In mammalian brain, glutamate dehydrogenase (GDH) is located predominantly in astrocytes, where is thought to play a role in transmitter glutamate's metabolism. Human GDH exists in GLUD1 (housekeeping) and GLUD2 (nerve tissue-specific) isoforms, which share all but 15 out of their 505 amino acids. The GLUD1 GDH is potently inhibited by GTP, whereas the GLUD2 enzyme is resistant to this compound. On the other hand, the GLUD2 isoform assumes in the absence of GTP a conformational state associated with little catalytic activity, but it remains amenable to full activation by ADP and/or L-leucine. Site-directed mutagenesis of the GLUD1 gene at sites that differ from the corresponding residues of the GLUD2 gene showed that replacement of Gly456 by Ala made the enzyme resistant to GTP (IC(50)=2.8+/-0.15 microM) compared to the wild-type GDH (IC(50)=0.19+/-0.01 microM). In addition, substitution of Ser for Arg443 virtually abolished basal activity and rendered the enzyme dependent on ADP for its function. These properties may permit the neural enzyme to be recruited under conditions of low energy charge (high ADP:ATP ratio), similar to those that prevail in synaptic astrocytes during intense glutamatergic transmission. Hence, substitution of Ser for Arg443 and Ala for Gly456 are the main evolutionary changes that led to the adaptation of the GLUD2 GDH to the unique metabolic needs of the nerve tissue.

Glutamate dehydrogenase (GDH), an enzyme central to glutamate metabolism, is located in the mitochondria although there is evidence for extramitochondrial localization of GDH. In the human, housekeeping and nerve tissue-specific isoforms, encoded by the GLUD1 and GLUD2 genes, have been identified. The two isoenzymes differ markedly in their baseline activities, allosteric regulation, and thermal stability. GTP potently inhibits GLUD1-derived GDH (IC(50) = 0.2 muM), whereas the GLUD2-derived isoenzyme is resistant to this compound. The GLUD2-derived GDH shows low basal activity and has the capacity to be activated fully by ADP or L-leucine. We used molecular biological tools to study the subcellular localization of GLUD1-derived GDH in cultured cells and the molecular basis of its regulation. COS7 cells, transfected with a GLUD1-pEGFP-N3 vector, revealed a GFP fluorescence pattern nearly identical to that of the mitochondrial marker pDsRed2-Mito. Site-directed mutagenesis of GLUD1 gene showed that replacement of Gly456 by Ala made the enzyme resistant to GTP (IC(50) = 2.8 +/- 0.15 microM) without altering its regulation by ADP. Substitution of Ser for Arg443 rendered the enzyme virtually inactive at its basal state, but fully responsive to ADP activation. The Arg443Ser mutant was more active at pH 7.0 than at pH 8.0. The Gly456Ala change therefore dissociated GLUD2-derived GDH function from GTP, whereas the Arg443Ser change made enzyme regulation possible without this inhibitor. These properties may allow the brain isoenzyme to function well under conditions of intracellular acidification and increased turnover of ATP to ADP, as occurs in synaptic astrocytes during excitatory transmission.

Glutamate dehydrogenase, an enzyme central to glutamate metabolism, is deficient in patients with heterogeneous neurological disorders characterized by multiple system atrophy. There is evidence for multiplicity of human glutamate dehydrogenase, which may account for the heterogeneity of the above disorders. However, only one mRNA that is encoded by an intron-containing gene (GLUD1) is presently known. Because blindness due to neuroretinal degeneration can occur in rare forms of multiple system atrophy, we searched for retina-specific GLUD mRNA(s) by screening a lambda gt10 library derived from human retina. A novel cDNA encoded by an X chromosome-linked intronless gene, designated GLUD2, was isolated and characterized. Reverse transcription-polymerase chain reaction analysis of human tissues revealed that the novel cDNA is expressed in human retina, testis, and, at lower levels, brain. In vitro translation of mRNAs derived from GLUD1 and GLUD2 genes generated proteins with distinct electrophoretic characteristics. The retinal cDNA was expressed in the baculovirus heterologous system, producing a protein capable of catalyzing the oxidative deamination of glutamate. The mobility of the expressed protein on SDS-polyacrylamide gel electrophoresis and its catalytic properties were very similar to those of the naturally occurring human brain glutamate dehydrogenases. The novel gene will be useful for understanding the biology of human neural and testicular tissues and in the study of X-linked neurodegenerative disorders.

Human glutamate dehydrogenase (GDH), an enzyme central to the metabolism of glutamate, is known to exist in housekeeping and nerve tissue-specific isoforms encoded by the GLUD1 and GLUD2 genes, respectively. As there is evidence that GDH function in vivo is regulated, and that regulatory mutations of human GDH are associated with metabolic abnormalities, we sought here to characterize further the functional properties of the two human isoenzymes. Each was obtained in recombinant form by expressing the corresponding cDNAs in Sf9 cells and studied with respect to its regulation by endogenous allosteric effectors, such as purine nucleotides and branched chain amino acids. Results showed that L-leucine, at 1.0 mM:, enhanced the activity of the nerve tissue-specific (GLUD2-derived) enzyme by approximately 1,600% and that of the GLUD1-derived GDH by approximately 75%. Concentrations of L-leucine similar to those present in human tissues ( approximately 0.1 mM:) had little effect on either isoenzyme. However, the presence of ADP (10-50 microM:) sensitized the two isoenzymes to L-leucine, permitting substantial enzyme activation at physiologically relevant concentrations of this amino acid. Nonactivated GLUD1 GDH was markedly inhibited by GTP (IC(50) = 0.20 microM:), whereas nonactivated GLUD2 GDH was totally insensitive to this compound (IC(50) > 5,000 microM:). In contrast, GLUD2 GDH activated by ADP and/or L-leucine was amenable to this inhibition, although at substantially higher GTP concentrations than the GLUD1 enzyme. ADP and L-leucine, acting synergistically, modified the cooperativity curves of the two isoenzymes. Kinetic studies revealed significant differences in the K:(m) values obtained for alpha-ketoglutarate and glutamate for the GLUD1- and the GLUD2-derived GDH, with the allosteric activators differentially altering these values. Hence, the activity of the two human GDH is regulated by distinct allosteric mechanisms, and these findings may have implications for the biologic functions of these isoenzymes.

Human glutamate dehydrogenase (GDH), an enzyme central to the metabolism of glutamate, is known to exist in housekeeping and nerve tissue-specific isoforms encoded by the GLUD1 and GLUD2 genes, respectively. As there is evidence that GDH function in vivo is regulated, and that regulatory mutations of human GDH are associated with metabolic abnormalities, we sought here to characterize further the functional properties of the two human isoenzymes. Each was obtained in recombinant form by expressing the corresponding cDNAs in Sf9 cells and studied with respect to its regulation by endogenous allosteric effectors, such as purine nucleotides and branched chain amino acids. Results showed that L-leucine, at 1.0 mM:, enhanced the activity of the nerve tissue-specific (GLUD2-derived) enzyme by approximately 1,600% and that of the GLUD1-derived GDH by approximately 75%. Concentrations of L-leucine similar to those present in human tissues ( approximately 0.1 mM:) had little effect on either isoenzyme. However, the presence of ADP (10-50 microM:) sensitized the two isoenzymes to L-leucine, permitting substantial enzyme activation at physiologically relevant concentrations of this amino acid. Nonactivated GLUD1 GDH was markedly inhibited by GTP (IC(50) = 0.20 microM:), whereas nonactivated GLUD2 GDH was totally insensitive to this compound (IC(50) > 5,000 microM:). In contrast, GLUD2 GDH activated by ADP and/or L-leucine was amenable to this inhibition, although at substantially higher GTP concentrations than the GLUD1 enzyme. ADP and L-leucine, acting synergistically, modified the cooperativity curves of the two isoenzymes. Kinetic studies revealed significant differences in the K:(m) values obtained for alpha-ketoglutarate and glutamate for the GLUD1- and the GLUD2-derived GDH, with the allosteric activators differentially altering these values. Hence, the activity of the two human GDH is regulated by distinct allosteric mechanisms, and these findings may have implications for the biologic functions of these isoenzymes.

In mammalian brain, glutamate dehydrogenase (GDH) is located predominantly in astrocytes, where is thought to play a role in transmitter glutamate's metabolism. Human GDH exists in GLUD1 (housekeeping) and GLUD2 (nerve tissue-specific) isoforms, which share all but 15 out of their 505 amino acids. The GLUD1 GDH is potently inhibited by GTP, whereas the GLUD2 enzyme is resistant to this compound. On the other hand, the GLUD2 isoform assumes in the absence of GTP a conformational state associated with little catalytic activity, but it remains amenable to full activation by ADP and/or L-leucine. Site-directed mutagenesis of the GLUD1 gene at sites that differ from the corresponding residues of the GLUD2 gene showed that replacement of Gly456 by Ala made the enzyme resistant to GTP (IC(50)=2.8+/-0.15 microM) compared to the wild-type GDH (IC(50)=0.19+/-0.01 microM). In addition, substitution of Ser for Arg443 virtually abolished basal activity and rendered the enzyme dependent on ADP for its function. These properties may permit the neural enzyme to be recruited under conditions of low energy charge (high ADP:ATP ratio), similar to those that prevail in synaptic astrocytes during intense glutamatergic transmission. Hence, substitution of Ser for Arg443 and Ala for Gly456 are the main evolutionary changes that led to the adaptation of the GLUD2 GDH to the unique metabolic needs of the nerve tissue.

Human glutamate dehydrogenase (GDH), an enzyme central to the metabolism of glutamate, is known to exist in housekeeping and nerve tissue-specific isoforms encoded by the GLUD1 and GLUD2 genes, respectively. As there is evidence that GDH function in vivo is regulated, and that regulatory mutations of human GDH are associated with metabolic abnormalities, we sought here to characterize further the functional properties of the two human isoenzymes. Each was obtained in recombinant form by expressing the corresponding cDNAs in Sf9 cells and studied with respect to its regulation by endogenous allosteric effectors, such as purine nucleotides and branched chain amino acids. Results showed that L-leucine, at 1.0 mM:, enhanced the activity of the nerve tissue-specific (GLUD2-derived) enzyme by approximately 1,600% and that of the GLUD1-derived GDH by approximately 75%. Concentrations of L-leucine similar to those present in human tissues ( approximately 0.1 mM:) had little effect on either isoenzyme. However, the presence of ADP (10-50 microM:) sensitized the two isoenzymes to L-leucine, permitting substantial enzyme activation at physiologically relevant concentrations of this amino acid. Nonactivated GLUD1 GDH was markedly inhibited by GTP (IC(50) = 0.20 microM:), whereas nonactivated GLUD2 GDH was totally insensitive to this compound (IC(50) > 5,000 microM:). In contrast, GLUD2 GDH activated by ADP and/or L-leucine was amenable to this inhibition, although at substantially higher GTP concentrations than the GLUD1 enzyme. ADP and L-leucine, acting synergistically, modified the cooperativity curves of the two isoenzymes. Kinetic studies revealed significant differences in the K:(m) values obtained for alpha-ketoglutarate and glutamate for the GLUD1- and the GLUD2-derived GDH, with the allosteric activators differentially altering these values. Hence, the activity of the two human GDH is regulated by distinct allosteric mechanisms, and these findings may have implications for the biologic functions of these isoenzymes.

Human glutamate dehydrogenase (GDH), an enzyme central to the metabolism of glutamate, is known to exist in housekeeping and nerve tissue-specific isoforms encoded by the GLUD1 and GLUD2 genes, respectively. As there is evidence that GDH function in vivo is regulated, and that regulatory mutations of human GDH are associated with metabolic abnormalities, we sought here to characterize further the functional properties of the two human isoenzymes. Each was obtained in recombinant form by expressing the corresponding cDNAs in Sf9 cells and studied with respect to its regulation by endogenous allosteric effectors, such as purine nucleotides and branched chain amino acids. Results showed that L-leucine, at 1.0 mM:, enhanced the activity of the nerve tissue-specific (GLUD2-derived) enzyme by approximately 1,600% and that of the GLUD1-derived GDH by approximately 75%. Concentrations of L-leucine similar to those present in human tissues ( approximately 0.1 mM:) had little effect on either isoenzyme. However, the presence of ADP (10-50 microM:) sensitized the two isoenzymes to L-leucine, permitting substantial enzyme activation at physiologically relevant concentrations of this amino acid. Nonactivated GLUD1 GDH was markedly inhibited by GTP (IC(50) = 0.20 microM:), whereas nonactivated GLUD2 GDH was totally insensitive to this compound (IC(50) > 5,000 microM:). In contrast, GLUD2 GDH activated by ADP and/or L-leucine was amenable to this inhibition, although at substantially higher GTP concentrations than the GLUD1 enzyme. ADP and L-leucine, acting synergistically, modified the cooperativity curves of the two isoenzymes. Kinetic studies revealed significant differences in the K:(m) values obtained for alpha-ketoglutarate and glutamate for the GLUD1- and the GLUD2-derived GDH, with the allosteric activators differentially altering these values. Hence, the activity of the two human GDH is regulated by distinct allosteric mechanisms, and these findings may have implications for the biologic functions of these isoenzymes.

Glutamate dehydrogenase, an enzyme central to glutamate metabolism, is deficient in patients with heterogeneous neurological disorders characterized by multiple system atrophy. There is evidence for multiplicity of human glutamate dehydrogenase, which may account for the heterogeneity of the above disorders. However, only one mRNA that is encoded by an intron-containing gene (GLUD1) is presently known. Because blindness due to neuroretinal degeneration can occur in rare forms of multiple system atrophy, we searched for retina-specific GLUD mRNA(s) by screening a lambda gt10 library derived from human retina. A novel cDNA encoded by an X chromosome-linked intronless gene, designated GLUD2, was isolated and characterized. Reverse transcription-polymerase chain reaction analysis of human tissues revealed that the novel cDNA is expressed in human retina, testis, and, at lower levels, brain. In vitro translation of mRNAs derived from GLUD1 and GLUD2 genes generated proteins with distinct electrophoretic characteristics. The retinal cDNA was expressed in the baculovirus heterologous system, producing a protein capable of catalyzing the oxidative deamination of glutamate. The mobility of the expressed protein on SDS-polyacrylamide gel electrophoresis and its catalytic properties were very similar to those of the naturally occurring human brain glutamate dehydrogenases. The novel gene will be useful for understanding the biology of human neural and testicular tissues and in the study of X-linked neurodegenerative disorders.

Glutamate dehydrogenase, an enzyme central to glutamate metabolism, is deficient in patients with heterogeneous neurological disorders characterized by multiple system atrophy. There is evidence for multiplicity of human glutamate dehydrogenase, which may account for the heterogeneity of the above disorders. However, only one mRNA that is encoded by an intron-containing gene (GLUD1) is presently known. Because blindness due to neuroretinal degeneration can occur in rare forms of multiple system atrophy, we searched for retina-specific GLUD mRNA(s) by screening a lambda gt10 library derived from human retina. A novel cDNA encoded by an X chromosome-linked intronless gene, designated GLUD2, was isolated and characterized. Reverse transcription-polymerase chain reaction analysis of human tissues revealed that the novel cDNA is expressed in human retina, testis, and, at lower levels, brain. In vitro translation of mRNAs derived from GLUD1 and GLUD2 genes generated proteins with distinct electrophoretic characteristics. The retinal cDNA was expressed in the baculovirus heterologous system, producing a protein capable of catalyzing the oxidative deamination of glutamate. The mobility of the expressed protein on SDS-polyacrylamide gel electrophoresis and its catalytic properties were very similar to those of the naturally occurring human brain glutamate dehydrogenases. The novel gene will be useful for understanding the biology of human neural and testicular tissues and in the study of X-linked neurodegenerative disorders.