in Neuro-Psychopharmacology & Biological Psychiatry 26 (2002) 427–435
occurs (Rajas and Rouset, 1993; Rajas et al., 1996); GDH possesses an RNA-binding activity and possibly is involved in the regulation of transcription (Preiss et al., 1993); GDH gene has been also identified as one of memory-related genes in the hippocampus (Cavallaro et al., 1997). A study of brain GDH is of particular interest because the enzyme has been found to be deficient in patients with some neurodegenerative disorders (Plaitakis et al., 1984; Hussain et al., 1989). It seems to be essential to have a detailed description of the various types of brain GDH that provides an approach to clarification of their roles in glutamate metabolism and participation in pathogenesis of diseases with nervous system pathology. The present work reports the first purification and characterization of two soluble and one membrane-associated isoforms of GDH from human brain. Polyclonal antisera to these GDH isoforms were obtained that enable to assess the levels of these isoforms in brain tissue extracts by immunoreactivity.
2. Methods 2.1. Materials Samples of brain, kidney, liver and heart obtained at autopsy from a person with no history of mental or neurological disorders were the starting material for enzyme purification. Permission of the Ethics Committee of the Mental Health Research Centre of the Russian Academy of Medical Sciences was obtained. Animal brains were purchased from a slaughterhouse. For the enzyme purification and immunochemical testing, all the samples were processed no later than 6 h after death. 2.2. Chemicals Phenyl-Sepharose 4B and DEAE-Sephacel were purchased from Amersham-Pharmacia Biotech (Sweden) and GTP-Agarose, a-oxoglutarate, NADH, ADP, EDTA and Triton X-100 were purchased from Sigma (USA). Rabbit polyclonal antiserum against bovine liver GDH was purchased from Biogenesis (England). Enzyme-chemiluminescent amplified (ECL) reagents were purchased from Amersham-Pharmacia Biotech. Goat antirabbit IgG antibody conjugate was obtained from Sigma. 2.3. Experimental procedures 2.3.1. GDH extraction Human brain tissue (total weight of 300 g) was cut onto small portions, frozen in liquid nitrogen and stored until use. After thawing, the brain tissue was minced with a scalpel and then homogenized with a Ultra-Turrax IKA-Werk knife
homogenizer (Germany) in two various media depending on further goals. The first medium was composed of following components: 10-mM Tris – HCl, pH 7.4, with 0.1-mM PMSF, 0.5-mM EDTA, 1% Triton X-100 and 0.1-M NaCl; the tissue/buffer ratio was 1:5 w/v. The second medium was used for protein extraction with the aim of further GDH purification. It comprised 10-mM Tris – HCl, pH 7.4, with 0.1-mM PMSF and 0.5-mM EDTA; the tissue/buffer ratio was 1:5 w/v. All purification steps were done at + 4 C. The homogenate was then centrifuged at 480 g for 10 min followed by centrifugation of the obtained supernatant at 100 000 g for 60 min. Both supernatant and pellet contained GDH enzymatic activity.
2.4. Purification of GDH forms Purification of GDH forms from human brain was performed using the same chromatography steps as described by Colon et al. (1986) and Hussain et al. (1989) in their schemes developed for GDH purification from rat and human brain correspondingly. The 100 000 g supernatant was used for purification of ‘‘readily solubilized’’ GDH forms. The 100 000 g pellet was used for purification of ‘‘membrane-associated’’ GDH. The 100 000 g pellet was resuspended in 10-mM Tris – HCl, pH 7.4, with 0.1-mM PMSF, 0.5-mM EDTA, 1% Triton X-100 and 0.5-M NaCl, and then centrifuged at 100 000 g for 60 min. The ‘‘membrane-associated’’ GDH III was purified to homogeneity from the obtained supernatant by modified method of Colon et al. (1986). The purification methods for both GDH ‘‘readily solubilized’’ and ‘‘membrane-associated’’ forms included ammonium sulfate fractionation (30 – 60% relative saturation), hydrophobic (Phenyl-Sepharose), ion exchange (DEAE-Sephacel) and affinity (GTP-Agarose) column chromatography. Following the ammonium sulfate fractionation of the 100 000 g supernatant or of the Triton X-100 extract obtained from the high-speed (100 000 g) pellet, the 30– 60% (NH4)2SO4 precipitate was dissolved in a minimum volume of 50-mM Tris – HCl buffer, pH 6.0, containing (NH4)2SO4 to 15% relative saturation and loaded onto the Phenyl-Sepharose 4B column (2.4 20 cm), equilibrated with the same buffer. The column was washed with two bed volumes of the starting buffer, followed by the elution with a total of 500 ml of a double gradient of 0 –90% ethylene glycol and 0 –15% ammonium sulfate in 50-mM Tris – HCl buffer, pH 6.0. Fractions containing GDH activity were pooled and dialyzed overnight against 25-mM Tris – HCl, pH 7.5, with 0.1-M KCl. Five chromatography runs were carried out for each the ‘‘readily solubilized’’ and ‘‘membraneassociated’’ GDH.
G.S. Burbaeva et al. / Progress in Neuro-Psychopharmacology & Biological Psychiatry 26 (2002) 427–435
Ion-exchange chromatography on a DEAE-Sephacel column (5 5 cm) was carried out in the same buffer as used for dialysis. The column was washed with two volumes of the starting buffer. The protein was eluted from the column with a 360-ml gradient of 0.1 –0.6-M KCl in 25-mM Tris – HCl, pH 7.5. Fractions with GDH activity were pooled. After 24-h dialysis against 2 l of 2-mM K-Pi, pH 7.2, with 1-mM EDTA and 0.1 mM PMSF, two changes, the protein was concentrated to the volume of 15 ml. Concentrated protein sample was applied onto the GTPAgarose column (0.9 6 cm), equilibrated with the same buffer. The column was washed with the same buffer until the protein peak was eluted. The proteins in this peak possessed no GDH activity. Then, active GDH was eluted with 200-mM KCl in the same buffer. 2.5. Preparation of brain and other organ tissue extracts for immunochemistry Preparation of brain and other organ tissue extracts for immunochemical testing included homogenization of 200 mg of tissue in 1 ml of 50-mM Tris – HCl buffer, pH 7.5,
429
with 1.4-mM 2-ME and 1% Triton using a glass-Teflon homogenizer (Braun, Germany); centrifugation at 1000 g for 15 min followed by centrifugation at 60 000 g for 1 h. The final supernatant was used for PAGE with subsequent ECL immunoblotting. 2.6. Determination of protein concentration Protein concentration was measured according to Lowry et al. (1951). 2.7. Gel electrophoresis Protein homogeneity was tested by SDS-PAGE in 10% polyacrylamide gel (PAG) by the Laemmli (1970) method. Gels were stained by Coomassie Brilliant Blue R-250. The amount of protein applied per lane was 3– 5 mg. Two-dimensional (2-D) electrophoresis was performed by the O’Farrell (1975) procedure. The pH range for isoelectrofocusing (IEF) was 5– 8.5; the pH gradient was adjusted using Pharmalytes (Pharmacia, Sweden).
Fig. 1. Chromatography of proteins extracted from human brain with 1% Triton X-100 + 0.5-M NaCl (a), readily solubilized GDH fraction (b) and membraneassociated GDH fraction (c) on Phenyl-Sepharose 4B column. Experimental details are given in Methods. Fractions of approximately 5 ml were collected and assayed for GDH activity (-5-5-) and protein (-.-.-), as described in Methods. The line -D-D- indicates gradient of ethylene glycol (EG).
430
G.S. Burbaeva et al. / Progress in Neuro-Psychopharmacology & Biological Psychiatry 26 (2002) 427–435
was defined as the amount of the GDH enzyme required to oxidize 1 mmol of NADH per 1 min. The reaction in reverse direction was determined by Fahien and Cohen (1970), by following the increase of absorbancy at 340 nm at 25 C, in a reaction mixture containing 50-mM Tris –HCl, pH 9.0, 2.6-mM EDTA, 1.5-mM NAD + /NADP + and 1-mM ADP. The reaction was started with the addition of L-glutamate to 15 mM. 2.9. Raising of polyclonal antibodies and Western immunoblotting
Fig. 2. PAG with purified GDH I + GDH II (a) and GDH III (b) stained by Coomassie Brilliant blue R-250. Low molecular mass markers are applied on the left, with MW given in kDa. Immunoblots of purified GDH I + GDH II stained by anti-GDH I + GDH II polyclonal antiserum (c) and by antiGDH III polyclonal antiserum (d); immunoblots of purified GDH III stained by anti-GDH III polyclonal antiserum (e) and by anti-GDH I + GDH II polyclonal antiserum (f).
Raising of antihuman GDH rabbit antiserum was fulfilled according to the following scheme (common for all studied forms of GDH): the first immunization, 100 mg subcutaneously with complete Freund’s adjuvant; the second in 2 weeks, 100 mg of GDH subcutaneously with incomplete Freund’s adjuvant; the third in 2 weeks, 100 mg of GDH subcutaneously with incomplete Freund’s adjuvant. ECL immunoblotting (Western blotting) was performed according to Towbin et al. (1979) and Amersham protocol using Hyperfilm ECL nitrocellulose membranes. Protein amount applied on the gel was 1 mg for pure enzymes and 30 mg for tissue extracts; first antibody dilution was 1:15 000 for antisera against GDH I + GDH II and GDH III and 1:25 000 for commercial anti-GDH antiserum. 2.10. Data analysis
2.8. GDH enzymatic activity determination GDH enzymatic activity was determined spectrophotometrically both in direction of reductive amination of aoxoglutarate and in reverse reaction of L -glutamate oxidation. The reaction in the first direction was evaluated by following the decrease of absorbancy at 340 nm in a reaction mixture containing 10-mM Tris – HCl, pH 8.0, 100mM ammonium chloride, 0.1-mM NADH, 2.6-mM EDTA and 1-mM ADP. The reaction was started with the addition of a-oxoglutarate to 10-mM final concentration at 25 C, according to Fahien and Cohen (1970). One unit of enzyme
Statistical analysis was performed using Excel and Microcal Origin 3.5 (Microsoft) software.
3. Results 3.1. Purification of GDH A human brain protein extract obtained after the tissue homogenization in the presence of 1% Triton X-100 and 0.1-M NaCl (see GDH extraction, the first medium) was applied onto the Phenyl-Sepharose 4B column. Three
Table 1 Purification of GDH I + GDH II from human brain GDH I + GDH II GDH activity Fraction
Protein (mg)
U
Specific activity (mmol/mg protein/min)
Purification fold
Brain tissue extract High-speed (100 000 g) supernatant 30 – 60% (NH4)2SO4 precipitate Phenyl-Sepharose DEAE-Sephacel GTP-Agarose
18 200.0 3081.0 236.8 177.5 3.7 1.2
399 600 263 425 23 180 18 500 11 080 5130
21.9 85.5 97.9 104.2 3019.1 4275.0
1 4 4.5 5 138 193
G.S. Burbaeva et al. / Progress in Neuro-Psychopharmacology & Biological Psychiatry 26 (2002) 427–435
431
Table 2 Purification of GDH III from human brain GDH activity Fraction
Protein (mg)
U
Specific activity (mmol/mg protein/min)
Purification fold
Brain tissue extract High-speed (100 000 g) precipitate 30 – 60% (NH4)2SO4 precipitate Phenyl-Sepharose DEAE-Sephacel GTP-Agarose
18 200 1680 710 74 20 0.9
399 600 73 416 37 133 35 860 20 600 5112
21.9 43.7 52.3 484.6 1030 5680
1 2 2.5 22 47 256
peaks of GDH activity was eluted from the column with 0 –90% ethylene glycol (Fig. 1a). Thus, GDH in human brain was supposed to be presented by three major forms differing in hydrophobicity. Then, a fractionation of human brain tissue homogenate (see GDH extraction, the second medium) was made, i.e., ‘‘readily solubilized’’ proteins were separated from ‘‘membrane-associated’’ ones. Two peaks of GDH activity were observed during the elution from Phenyl-Sepharose column in the case of 100 000 g supernatant (Fig. 1b). The first fraction of GDH activity (Peak 1 was eluted at about 40% ethylene glycol) substantially overlapped with the second fraction of GDH activity (Peak 2 was eluted at about 50% ethylene glycol). The attempts to separate GDH I from GDH II on Phenyl-Sepharose column have failed due to similarity of their hydrophobic properties and, hence, highly overlapping fractions. On the other hand, when the Triton X-100 extract from 100 000 g pellet was applied onto the same PhenylSepharose column under the same conditions as in the case of GDH I and II, the elution with 0 – 90% gradient of ethylene glycol led to one peak of GDH activity corresponding to 60% of ethylene glycol (Fig. 1c). The fractions with GDH activity from Peaks 1 and 2 were pooled, and the ethylene glycol and ammonium sulfate were removed by dialysis against 25-mM Tris – HCl, pH 7.5, with 0.1-M KCl before ion-exchange chromatography. Further fractionation of Peaks 1 and 2 was carried in accordance with the procedure proposed by Colon et al. (1986) for soluble GDH from rat brain and also using GTP-Agarose column chromatography step proposed by Hussain et al. (1989) and described in Methods. The final fractions obtained from GTP-Agarose column contained GDH, which gave doubled protein band on SDSPAGE corresponding to 58 ± 1 and 56 ± 1 kDa (we have termed the proteins correspondingly GDH I and GDH II) (Fig. 2a). Subsequent chromatography of GDH peak originated from the Triton X-100 extract on GTP-Agarose column has allowed obtaining apparently homogeneous (as judge from SDS-PAGE) protein possessing GDH activity, termed as GDH III. GDH III was presented by one protein band of 56 ± 1 kDa on SDS-PAGE (Fig. 2b). The results of GDH
I + GDH II and GDH III purification are summarized in Tables 1 and 2. 3.1.1. Properties of GDH Properties of purified GDH I + GDH II and GDH III were compared. The isoenzymes were found to differ in hydrophobicity as indicated by differing affinity to Phenyl-Sepharose (see above). They also show different stability when heated: the GDH III is more heat stabile than GDH I + GDH II, which can be referred as heat labile one. After 30 min incubation at 45 C, the GDH I + GDH II activity was reduced by 60%, whereas the GDH III activity remained on the 90% level of the initial value (Fig. 3). On the other hand, the pH dependencies of activities for GDH I + GDH II and GDH III were similar. Slight differences were observed only in marginal acidic and basic ranges (data not shown). Both GDH I + GDH II and GDH III activities in direct and reversed reactions (see Methods) are stimulated by ADP
Fig. 3. Dependencies of GDH I + GDH II (a) and GDH III (b) activities on time of incubation at 45 C.
432
G.S. Burbaeva et al. / Progress in Neuro-Psychopharmacology & Biological Psychiatry 26 (2002) 427–435
Table 3 Kinetic parameters (KM) for human brain GDH III
Constant substrates
Varying substrate
NH4 + , NADH NH4 + , a-oxoglutarate a-oxoglutarate, NADH NAD + L-glutamate
a-oxoglutarate
Range of concentration for varying substrate (mM)
GDH III shows also microheterogeneity in 2-D PAGE (Fig. 4b): at least five spots ranged in pI range from 7.5 to 6.6. Km (mM)
0.8 – 12.0
1.51 ± 0.12
NADH
0.01 – 0.10
0.11 ± 0.02
NH4 +
10 – 100
24.47 ± 2.20
L-Glutamate NAD + NADP +
0.8 – 16.0 0.2 – 1.8 0.4 – 1.5
3.03 ± 0.20 0.63 ± 0.03 0.72 ± 0.05
to the same extent (about 150% of initial activity was observed in the presence of 0.25 – 0.5-mM ADP in the reaction mixture). Kinetic data were obtained for GDH III (Table 3). Concentrations of constant substrates and pH values of reaction mixtures were as given in Methods. Additional characteristics of subunits of GDH I, GDH II and GDH III forms were obtained using 2-D PAGE. Fig. 4a shows the gel stained by Coomassie Brilliant Blue R-250 after 2-D separation (first dimension: IEF and second dimension: SDS-PAGE) of the purified GDH I + GDH II. The pH gradient during the IEF step is shown along the bottom (beneath the gel); the MW scale is given on the left. GDH I is represented by a train of several spots (at least five) corresponding to MW of 58 ± 1 kDa with pI in a range 7.5 – 6.6. GDH II is represented by a train of at least six spots corresponding to MW of 56 ± 1 kDa with pI ranging from 7.3 to 6.6.
3.1.2. Characterization of antisera to GDH I+GDH II and GDH III Polyclonal rabbit antisera were raised to GDH I + GDH II and GDH III. The specificity of each antiserum was evaluated by ECL immunoblotting using the purified GDH I + GDH II and GDH III samples. Among four antisera raised against GDH I + GDH II, the only antiserum possesses high selectivity to GDH I + GDH II (Fig. 2c) and gives no reaction with GDH III (Fig. 2d). We have also succeeded in obtaining of one specific antiserum to GDH III, which reacts specifically with GDH III (Fig. 2e) and gives extremely low staining with GDH I + GDH II (Fig. 2f). The commercial antiserum raised against bovine liver GDH recognizes all three purified isoforms of human brain GDH (data not shown). 3.1.3. Revealing of immunoreactive proteins in various human tissues using anti-GDH I+GDH II, anti-GDH III and commercial antisera Patterns of immunoreactive proteins stained by antiserum to GDH I + GDH II, antiserum to GDH III and commercial antiserum to bovine liver GDH in extracts (see Methods) of various human tissues (brain, heart, liver and kidney) are shown in the Fig. 5. Antiserum to GDH I + GDH II stained 58- and 56-kDa protein bands in extracts from all tested organs (Fig. 5a), while the antiserum to GDH III (Fig. 5b) and the commercial antiserum (Fig. 5c) revealed 56-kDa proteins and also an unknown 66-kDa protein. 3.1.4. Revealing of immunoreactive proteins in extracts from brains of various species using anti-GDH I+GDH II, anti-GDH III and commercial antisera The abilities of the antisera to reveal GDH in brain extracts of various species were compared (Fig. 6). The antiserum to human GDH I + GDH II revealed a single protein band corresponding to MW 58 kDa in bovine and pig brain extracts (Fig. 6a). The pattern of immuno-
Fig. 4. PAG after 2-D electrophoresis of GDH I + GDH II (a) and GDH III (b) stained by Coomassie Brilliant blue R-250.
Fig. 5. Detection of GDH in protein extracts from various human organs by ECL-Western immunoblotting. (a) revealed by antiserum to GDH I + GDH II, (b) revealed by antiserum to GDH III, (c) revealed by commercial antiserum to bull liver GDH. B: brain; H: heart; L: liver; K: kidney.
G.S. Burbaeva et al. / Progress in Neuro-Psychopharmacology & Biological Psychiatry 26 (2002) 427–435
433
Thus, on the last stage of purification (GTP-Agarose column chromatography in each the case), there were obtained correspondingly GDH I and GDH II from ‘‘soluble’’ fraction and GDH III from ‘‘membrane-associated’’ fraction. 4.2. Characterization of GDH subunit compositions Fig. 6. Detection of GDH in extracts from brains of various species by ECLWestern immunoblotting. (a) revealed by antiserum to GDH I + GDH II, (b) revealed by antiserum to GDH III, (c) revealed by commercial antiserum to bull liver GDH; H: human; P: pig; B: bull; R: rat.
positive protein bands revealed by the antiserum to GDH I + GDH II in rat brain differed from that of human, bovine and pig brain: two protein bands corresponding to MW 56 and 66 kDa were detected. The antiserum to GDH III revealed a single protein band corresponding to MW 56 kDa in extracts from bovine, pig and rat brain but revealed two protein bands corresponding to 56 and 66 kDa in extract from human brain (Fig. 6b). As for commercial antiserum to bovine liver GDH, it revealed 56- and 66-kDa protein bands in extracts from human, bovine, pig and rat brains (Fig. 6c).
4. Discussion 4.1. Purification of GDH isoforms During Phenyl-Sepharose 4B column chromatography of human brain protein extract, the presence of three GDH forms of various hydrophobicity in human brain was found. Keeping in mind the existence of soluble and particulate isoforms of GDH (Colon et al., 1986), the authors of present paper separated ‘‘soluble’’ and ‘‘membrane-associated’’ protein fractions on the first stage of GDH purification, i.e., on the stage of protein extraction from brain tissue. The ‘‘soluble’’ fraction was obtained by extraction with neutral buffer of low ionic strength. For the extraction of ‘‘membrane-associated’’ proteins from 100 000 g pellet (contained mitochondrial and other membranes), we used 1% Triton X-100 and 0.5-M NaCl as proposed by Shashidharan et al. (1994). The following GDH purification steps from ‘‘soluble’’ and ‘‘membrane-associated’’ fractions were similar. During the purification on Phenyl-Sepharose, three isoforms of GDH in human brain were discovered: GDH I, GDH II and GDH III. These proteins differ in their affinity to Phenyl-Sepharose inasmuch their different hydrophobicity. While the affinities of GDH I and GDH II to the resin differ not sufficiently for their separation, this step of purification gives substantial gain in GDH III purification.
GDH I consists of 58-kDa subunits, whereas GDH II and GDH III consist of 56-kDa subunits. Subunits of all three GDH isoenzymes are heterogeneous and give several spots (‘‘trains’’) on 2-D PAGE. 2-D PAGE pattern of GDH III purified in the present paper reminds that obtained for GDH purified from human cerebellum (Hussain et al., 1989). The authors aimed to purify ‘‘particulate’’ GDH form. The microheterogeneity observed in GDH may be accounted for sequential Nterminal amino acid cleavage during the transport of GDH into mitochondrial matrix (Mihara et al., 1982; Shashidharan et al., 1997; Hussain et al., 1989). 4.3. Comparison of biochemical properties of purified GDH forms with known from earlier works Kinetic parameters such as KM for a-oxoglutarate and reduced pyridine nucleotides are similar for GDH III and ‘‘particulate’’ GDH as described by Shashidharan et al. (1994) and Hussain et al. (1989). Whereas KM for Lglutamate, determined in this work, differs from that in earlier works, it is threefold less than KM determined by Shashidharan et al. (1994) and sixfold less than KM determined by Hussain et al. (1989). Thus, the affinity of ‘‘particulate’’ GDH III to L-glutamate is higher than that in ‘‘particulate’’ enzymes described by Shashidharan et al. (1994) and Hussain et al. (1989). As for soluble forms of GDH, Cho et al. (1995) and Cho and Lee (1996) have purified two GDH isoenzymes from bovine brain, but there were no data in literature for the presence of several soluble GDH isoforms in human brain. Bovine brain isoproteins, unlike GDH I and GDH II, discovered in our work, showed similarity in MW of their subunits and were presented by one 57.5-kDa protein band when subjected to SDS-PAGE. Plaitakis et al. (2000) in their paper described properties of two GDH recombinant forms, ‘‘nerve tissue-specific’’ GDH and ‘‘housekeeping’’ one synthesized in the baculovirus expression system from corresponding expressed human genes GLUD1 and GLUD2. The results revealed the differences in heat stability of the expressed enzymes. GDH III purified in present work reminds ‘‘housekeeping’’ GDH, which is relatively more heat stable. At the same time, it cannot be said unambiguously, what of two components, GDH I or GDH II, is responsible for heat lability of GDH I + GDH II sample (it is heat lability— the property that was noted as characteristic feature of one
434
G.S. Burbaeva et al. / Progress in Neuro-Psychopharmacology & Biological Psychiatry 26 (2002) 427–435
of two ‘‘soluble’’ GDH forms purified from bovine brain by Cho et al., 1995). Magnitude of ADP activation of GDH I + GDH II and GDH III has reached 1.5 folds, which is similar to the activation extent described for ‘‘housekeeping’’ GDH, while nervous tissue-specific GDH stimulation reached thousand folds (Plaitakis et al., 2000; Shashidharan et al., 1997). Thus, biochemical properties such as ATP stimulation of enzymatic activity and heat stability of GDH I, II and III evidence that they are similar to these in ‘‘housekeeping’’ GDH. 4.4. Specificity of polyclonal antisera against various GDH forms The method for quantification of GDH forms in brain extracts by immunoreactivity was developed in the present work. As known, high homology exists between GDH primary structures from various tissues and species (Cho et al., 1995), providing a possibility to use antiserum against bovine liver GDH in immunochemical studies of brain GDH and, on the other hand, making difficult to obtain specific antibodies to various closely related isoforms. Nevertheless, Choi et al. (1999) have succeeded in revealing different antigen reactivity in bovine brain GDH isoproteins. Monoclonal antibody obtained to GDH II in his work gave no reaction with GDH I. In present work, the authors succeeded in obtaining the antiserum to GDH III, which did not recognized purified GDH I + GDH II and reacted with purified GDH III, whereas the antiserum to GDH I + GDH II recognized purified GDH I + GDH II but did not recognized purified GDH III when tested by Western immunoblotting. The antiserum to GDH I + GDH II when tested with extracts from human brain revealed two protein bands, MW 58 (GDH I) and 56 (GDH II) kDa on immunoblots, whereas the antiserum to GDH III revealed a protein band with MW 56 kDa (GDH III) and a protein band with MW 66 kDa. A pattern similar to that observed for antiserum to GDH III was obtained using a commercial antiserum to bovine liver GDH. The fact that two different GDH isoenzymes (one is readily solubilized and another is membrane associated) consist of subunits of the same MW on SDS-PAGE (56 kDa) should be noted. The difference between these isoenzymes was demonstrated by specific staining of 56-kDa protein bands by corresponding antisera using purified GDH I + GDH II and GDH III. The difference in hydrophobicity between the 56-kDa proteins is obvious from their distinct affinity to Phenyl-Sepharose 4B. In fact, three peaks of GDH activity were observed during chromatography of brain tissue extracts obtained in the presence of 1% Triton X-100 + 0.1-M NaCl— two of them contained 56-kDa GDH and each was revealed by appropriate antiserum. In other examined human organs such as heart, liver and kidney, the pattern of stained protein bands revealed by
antiserum to GDH I + GDH II or antiserum to GDH III looked like the corresponding pattern in brain. Interestingly, some differences were observed in GDH pattern revealed by antiserum to GDH I + GDH II in brain extracts of various species. So, two proteins (56 and 58 kDa) were detected in human brain extracts, as mentioned above, but only one protein band (58 kDa) in pig and bovine brain extracts and two proteins (57 and 66 kDa) in rat brain. Thus, the spectrum of proteins detected by the antiserum to GDH I + GDH II depends on species. The pattern revealed by antiserum to GDH III also depends on species. So, antiserum to GDH III revealed two protein bands with MW 56 and 66 kDa on immunoblots of human brain extracts, while on immunoblots of pig, bovine and rat brain extracts, it revealed only one band with MW 56 kDa. Commercial antiserum to bovine liver GDH revealed two protein bands with MW 56 and 66 kDa in all examined brain extracts from various species: human, pig, bovine and rat. Thus, GDH I, GDH II and GDH III described in present work seem to be not nervous tissue-specific proteins because the antisera directed to these proteins reveal protein bands (corresponding to GDH I, II and III) not only in brain but also in human heart, liver and kidney. GDH I and GDH II may correspond to soluble GDH isoforms discovered by Cho et al. (1995) in bovine brain. GDH III may correspond to ‘‘housekeeping’’ GDH described by Shashidharan et al. (1994, 1997) and to particulate form studied by Hussain et al. (1989). As for 66-kDa protein recognizable by polyclonal antisera to GDH III and commercial antiserum, the 66-kDa protein was separated from protein fractions possessing GDH activity after Phenyl-Sepharose 4B chromatography (the first step of GDH purification) and was eluted in fractions with no GDH enzymatic activity. Also, the monitoring of GDH enzymatic activity during GDH chromatography showed that 66-kDa protein immunoreactivity was absent in fractions with GDH enzymatic activity during chromatography steps. Hence, 66-kDa protein is not likely to be a GDH isoform. However, GDH and 66-kDa protein carry some epitope(s) with similar structure. Our results confirm the heterogeneity of GDH that may be the cause of contradictory data in brain GDH studies. The GDH activity determined in brain extract is the sum of enzymatic activities at least of three GDH forms and measurement of bulk activity gives no information on the level of each GDH isoform. The use of obtained in the present work antisera to GDH isoforms provide the possibility to evaluate the content of GDH I and GDH II separately and also GDH III by the level of their immunoreactivity in ECL-Western immunoblotting. We suppose that the revealing of any differential alteration in these constituents in brain with nervous pathology offer some additional insights into the pathogenesis of these disorders.
G.S. Burbaeva et al. / Progress in Neuro-Psychopharmacology & Biological Psychiatry 26 (2002) 427–435
5. Conclusion Three isoenzymes of GDH are isolated from human brain and are characterized—two of them are readily solubilized and one is a membrane-associated isoform. Polyclonal antibodies obtained from these isoenzymes enable to conduct selective revealing of these isoforms and evaluation of their levels in brain tissue extracts by ECL-Western immunoblotting.
Acknowledgments The study is supported by Theodore and Vada Stanley Foundation (USA).
References Aoki, C., Milner, T.A., Berger, S.B., Sheu, K.-F.R., Blass, J.P., Pickel, V.M., 1987. Regional distribution of astrocytes with intense immunoreactivity for glutamate dehydrogenase in rat brain: implications for neuron – glia interactions in glutamate transmission. J. Neurosci. 7, 2214 – 2231. Aree, C., Canadas, S., Oset-Gasque, M.J., Castro, E., Gonzalez, M.P., 1990. Glutamate dehydrogenase: some properties of the rat brain enzyme from different cellular compartments. Comp. Biochem. Physiol. C97, 265 – 267. Cavallaro, S., Meiri, N., Yi, C.-L., Musco, S., Ma, W., Goldberg, J., Alkon, D.L., 1997. Late memory-related genes in the hippocampus revealed by RNA fingerprinting. Proc. Natl. Acad. Sci. USA Cell Biol. 94, 9669 – 9673. Cho, S.-W., Lee, J.E., 1996. Modification of brain glutamate dehydrogenase isoproteins with pyridoxal 50-phosphate. Biochimie 78, 817 – 821. Cho, S.-W., Lee, J., Choi, S.Y., 1995. Two soluble forms of glutamate dehydrogenase isoproteins from bovine brain. Eur. J. Biochem. 233, 340 – 346. Choi, S.Y., Hong, J.W., Song, M.-S., Jeon, S.G., Bahn, J.H., Lee, B.R., Ahn, J.-Y., Cho, S.-W., 1999. Different antigenic reactivities of bovine brain glutamate dehydrogenase isoproteins. J. Neurochem. 72, 2162 – 2169. Colon, A.D., Plaitakis, A., Perakis, A., Berl, S., Clarke, D.D., 1986. Purification and characterization of a soluble and a particulate glutamate dehydrogenase from rat brain. J. Neurochem. 46, 1811 – 1819. Fahien, L.A., Cohen, P.P., 1970. L-Glutamate dehydrogenase from frog and tadpole liver. Enzymology 117, 839. Hussain, M.H., Zannis, V.I., Plaitakis, A., 1989. Characterization of glutamate dehydrogenase isoproteins purified from the cerebellum of normal subjects and patients with degenerative neurological disorders, and from human neoplastic cell lines. J. Biol. Chem. 264, 20730 – 20735. Kanamori, K., Ross, B.D., 1995. Steady-state in vivo glutamate dehydrogenase activity in rat brain measured by N NMR. J. Biol. Chem. 270, 24805 – 24809. Kaneko, T., Akiyama, H., Mizuno, N., 1987. Immunohistochemical demonstration of glutamate dehydrogenase in astrocytes. Neurosci. Lett. 77, 171 – 175. Kugler, P., 1993. Enzymes involved in glutamatergic and GABAergic neurotransmission. Int. Rev. Cytol. 147, 285 – 336.
435
Laemmli, U., 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680 – 685. Lowry, O.H., Rosenbrough, N.J., Farr, A.L., Randall, R.J., 1951. Protein measurement with the folin phenol reagent. J. Biol. Chem. 193, 265 – 275. Mavrothalassitis, G., Tzimagiorgis, G., Mitsialis, A., Zannis, V.I., Plaitakis, A., Papamatheakis, J., Moschonas, N.K., 1988. Isolation and characterization of cDNA clones encoding human liver GDH, evidence for a smaller gene family. PNAS USA 85, 3494 – 3498. Michaelidis, T.M., Tzimagiorgis, G., Moschonas, N., Papamatheakis, J., 1993. The human glutamate dehydrogenase gene family, gene organization and structural characterization. Genomics 16, 150 – 160. Mihara, K., Omura, T., Harano, T., Brenner, S., Fleisher, S., Rajagopalank, V., Blodel, G., 1982. Rat liver L-glutamate dehydrogenase, D-bhydroxybutyrate dehydrogenase, malate dehydrogenase and sulfite oxidase are synthesized as larger precursors by cytoplasmic-free polysomes. J. Biol. Chem. 257, 3355 – 3358. O’Farrell, P.H., 1975. High-resolution two-dimensional electrophoresis of proteins. J. Biol. Chem. 250, 4007 – 4021. Plaitakis, A., Berl, S., Yahr, M.D., 1984. Neurological disorders associated with deficiency of GDH. Ann. Neurol. 15, 144 – 153. Plaitakis, A., Flessas, P., Natsiou, A.B., Shashidharan, P., 1993. Glutamate dehydrogenase deficiency in cerebellar degenerations, clinical, biochemical and molecular genetic aspects. Can. J. Neurol. Sci. 3, S109 – S116 (Suppl.). Plaitakis, A., Metaxari, M., Shashidharan, P., 2000. Nerve tissue-specific (GLUD2) and housekeeping (GLUD1) human glutamate dehydrogenases are regulated by distinct allosteric mechanisms. J. Neurochem. 75, 1862 – 1869. Preiss, T., Hall, A.G., Lightowlers, R.N., 1993. Identification of bovine glutamate dehydrogenase as an RNA-binding protein. J. Biol. Chem. 268, 24523 – 24526. Rajas, F., Rouset, B., 1993. A membrane-bound form of glutamate dehydrogenase possesses an ATP-dependent high-affinity microtubulebinding activity. Biochem. J. 295, 447 – 455. Rajas, F., Gire, V., Rousset, B., 1996. Involvement of a membrane-bound form of glutamate dehydrogenase in the association of lysosomes to microtubules. J. Biol. Chem. 271, 29882 – 29890. Rothe, F., Wolf, G., Schunzel, G., 1990. Immunohistochemical demonstration of glutamate dehydrogenase in the postnatally developing rat hippocampal formation and cerebellar cortex, comparison to activity staining. Neuroscience 39, 419 – 429. Schmitt, A., Kugler, P., 1999. Cellular and regional expression of glutamate dehydrogenase in the rat nervous system, non-radioactive in situ hybridization and comparative immunocytochemistry. Neuroscience 92, 293 – 308. Shashidharan, P., Michaelidis, M., Robakis, N.K., Kresovali, A., Papamatheakis, J., Plaitakis, A., 1994. Novel human GDH expressed in neural and testicular tissues and encoded by an X-linked intronless gene. J. Biol. Chem. 269, 16971 – 16976. Shashidharan, P., Clarke, D.D., Ahmed, N., Moschonas, N., Plaitakis, A., 1997. Nerve tissue-specific human GDH that is thermolabile and highly regulated by ADP. J. Neurochem. 68, 1804 – 1811. Subbalakshmi, G.Y., Murthy, C.R., 1985. Isolation of astrocytes, neurons, and synaptosomes of rat brain cortex, distribution of enzymes of glutamate metabolism. Neurochem. Res. 10, 239 – 250. Towbin, H., Staehelin, T., Gordon, J., 1979. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets, procedure and some applications. Proc. Natl. Acad. Sci. USA 76, 4350 – 4354. Yudkoff, M., Nissim, I., Nelson, D., Lin, Z.P., Erecinska, M., 1991. Glutamate dehydrogenase reaction as a source of glutamic acid in synaptosomes. J. Neurochem. 57, 153 – 160.
Article
Diversity of glutamate dehydrogenase in human brain Gulnur Sh. Burbaeva*, Marina S. Turishcheva, Elena A. Vorobyeva, Olga K. Savushkina, Elena B. Tereshkina, Irina S. Boksha Laboratory of Neurochemistry, Mental Health Research Center RAMS, 113152 Zagorodnoje shosse 2-2, Moscow, Russia
Abstract Three forms of glutamate dehydrogenase (GDH, EC 1.4.1.3) are purified from human brain tissue. Two of them, named GDH I (consisting of 58 ± 1-kDa subunit) and GDH II (consisting of 56 ± 1-kDa subunit), are readily solubilized and the third one, GDH III (consisting of 56 ± 1-kDa subunit), is a membrane-associated (particulate bound) isoform. Kinetic constants were determined for GDH III. These GDH forms were found to differ in hydrophobicity as indicated by different affinity to Phenyl-Sepharose. All three GDH forms showed microheterogeneity on two-dimensional (2-D) gel electrophoresis. Specific polyclonal antibodies, which enable to determine the levels of immunoreactivities of all the GDH forms in human brain extracts by enzyme-chemiluminescent amplified (ECL)-Western immunoblotting, were obtained. D 2001 Elsevier Science Inc. All rights reserved. Keywords: Antiglutamate dehydrogenase polyclonal antibodies; Glutamate dehydrogenase isoenzymes; Human brain; Western immunoblotting
1. Introduction Glutamate is the major excitatory neurotransmitter in mammalian brain, and glutamate dehydrogenase (GDH, EC 1.4.1.3) is one of key enzymes of glutamate metabolism. GDH catalyses reversible oxidative deamination of L-glutamate to a-oxoglutarate using NAD/NADP as coenzymes. There is some discrepancy in respect of the role of GDH in glutamate biosynthesis/degradation in vivo (Yudkoff et al., 1991; Kugler, 1993; Kanamori and Ross, 1995; Schmitt and Kugler, 1999), and there are also controversies in results of investigations of GDH cellular localization in central nervous system. According to most biochemical studies, GDH is ubiquitously detected in neurons and glia (Aree et al., 1990; Subbalakshmi and Murthy, 1985). In contrast, immunohistochemical studies have shown that GDH is enriched or exclusively localized in astrocytes (Aoki et al., 1987; Kaneko et al., 1987; Rothe et al., 1990), whereas a significant expression of GDH mRNA in neurons was observed (Schmitt and Kugler, 1999). The contradictory data obtained in brain GDH studies may be due to heterogeneity of this enzyme. Actually, the Abbreviations: GDH, glutamate dehydrogenase; PAG, polyacrylamide gel. * Corresponding author. Tel.: +7-95-952-9129; fax: +7-95-952-8940. E-mail address: [email protected] (G.S. Burbaeva).
existence of soluble and particulate isoproteins of brain GDH has been discovered (Colon et al., 1986; Plaitakis et al., 1993; Rajas and Rouset, 1993). Cho et al. (1995) has detected two different GDH isoproteins (GDH I and GDH II) in bovine brain that were readily solubilized, and no detergents were required for their extraction. Soluble and particulate isoforms of GDH, and also GDH I and GDH II, differ in relative resistance to thermal inactivation and allosteric regulation. The described GDH isoforms may represent the products of different genes. Alternatively, they may be translated from different GDH mRNA species generated by alternative splicing or may arise due to posttranslation modifications. Multiple GDH genes were shown to exist in human (Michaelidis et al., 1993). At least two of them are functional (Mavrothalassitis et al., 1988; Shashidharan et al., 1994). The mRNA encoded by the GLUD1 gene (Michaelidis et al., 1993) is expressed widely (housekeeping), whereas mRNA of the second one, encoded by the GLUD2 gene, is specifically expressed in neural and testicular tissues (Shashidharan et al., 1994). GLUD1 and GLUD2 isoforms of GDH are regulated by distinct allosteric mechanisms (Plaitakis et al., 2000). Besides GDH enzymatic activity, other roles of GDHs have been reported: a particulate GDH form possesses a microtubule-binding activity and acts as a molecule by means of which an of attachment of lysosomes to microtubules
0278-5846/01/$ – see front matter D 2001 Elsevier Science Inc. All rights reserved. PII: S 0 2 7 8 - 5 8 4 6 ( 0 1 ) 0 0 2 7 3 - 1
428
G.S. Burbaeva et al. / Progress in Neuro-Psychopharmacology & Biological Psychiatry 26 (2002) 427–435
occurs (Rajas and Rouset, 1993; Rajas et al., 1996); GDH possesses an RNA-binding activity and possibly is involved in the regulation of transcription (Preiss et al., 1993); GDH gene has been also identified as one of memory-related genes in the hippocampus (Cavallaro et al., 1997). A study of brain GDH is of particular interest because the enzyme has been found to be deficient in patients with some neurodegenerative disorders (Plaitakis et al., 1984; Hussain et al., 1989). It seems to be essential to have a detailed description of the various types of brain GDH that provides an approach to clarification of their roles in glutamate metabolism and participation in pathogenesis of diseases with nervous system pathology. The present work reports the first purification and characterization of two soluble and one membrane-associated isoforms of GDH from human brain. Polyclonal antisera to these GDH isoforms were obtained that enable to assess the levels of these isoforms in brain tissue extracts by immunoreactivity.
2. Methods 2.1. Materials Samples of brain, kidney, liver and heart obtained at autopsy from a person with no history of mental or neurological disorders were the starting material for enzyme purification. Permission of the Ethics Committee of the Mental Health Research Centre of the Russian Academy of Medical Sciences was obtained. Animal brains were purchased from a slaughterhouse. For the enzyme purification and immunochemical testing, all the samples were processed no later than 6 h after death. 2.2. Chemicals Phenyl-Sepharose 4B and DEAE-Sephacel were purchased from Amersham-Pharmacia Biotech (Sweden) and GTP-Agarose, a-oxoglutarate, NADH, ADP, EDTA and Triton X-100 were purchased from Sigma (USA). Rabbit polyclonal antiserum against bovine liver GDH was purchased from Biogenesis (England). Enzyme-chemiluminescent amplified (ECL) reagents were purchased from Amersham-Pharmacia Biotech. Goat antirabbit IgG antibody conjugate was obtained from Sigma. 2.3. Experimental procedures 2.3.1. GDH extraction Human brain tissue (total weight of 300 g) was cut onto small portions, frozen in liquid nitrogen and stored until use. After thawing, the brain tissue was minced with a scalpel and then homogenized with a Ultra-Turrax IKA-Werk knife
homogenizer (Germany) in two various media depending on further goals. The first medium was composed of following components: 10-mM Tris – HCl, pH 7.4, with 0.1-mM PMSF, 0.5-mM EDTA, 1% Triton X-100 and 0.1-M NaCl; the tissue/buffer ratio was 1:5 w/v. The second medium was used for protein extraction with the aim of further GDH purification. It comprised 10-mM Tris – HCl, pH 7.4, with 0.1-mM PMSF and 0.5-mM EDTA; the tissue/buffer ratio was 1:5 w/v. All purification steps were done at + 4 C. The homogenate was then centrifuged at 480 g for 10 min followed by centrifugation of the obtained supernatant at 100 000 g for 60 min. Both supernatant and pellet contained GDH enzymatic activity.
2.4. Purification of GDH forms Purification of GDH forms from human brain was performed using the same chromatography steps as described by Colon et al. (1986) and Hussain et al. (1989) in their schemes developed for GDH purification from rat and human brain correspondingly. The 100 000 g supernatant was used for purification of ‘‘readily solubilized’’ GDH forms. The 100 000 g pellet was used for purification of ‘‘membrane-associated’’ GDH. The 100 000 g pellet was resuspended in 10-mM Tris – HCl, pH 7.4, with 0.1-mM PMSF, 0.5-mM EDTA, 1% Triton X-100 and 0.5-M NaCl, and then centrifuged at 100 000 g for 60 min. The ‘‘membrane-associated’’ GDH III was purified to homogeneity from the obtained supernatant by modified method of Colon et al. (1986). The purification methods for both GDH ‘‘readily solubilized’’ and ‘‘membrane-associated’’ forms included ammonium sulfate fractionation (30 – 60% relative saturation), hydrophobic (Phenyl-Sepharose), ion exchange (DEAE-Sephacel) and affinity (GTP-Agarose) column chromatography. Following the ammonium sulfate fractionation of the 100 000 g supernatant or of the Triton X-100 extract obtained from the high-speed (100 000 g) pellet, the 30– 60% (NH4)2SO4 precipitate was dissolved in a minimum volume of 50-mM Tris – HCl buffer, pH 6.0, containing (NH4)2SO4 to 15% relative saturation and loaded onto the Phenyl-Sepharose 4B column (2.4 20 cm), equilibrated with the same buffer. The column was washed with two bed volumes of the starting buffer, followed by the elution with a total of 500 ml of a double gradient of 0 –90% ethylene glycol and 0 –15% ammonium sulfate in 50-mM Tris – HCl buffer, pH 6.0. Fractions containing GDH activity were pooled and dialyzed overnight against 25-mM Tris – HCl, pH 7.5, with 0.1-M KCl. Five chromatography runs were carried out for each the ‘‘readily solubilized’’ and ‘‘membraneassociated’’ GDH.
G.S. Burbaeva et al. / Progress in Neuro-Psychopharmacology & Biological Psychiatry 26 (2002) 427–435
Ion-exchange chromatography on a DEAE-Sephacel column (5 5 cm) was carried out in the same buffer as used for dialysis. The column was washed with two volumes of the starting buffer. The protein was eluted from the column with a 360-ml gradient of 0.1 –0.6-M KCl in 25-mM Tris – HCl, pH 7.5. Fractions with GDH activity were pooled. After 24-h dialysis against 2 l of 2-mM K-Pi, pH 7.2, with 1-mM EDTA and 0.1 mM PMSF, two changes, the protein was concentrated to the volume of 15 ml. Concentrated protein sample was applied onto the GTPAgarose column (0.9 6 cm), equilibrated with the same buffer. The column was washed with the same buffer until the protein peak was eluted. The proteins in this peak possessed no GDH activity. Then, active GDH was eluted with 200-mM KCl in the same buffer. 2.5. Preparation of brain and other organ tissue extracts for immunochemistry Preparation of brain and other organ tissue extracts for immunochemical testing included homogenization of 200 mg of tissue in 1 ml of 50-mM Tris – HCl buffer, pH 7.5,
429
with 1.4-mM 2-ME and 1% Triton using a glass-Teflon homogenizer (Braun, Germany); centrifugation at 1000 g for 15 min followed by centrifugation at 60 000 g for 1 h. The final supernatant was used for PAGE with subsequent ECL immunoblotting. 2.6. Determination of protein concentration Protein concentration was measured according to Lowry et al. (1951). 2.7. Gel electrophoresis Protein homogeneity was tested by SDS-PAGE in 10% polyacrylamide gel (PAG) by the Laemmli (1970) method. Gels were stained by Coomassie Brilliant Blue R-250. The amount of protein applied per lane was 3– 5 mg. Two-dimensional (2-D) electrophoresis was performed by the O’Farrell (1975) procedure. The pH range for isoelectrofocusing (IEF) was 5– 8.5; the pH gradient was adjusted using Pharmalytes (Pharmacia, Sweden).
Fig. 1. Chromatography of proteins extracted from human brain with 1% Triton X-100 + 0.5-M NaCl (a), readily solubilized GDH fraction (b) and membraneassociated GDH fraction (c) on Phenyl-Sepharose 4B column. Experimental details are given in Methods. Fractions of approximately 5 ml were collected and assayed for GDH activity (-5-5-) and protein (-.-.-), as described in Methods. The line -D-D- indicates gradient of ethylene glycol (EG).
430
G.S. Burbaeva et al. / Progress in Neuro-Psychopharmacology & Biological Psychiatry 26 (2002) 427–435
was defined as the amount of the GDH enzyme required to oxidize 1 mmol of NADH per 1 min. The reaction in reverse direction was determined by Fahien and Cohen (1970), by following the increase of absorbancy at 340 nm at 25 C, in a reaction mixture containing 50-mM Tris –HCl, pH 9.0, 2.6-mM EDTA, 1.5-mM NAD + /NADP + and 1-mM ADP. The reaction was started with the addition of L-glutamate to 15 mM. 2.9. Raising of polyclonal antibodies and Western immunoblotting
Fig. 2. PAG with purified GDH I + GDH II (a) and GDH III (b) stained by Coomassie Brilliant blue R-250. Low molecular mass markers are applied on the left, with MW given in kDa. Immunoblots of purified GDH I + GDH II stained by anti-GDH I + GDH II polyclonal antiserum (c) and by antiGDH III polyclonal antiserum (d); immunoblots of purified GDH III stained by anti-GDH III polyclonal antiserum (e) and by anti-GDH I + GDH II polyclonal antiserum (f).
Raising of antihuman GDH rabbit antiserum was fulfilled according to the following scheme (common for all studied forms of GDH): the first immunization, 100 mg subcutaneously with complete Freund’s adjuvant; the second in 2 weeks, 100 mg of GDH subcutaneously with incomplete Freund’s adjuvant; the third in 2 weeks, 100 mg of GDH subcutaneously with incomplete Freund’s adjuvant. ECL immunoblotting (Western blotting) was performed according to Towbin et al. (1979) and Amersham protocol using Hyperfilm ECL nitrocellulose membranes. Protein amount applied on the gel was 1 mg for pure enzymes and 30 mg for tissue extracts; first antibody dilution was 1:15 000 for antisera against GDH I + GDH II and GDH III and 1:25 000 for commercial anti-GDH antiserum. 2.10. Data analysis
2.8. GDH enzymatic activity determination GDH enzymatic activity was determined spectrophotometrically both in direction of reductive amination of aoxoglutarate and in reverse reaction of L -glutamate oxidation. The reaction in the first direction was evaluated by following the decrease of absorbancy at 340 nm in a reaction mixture containing 10-mM Tris – HCl, pH 8.0, 100mM ammonium chloride, 0.1-mM NADH, 2.6-mM EDTA and 1-mM ADP. The reaction was started with the addition of a-oxoglutarate to 10-mM final concentration at 25 C, according to Fahien and Cohen (1970). One unit of enzyme
Statistical analysis was performed using Excel and Microcal Origin 3.5 (Microsoft) software.
3. Results 3.1. Purification of GDH A human brain protein extract obtained after the tissue homogenization in the presence of 1% Triton X-100 and 0.1-M NaCl (see GDH extraction, the first medium) was applied onto the Phenyl-Sepharose 4B column. Three
Table 1 Purification of GDH I + GDH II from human brain GDH I + GDH II GDH activity Fraction
Protein (mg)
U
Specific activity (mmol/mg protein/min)
Purification fold
Brain tissue extract High-speed (100 000 g) supernatant 30 – 60% (NH4)2SO4 precipitate Phenyl-Sepharose DEAE-Sephacel GTP-Agarose
18 200.0 3081.0 236.8 177.5 3.7 1.2
399 600 263 425 23 180 18 500 11 080 5130
21.9 85.5 97.9 104.2 3019.1 4275.0
1 4 4.5 5 138 193
G.S. Burbaeva et al. / Progress in Neuro-Psychopharmacology & Biological Psychiatry 26 (2002) 427–435
431
Table 2 Purification of GDH III from human brain GDH activity Fraction
Protein (mg)
U
Specific activity (mmol/mg protein/min)
Purification fold
Brain tissue extract High-speed (100 000 g) precipitate 30 – 60% (NH4)2SO4 precipitate Phenyl-Sepharose DEAE-Sephacel GTP-Agarose
18 200 1680 710 74 20 0.9
399 600 73 416 37 133 35 860 20 600 5112
21.9 43.7 52.3 484.6 1030 5680
1 2 2.5 22 47 256
peaks of GDH activity was eluted from the column with 0 –90% ethylene glycol (Fig. 1a). Thus, GDH in human brain was supposed to be presented by three major forms differing in hydrophobicity. Then, a fractionation of human brain tissue homogenate (see GDH extraction, the second medium) was made, i.e., ‘‘readily solubilized’’ proteins were separated from ‘‘membrane-associated’’ ones. Two peaks of GDH activity were observed during the elution from Phenyl-Sepharose column in the case of 100 000 g supernatant (Fig. 1b). The first fraction of GDH activity (Peak 1 was eluted at about 40% ethylene glycol) substantially overlapped with the second fraction of GDH activity (Peak 2 was eluted at about 50% ethylene glycol). The attempts to separate GDH I from GDH II on Phenyl-Sepharose column have failed due to similarity of their hydrophobic properties and, hence, highly overlapping fractions. On the other hand, when the Triton X-100 extract from 100 000 g pellet was applied onto the same PhenylSepharose column under the same conditions as in the case of GDH I and II, the elution with 0 – 90% gradient of ethylene glycol led to one peak of GDH activity corresponding to 60% of ethylene glycol (Fig. 1c). The fractions with GDH activity from Peaks 1 and 2 were pooled, and the ethylene glycol and ammonium sulfate were removed by dialysis against 25-mM Tris – HCl, pH 7.5, with 0.1-M KCl before ion-exchange chromatography. Further fractionation of Peaks 1 and 2 was carried in accordance with the procedure proposed by Colon et al. (1986) for soluble GDH from rat brain and also using GTP-Agarose column chromatography step proposed by Hussain et al. (1989) and described in Methods. The final fractions obtained from GTP-Agarose column contained GDH, which gave doubled protein band on SDSPAGE corresponding to 58 ± 1 and 56 ± 1 kDa (we have termed the proteins correspondingly GDH I and GDH II) (Fig. 2a). Subsequent chromatography of GDH peak originated from the Triton X-100 extract on GTP-Agarose column has allowed obtaining apparently homogeneous (as judge from SDS-PAGE) protein possessing GDH activity, termed as GDH III. GDH III was presented by one protein band of 56 ± 1 kDa on SDS-PAGE (Fig. 2b). The results of GDH
I + GDH II and GDH III purification are summarized in Tables 1 and 2. 3.1.1. Properties of GDH Properties of purified GDH I + GDH II and GDH III were compared. The isoenzymes were found to differ in hydrophobicity as indicated by differing affinity to Phenyl-Sepharose (see above). They also show different stability when heated: the GDH III is more heat stabile than GDH I + GDH II, which can be referred as heat labile one. After 30 min incubation at 45 C, the GDH I + GDH II activity was reduced by 60%, whereas the GDH III activity remained on the 90% level of the initial value (Fig. 3). On the other hand, the pH dependencies of activities for GDH I + GDH II and GDH III were similar. Slight differences were observed only in marginal acidic and basic ranges (data not shown). Both GDH I + GDH II and GDH III activities in direct and reversed reactions (see Methods) are stimulated by ADP
Fig. 3. Dependencies of GDH I + GDH II (a) and GDH III (b) activities on time of incubation at 45 C.
432
G.S. Burbaeva et al. / Progress in Neuro-Psychopharmacology & Biological Psychiatry 26 (2002) 427–435
Table 3 Kinetic parameters (KM) for human brain GDH III
Constant substrates
Varying substrate
NH4 + , NADH NH4 + , a-oxoglutarate a-oxoglutarate, NADH NAD + L-glutamate
a-oxoglutarate
Range of concentration for varying substrate (mM)
GDH III shows also microheterogeneity in 2-D PAGE (Fig. 4b): at least five spots ranged in pI range from 7.5 to 6.6. Km (mM)
0.8 – 12.0
1.51 ± 0.12
NADH
0.01 – 0.10
0.11 ± 0.02
NH4 +
10 – 100
24.47 ± 2.20
L-Glutamate NAD + NADP +
0.8 – 16.0 0.2 – 1.8 0.4 – 1.5
3.03 ± 0.20 0.63 ± 0.03 0.72 ± 0.05
to the same extent (about 150% of initial activity was observed in the presence of 0.25 – 0.5-mM ADP in the reaction mixture). Kinetic data were obtained for GDH III (Table 3). Concentrations of constant substrates and pH values of reaction mixtures were as given in Methods. Additional characteristics of subunits of GDH I, GDH II and GDH III forms were obtained using 2-D PAGE. Fig. 4a shows the gel stained by Coomassie Brilliant Blue R-250 after 2-D separation (first dimension: IEF and second dimension: SDS-PAGE) of the purified GDH I + GDH II. The pH gradient during the IEF step is shown along the bottom (beneath the gel); the MW scale is given on the left. GDH I is represented by a train of several spots (at least five) corresponding to MW of 58 ± 1 kDa with pI in a range 7.5 – 6.6. GDH II is represented by a train of at least six spots corresponding to MW of 56 ± 1 kDa with pI ranging from 7.3 to 6.6.
3.1.2. Characterization of antisera to GDH I+GDH II and GDH III Polyclonal rabbit antisera were raised to GDH I + GDH II and GDH III. The specificity of each antiserum was evaluated by ECL immunoblotting using the purified GDH I + GDH II and GDH III samples. Among four antisera raised against GDH I + GDH II, the only antiserum possesses high selectivity to GDH I + GDH II (Fig. 2c) and gives no reaction with GDH III (Fig. 2d). We have also succeeded in obtaining of one specific antiserum to GDH III, which reacts specifically with GDH III (Fig. 2e) and gives extremely low staining with GDH I + GDH II (Fig. 2f). The commercial antiserum raised against bovine liver GDH recognizes all three purified isoforms of human brain GDH (data not shown). 3.1.3. Revealing of immunoreactive proteins in various human tissues using anti-GDH I+GDH II, anti-GDH III and commercial antisera Patterns of immunoreactive proteins stained by antiserum to GDH I + GDH II, antiserum to GDH III and commercial antiserum to bovine liver GDH in extracts (see Methods) of various human tissues (brain, heart, liver and kidney) are shown in the Fig. 5. Antiserum to GDH I + GDH II stained 58- and 56-kDa protein bands in extracts from all tested organs (Fig. 5a), while the antiserum to GDH III (Fig. 5b) and the commercial antiserum (Fig. 5c) revealed 56-kDa proteins and also an unknown 66-kDa protein. 3.1.4. Revealing of immunoreactive proteins in extracts from brains of various species using anti-GDH I+GDH II, anti-GDH III and commercial antisera The abilities of the antisera to reveal GDH in brain extracts of various species were compared (Fig. 6). The antiserum to human GDH I + GDH II revealed a single protein band corresponding to MW 58 kDa in bovine and pig brain extracts (Fig. 6a). The pattern of immuno-
Fig. 4. PAG after 2-D electrophoresis of GDH I + GDH II (a) and GDH III (b) stained by Coomassie Brilliant blue R-250.
Fig. 5. Detection of GDH in protein extracts from various human organs by ECL-Western immunoblotting. (a) revealed by antiserum to GDH I + GDH II, (b) revealed by antiserum to GDH III, (c) revealed by commercial antiserum to bull liver GDH. B: brain; H: heart; L: liver; K: kidney.
G.S. Burbaeva et al. / Progress in Neuro-Psychopharmacology & Biological Psychiatry 26 (2002) 427–435
433
Thus, on the last stage of purification (GTP-Agarose column chromatography in each the case), there were obtained correspondingly GDH I and GDH II from ‘‘soluble’’ fraction and GDH III from ‘‘membrane-associated’’ fraction. 4.2. Characterization of GDH subunit compositions Fig. 6. Detection of GDH in extracts from brains of various species by ECLWestern immunoblotting. (a) revealed by antiserum to GDH I + GDH II, (b) revealed by antiserum to GDH III, (c) revealed by commercial antiserum to bull liver GDH; H: human; P: pig; B: bull; R: rat.
positive protein bands revealed by the antiserum to GDH I + GDH II in rat brain differed from that of human, bovine and pig brain: two protein bands corresponding to MW 56 and 66 kDa were detected. The antiserum to GDH III revealed a single protein band corresponding to MW 56 kDa in extracts from bovine, pig and rat brain but revealed two protein bands corresponding to 56 and 66 kDa in extract from human brain (Fig. 6b). As for commercial antiserum to bovine liver GDH, it revealed 56- and 66-kDa protein bands in extracts from human, bovine, pig and rat brains (Fig. 6c).
4. Discussion 4.1. Purification of GDH isoforms During Phenyl-Sepharose 4B column chromatography of human brain protein extract, the presence of three GDH forms of various hydrophobicity in human brain was found. Keeping in mind the existence of soluble and particulate isoforms of GDH (Colon et al., 1986), the authors of present paper separated ‘‘soluble’’ and ‘‘membrane-associated’’ protein fractions on the first stage of GDH purification, i.e., on the stage of protein extraction from brain tissue. The ‘‘soluble’’ fraction was obtained by extraction with neutral buffer of low ionic strength. For the extraction of ‘‘membrane-associated’’ proteins from 100 000 g pellet (contained mitochondrial and other membranes), we used 1% Triton X-100 and 0.5-M NaCl as proposed by Shashidharan et al. (1994). The following GDH purification steps from ‘‘soluble’’ and ‘‘membrane-associated’’ fractions were similar. During the purification on Phenyl-Sepharose, three isoforms of GDH in human brain were discovered: GDH I, GDH II and GDH III. These proteins differ in their affinity to Phenyl-Sepharose inasmuch their different hydrophobicity. While the affinities of GDH I and GDH II to the resin differ not sufficiently for their separation, this step of purification gives substantial gain in GDH III purification.
GDH I consists of 58-kDa subunits, whereas GDH II and GDH III consist of 56-kDa subunits. Subunits of all three GDH isoenzymes are heterogeneous and give several spots (‘‘trains’’) on 2-D PAGE. 2-D PAGE pattern of GDH III purified in the present paper reminds that obtained for GDH purified from human cerebellum (Hussain et al., 1989). The authors aimed to purify ‘‘particulate’’ GDH form. The microheterogeneity observed in GDH may be accounted for sequential Nterminal amino acid cleavage during the transport of GDH into mitochondrial matrix (Mihara et al., 1982; Shashidharan et al., 1997; Hussain et al., 1989). 4.3. Comparison of biochemical properties of purified GDH forms with known from earlier works Kinetic parameters such as KM for a-oxoglutarate and reduced pyridine nucleotides are similar for GDH III and ‘‘particulate’’ GDH as described by Shashidharan et al. (1994) and Hussain et al. (1989). Whereas KM for Lglutamate, determined in this work, differs from that in earlier works, it is threefold less than KM determined by Shashidharan et al. (1994) and sixfold less than KM determined by Hussain et al. (1989). Thus, the affinity of ‘‘particulate’’ GDH III to L-glutamate is higher than that in ‘‘particulate’’ enzymes described by Shashidharan et al. (1994) and Hussain et al. (1989). As for soluble forms of GDH, Cho et al. (1995) and Cho and Lee (1996) have purified two GDH isoenzymes from bovine brain, but there were no data in literature for the presence of several soluble GDH isoforms in human brain. Bovine brain isoproteins, unlike GDH I and GDH II, discovered in our work, showed similarity in MW of their subunits and were presented by one 57.5-kDa protein band when subjected to SDS-PAGE. Plaitakis et al. (2000) in their paper described properties of two GDH recombinant forms, ‘‘nerve tissue-specific’’ GDH and ‘‘housekeeping’’ one synthesized in the baculovirus expression system from corresponding expressed human genes GLUD1 and GLUD2. The results revealed the differences in heat stability of the expressed enzymes. GDH III purified in present work reminds ‘‘housekeeping’’ GDH, which is relatively more heat stable. At the same time, it cannot be said unambiguously, what of two components, GDH I or GDH II, is responsible for heat lability of GDH I + GDH II sample (it is heat lability— the property that was noted as characteristic feature of one
434
G.S. Burbaeva et al. / Progress in Neuro-Psychopharmacology & Biological Psychiatry 26 (2002) 427–435
of two ‘‘soluble’’ GDH forms purified from bovine brain by Cho et al., 1995). Magnitude of ADP activation of GDH I + GDH II and GDH III has reached 1.5 folds, which is similar to the activation extent described for ‘‘housekeeping’’ GDH, while nervous tissue-specific GDH stimulation reached thousand folds (Plaitakis et al., 2000; Shashidharan et al., 1997). Thus, biochemical properties such as ATP stimulation of enzymatic activity and heat stability of GDH I, II and III evidence that they are similar to these in ‘‘housekeeping’’ GDH. 4.4. Specificity of polyclonal antisera against various GDH forms The method for quantification of GDH forms in brain extracts by immunoreactivity was developed in the present work. As known, high homology exists between GDH primary structures from various tissues and species (Cho et al., 1995), providing a possibility to use antiserum against bovine liver GDH in immunochemical studies of brain GDH and, on the other hand, making difficult to obtain specific antibodies to various closely related isoforms. Nevertheless, Choi et al. (1999) have succeeded in revealing different antigen reactivity in bovine brain GDH isoproteins. Monoclonal antibody obtained to GDH II in his work gave no reaction with GDH I. In present work, the authors succeeded in obtaining the antiserum to GDH III, which did not recognized purified GDH I + GDH II and reacted with purified GDH III, whereas the antiserum to GDH I + GDH II recognized purified GDH I + GDH II but did not recognized purified GDH III when tested by Western immunoblotting. The antiserum to GDH I + GDH II when tested with extracts from human brain revealed two protein bands, MW 58 (GDH I) and 56 (GDH II) kDa on immunoblots, whereas the antiserum to GDH III revealed a protein band with MW 56 kDa (GDH III) and a protein band with MW 66 kDa. A pattern similar to that observed for antiserum to GDH III was obtained using a commercial antiserum to bovine liver GDH. The fact that two different GDH isoenzymes (one is readily solubilized and another is membrane associated) consist of subunits of the same MW on SDS-PAGE (56 kDa) should be noted. The difference between these isoenzymes was demonstrated by specific staining of 56-kDa protein bands by corresponding antisera using purified GDH I + GDH II and GDH III. The difference in hydrophobicity between the 56-kDa proteins is obvious from their distinct affinity to Phenyl-Sepharose 4B. In fact, three peaks of GDH activity were observed during chromatography of brain tissue extracts obtained in the presence of 1% Triton X-100 + 0.1-M NaCl— two of them contained 56-kDa GDH and each was revealed by appropriate antiserum. In other examined human organs such as heart, liver and kidney, the pattern of stained protein bands revealed by
antiserum to GDH I + GDH II or antiserum to GDH III looked like the corresponding pattern in brain. Interestingly, some differences were observed in GDH pattern revealed by antiserum to GDH I + GDH II in brain extracts of various species. So, two proteins (56 and 58 kDa) were detected in human brain extracts, as mentioned above, but only one protein band (58 kDa) in pig and bovine brain extracts and two proteins (57 and 66 kDa) in rat brain. Thus, the spectrum of proteins detected by the antiserum to GDH I + GDH II depends on species. The pattern revealed by antiserum to GDH III also depends on species. So, antiserum to GDH III revealed two protein bands with MW 56 and 66 kDa on immunoblots of human brain extracts, while on immunoblots of pig, bovine and rat brain extracts, it revealed only one band with MW 56 kDa. Commercial antiserum to bovine liver GDH revealed two protein bands with MW 56 and 66 kDa in all examined brain extracts from various species: human, pig, bovine and rat. Thus, GDH I, GDH II and GDH III described in present work seem to be not nervous tissue-specific proteins because the antisera directed to these proteins reveal protein bands (corresponding to GDH I, II and III) not only in brain but also in human heart, liver and kidney. GDH I and GDH II may correspond to soluble GDH isoforms discovered by Cho et al. (1995) in bovine brain. GDH III may correspond to ‘‘housekeeping’’ GDH described by Shashidharan et al. (1994, 1997) and to particulate form studied by Hussain et al. (1989). As for 66-kDa protein recognizable by polyclonal antisera to GDH III and commercial antiserum, the 66-kDa protein was separated from protein fractions possessing GDH activity after Phenyl-Sepharose 4B chromatography (the first step of GDH purification) and was eluted in fractions with no GDH enzymatic activity. Also, the monitoring of GDH enzymatic activity during GDH chromatography showed that 66-kDa protein immunoreactivity was absent in fractions with GDH enzymatic activity during chromatography steps. Hence, 66-kDa protein is not likely to be a GDH isoform. However, GDH and 66-kDa protein carry some epitope(s) with similar structure. Our results confirm the heterogeneity of GDH that may be the cause of contradictory data in brain GDH studies. The GDH activity determined in brain extract is the sum of enzymatic activities at least of three GDH forms and measurement of bulk activity gives no information on the level of each GDH isoform. The use of obtained in the present work antisera to GDH isoforms provide the possibility to evaluate the content of GDH I and GDH II separately and also GDH III by the level of their immunoreactivity in ECL-Western immunoblotting. We suppose that the revealing of any differential alteration in these constituents in brain with nervous pathology offer some additional insights into the pathogenesis of these disorders.
G.S. Burbaeva et al. / Progress in Neuro-Psychopharmacology & Biological Psychiatry 26 (2002) 427–435
5. Conclusion Three isoenzymes of GDH are isolated from human brain and are characterized—two of them are readily solubilized and one is a membrane-associated isoform. Polyclonal antibodies obtained from these isoenzymes enable to conduct selective revealing of these isoforms and evaluation of their levels in brain tissue extracts by ECL-Western immunoblotting.
Acknowledgments The study is supported by Theodore and Vada Stanley Foundation (USA).
References Aoki, C., Milner, T.A., Berger, S.B., Sheu, K.-F.R., Blass, J.P., Pickel, V.M., 1987. Regional distribution of astrocytes with intense immunoreactivity for glutamate dehydrogenase in rat brain: implications for neuron – glia interactions in glutamate transmission. J. Neurosci. 7, 2214 – 2231. Aree, C., Canadas, S., Oset-Gasque, M.J., Castro, E., Gonzalez, M.P., 1990. Glutamate dehydrogenase: some properties of the rat brain enzyme from different cellular compartments. Comp. Biochem. Physiol. C97, 265 – 267. Cavallaro, S., Meiri, N., Yi, C.-L., Musco, S., Ma, W., Goldberg, J., Alkon, D.L., 1997. Late memory-related genes in the hippocampus revealed by RNA fingerprinting. Proc. Natl. Acad. Sci. USA Cell Biol. 94, 9669 – 9673. Cho, S.-W., Lee, J.E., 1996. Modification of brain glutamate dehydrogenase isoproteins with pyridoxal 50-phosphate. Biochimie 78, 817 – 821. Cho, S.-W., Lee, J., Choi, S.Y., 1995. Two soluble forms of glutamate dehydrogenase isoproteins from bovine brain. Eur. J. Biochem. 233, 340 – 346. Choi, S.Y., Hong, J.W., Song, M.-S., Jeon, S.G., Bahn, J.H., Lee, B.R., Ahn, J.-Y., Cho, S.-W., 1999. Different antigenic reactivities of bovine brain glutamate dehydrogenase isoproteins. J. Neurochem. 72, 2162 – 2169. Colon, A.D., Plaitakis, A., Perakis, A., Berl, S., Clarke, D.D., 1986. Purification and characterization of a soluble and a particulate glutamate dehydrogenase from rat brain. J. Neurochem. 46, 1811 – 1819. Fahien, L.A., Cohen, P.P., 1970. L-Glutamate dehydrogenase from frog and tadpole liver. Enzymology 117, 839. Hussain, M.H., Zannis, V.I., Plaitakis, A., 1989. Characterization of glutamate dehydrogenase isoproteins purified from the cerebellum of normal subjects and patients with degenerative neurological disorders, and from human neoplastic cell lines. J. Biol. Chem. 264, 20730 – 20735. Kanamori, K., Ross, B.D., 1995. Steady-state in vivo glutamate dehydrogenase activity in rat brain measured by N NMR. J. Biol. Chem. 270, 24805 – 24809. Kaneko, T., Akiyama, H., Mizuno, N., 1987. Immunohistochemical demonstration of glutamate dehydrogenase in astrocytes. Neurosci. Lett. 77, 171 – 175. Kugler, P., 1993. Enzymes involved in glutamatergic and GABAergic neurotransmission. Int. Rev. Cytol. 147, 285 – 336.
435
Laemmli, U., 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680 – 685. Lowry, O.H., Rosenbrough, N.J., Farr, A.L., Randall, R.J., 1951. Protein measurement with the folin phenol reagent. J. Biol. Chem. 193, 265 – 275. Mavrothalassitis, G., Tzimagiorgis, G., Mitsialis, A., Zannis, V.I., Plaitakis, A., Papamatheakis, J., Moschonas, N.K., 1988. Isolation and characterization of cDNA clones encoding human liver GDH, evidence for a smaller gene family. PNAS USA 85, 3494 – 3498. Michaelidis, T.M., Tzimagiorgis, G., Moschonas, N., Papamatheakis, J., 1993. The human glutamate dehydrogenase gene family, gene organization and structural characterization. Genomics 16, 150 – 160. Mihara, K., Omura, T., Harano, T., Brenner, S., Fleisher, S., Rajagopalank, V., Blodel, G., 1982. Rat liver L-glutamate dehydrogenase, D-bhydroxybutyrate dehydrogenase, malate dehydrogenase and sulfite oxidase are synthesized as larger precursors by cytoplasmic-free polysomes. J. Biol. Chem. 257, 3355 – 3358. O’Farrell, P.H., 1975. High-resolution two-dimensional electrophoresis of proteins. J. Biol. Chem. 250, 4007 – 4021. Plaitakis, A., Berl, S., Yahr, M.D., 1984. Neurological disorders associated with deficiency of GDH. Ann. Neurol. 15, 144 – 153. Plaitakis, A., Flessas, P., Natsiou, A.B., Shashidharan, P., 1993. Glutamate dehydrogenase deficiency in cerebellar degenerations, clinical, biochemical and molecular genetic aspects. Can. J. Neurol. Sci. 3, S109 – S116 (Suppl.). Plaitakis, A., Metaxari, M., Shashidharan, P., 2000. Nerve tissue-specific (GLUD2) and housekeeping (GLUD1) human glutamate dehydrogenases are regulated by distinct allosteric mechanisms. J. Neurochem. 75, 1862 – 1869. Preiss, T., Hall, A.G., Lightowlers, R.N., 1993. Identification of bovine glutamate dehydrogenase as an RNA-binding protein. J. Biol. Chem. 268, 24523 – 24526. Rajas, F., Rouset, B., 1993. A membrane-bound form of glutamate dehydrogenase possesses an ATP-dependent high-affinity microtubulebinding activity. Biochem. J. 295, 447 – 455. Rajas, F., Gire, V., Rousset, B., 1996. Involvement of a membrane-bound form of glutamate dehydrogenase in the association of lysosomes to microtubules. J. Biol. Chem. 271, 29882 – 29890. Rothe, F., Wolf, G., Schunzel, G., 1990. Immunohistochemical demonstration of glutamate dehydrogenase in the postnatally developing rat hippocampal formation and cerebellar cortex, comparison to activity staining. Neuroscience 39, 419 – 429. Schmitt, A., Kugler, P., 1999. Cellular and regional expression of glutamate dehydrogenase in the rat nervous system, non-radioactive in situ hybridization and comparative immunocytochemistry. Neuroscience 92, 293 – 308. Shashidharan, P., Michaelidis, M., Robakis, N.K., Kresovali, A., Papamatheakis, J., Plaitakis, A., 1994. Novel human GDH expressed in neural and testicular tissues and encoded by an X-linked intronless gene. J. Biol. Chem. 269, 16971 – 16976. Shashidharan, P., Clarke, D.D., Ahmed, N., Moschonas, N., Plaitakis, A., 1997. Nerve tissue-specific human GDH that is thermolabile and highly regulated by ADP. J. Neurochem. 68, 1804 – 1811. Subbalakshmi, G.Y., Murthy, C.R., 1985. Isolation of astrocytes, neurons, and synaptosomes of rat brain cortex, distribution of enzymes of glutamate metabolism. Neurochem. Res. 10, 239 – 250. Towbin, H., Staehelin, T., Gordon, J., 1979. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets, procedure and some applications. Proc. Natl. Acad. Sci. USA 76, 4350 – 4354. Yudkoff, M., Nissim, I., Nelson, D., Lin, Z.P., Erecinska, M., 1991. Glutamate dehydrogenase reaction as a source of glutamic acid in synaptosomes. J. Neurochem. 57, 153 – 160.