Glutamate Metabolism in Rat Cortical Astrocyte Cultures

Glutamate metabolism in rat cortical astrocyte cultures was studied to evaluate the relative rates of flux of glutamate carbon through oxidative pathways and through glutamine synthetase (GS). Rates of 14CO2 production from [1-14C]glutamate were determined, as was the metabolic fate of [14C(U)]glutamate in the presence and absence of the transaminase inhibitor aminooxyacetic acid and of methionine sulfoximine, an irreversible inhibitor of GS. The effects of subculturing and dibutyryl cyclic AMP treatment of astrocytes on these parameters were also examined. The vast majority of exogenously added glutamate was converted to glutamine and exported into the extracellular medium. Inhibition of GS led to a sustained and greatly elevated intracellular glutamate level, thereby demonstrating the predominance of this pathway in the astrocytic metabolism of glutamate. Nevertheless, there was some glutamate oxidation in the astrocyte culture, as evidenced by aspartate production and labeling of intracellular aspartate pools. Inhibition of aspartate aminotransferase caused a greater than 70% decrease in 14CO2 production from [1-14C]glutamate. Inhibition of GS caused an increase in aspartate production. It is concluded that transamination of glutamate rather than oxidative deamination catalyzed by glutamate dehydrogenase is the first step in the entry of glutamate carbon into the citric acid cycle in cultured astrocytes. This scheme of glutamate metabolism was not qualitatively altered by subculturing or by treatment of the cultures with dibutyryl cyclic AMP.     

The pathways of ammonium assimilation in Rhizobium meliloti

Two pathways of ammonium assimilation are known in bacteria, one mediated by glutamate dehydrogenase, the other by glutamine synthetase and glutamate synthase. The activities of these three enzymes were measured in crude extracts from four Rhizobium meliloti wild-type strains, 2011, M15S, 444 and 12. All the strains had active glutamine synthetase and NADP-linked glutamate synthase. Assimilatory glutamate dehydrogenase activity was present in strains 2011, M15S, 444, but not in strain 12. Three glutamate synthase deficient mutants were isolated from strain 2011. They were unable to use 1 mM ammonium as a sole nitrogen source. However, increased ammonium concentration allowed these mutants to assimilate ammonium via glutamate dehydrogenase. It was found that the sole mode of ammonium assimilation in strain 12 is the glutamine synthetase-glutamate synthase route; whereas the two pathways are functional in strain 2011.Abbreviations GS glutamine synthetase - GOGAT glutamate synthase - GDH glutamate dehydrogenase     

A Rhizobium meliloti mutant, lacking a functional γ-aminobutyrate (GABA) bypass, is defective in glutamate catabolism and symbiotic nitrogen fixation

Abstract A Rhizobium meliloti mutant, CMF1 2:38, was isolated which was specifically defective in the degradation of glutamate as sole carbon and nitrogen source. Biochemical analysis of CMF1 2:38 revealed a reduction in succinic semialdehyde dehydrogenase (SSDH) activity, the third enzyme of the γ-aminobutyrate (GABA) bypass. Evidence is presented which suggests that the Tn 5-induced mutation in CMF1 2:38 exists in a regulatory gene governing the expression of both NAD and NADP-linked SSDH activity. CMF1 2:38 nodulated alfalfa plants, but was reduced in its nitrogen fixation activity and biomass accumulating ability relative to the wild-type strain. The results presented in this study indicate that the GABA bypass is a major mechanism of glutamate degradation in R. meliloti CMF1 and that glutamate catabolism via this pathway may play an important role in the symbiotic nitrogen fixation process.     

Activation of Glutamate Dehydrogenase by Leucine and Its Nonmetabolizable Analogue in Rat Brain Synaptosomes

Leucine and beta-(+/-)-2-aminobicyclo[2.2.1]heptane-2-carboxylic acid (BCH) stimulated, in a dose-dependent manner, reductive amination of 2-oxoglutarate in rat brain synaptosomes treated with Triton X-100. The concentration dependence curves were sigmoid, with 10-15-fold stimulations at 15 mM leucine (or BCH); oxidative deamination of glutamate also was enhanced, albeit less. In intact synaptosomes, leucine and BCH elevated oxygen uptake and increased ammonia formation, consistent with stimulation of glutamate dehydrogenase (GDH). Enhancement of oxidative deamination was seen with endogenous as well as exogenous glutamate and with glutamate generated inside synaptosomes from added glutamine. With endogenous glutamate, the stimulation of oxidative deamination was accompanied by a decrease in aspartate formation, which suggests a concomitant reduction in flux through aspartate aminotransferase. Activation of reductive amination of 2-oxoglutarate by BCH or leucine could not be demonstrated even in synaptosomes depleted of internal glutamate. It is suggested that GDH in synaptosomes functions in the direction of glutamate oxidation, and that leucine may act as an endogenous activator of GDH in brain in vivo.     

Transport and catabolism of proline betaine in salt-stressed Rhizobium meliloti

Exogenous proline betaine (N,N-dimethylproline or stachydrine) highly stimulated the growth rate of Rhizobium meliloti, in media of inhibitory concentration of NaCl whereas proline was ineffective. High levels of proline betaine uptake occurred in cells grown in media of elevated osmotic strength; on the contrary, only low activity was found in cells grown in minimal medium. The apparent K _m was 10 M with a maximal transport rate of 25 nmol min^-1 mg^-1 of protein in 0.3 M NaCl-grown cells. The concentrative transport was totally abolished by KCN (2 mM), 2,4-dinitrophenol (2 mM), and carbonyl cyanide-m-chlorophenyl hydrazone (CCCP 10 M) but was insensitive to arsenate (5 mM). Glycine betaine was a very potent inhibitor of proline betaine uptake while proline was not. Proline betaine transport was not reduced in osmotically shocked cells and no proline betaine binding activity was detected in the crude periplasmic shock fluid. In the absence of salt stress, Rhizobium meliloti actively catabolized proline betaine but this catabolism was blocked by increasing the osmotic strength of the medium. The osmolarity in the growth medium regulates the use of proline betaine either as a carbon and nitrogen source or as an osmoprotectant.Abbreviations LAS lactate-aspartate-salts - MSY mannitol-salts-yeast - CCCP carbonyl cyanide-m-chlorophenyl hydrazone - DCCD dicyclohexylcarbodiimide - KCN potassium cyanide - Hepes 4-(2-hydroxyethyl)-1-piperzine-ethanesulphonic acid     

Nerve Tissue-Specific Human Glutamate Dehydrogenase that Is Thermolabile and Highly Regulated by ADP

Abstract: Glutamate dehydrogenase (GDH), an enzyme that is central to the metabolism of glutamate, is present at high levels in the mammalian brain. Studies on human leukocytes and rat brain suggested the presence of two GDH activities differing in thermal stability and allosteric regulation, but molecular biological investigations led to the cloning of two human GDH-specific genes encoding highly homologous polypeptides. The first gene, designated GLUD1, is expressed in all tissues (housekeeping GDH), whereas the second gene, designated GLUD2, is expressed specifically in neural and testicular tissues. In this study, we obtained both GDH isoenzymes in pure form by expressing a GLUD1 cDNA and a GLUD2 cDNA in Sf9 cells and studied their properties. The enzymes generated showed comparable catalytic properties when fully activated by 1 mM ADP. However, in the absence of ADP, the nerve tissue-specific GDH showed only 5% of its maximal activity, compared with ～40% showed by the housekeeping enzyme. Low physiological levels of ADP (0.05–0.25 mM) induced a concentration-dependent enhancement of enzyme activity that was proportionally greater for the nerve tissue GDH (by 550–1,300%) than of the housekeeping enzyme (by 120–150%). Magnesium chloride (1–2 mM) inhibited the nonactivated housekeeping GDH (by 45–64%); this inhibition was reversed almost completely by ADP. In contrast, Mg²⁺ did not affect the nonstimulated nerve tissue-specific GDH, although the cation prevented much of the allosteric activation of the enzyme at low ADP levels (0.05–0.25 mM). Heat-inactivation experiments revealed that the half-life of the housekeeping and nerve tissue-specific GDH was 3.5 and 0.5 h, respectively. Hence, the nerve tissue-specific GDH is relatively thermolabile and has evolved into a highly regulated enzyme. These allosteric properties may be of importance for regulating brain glutamate fluxes in vivo under changing energy demands.     

Glutamate Dehydrogenase in Human Brain: Regional Distribution and Properties

Glutamate dehydrogenase (GDH) activity was studied in 17 regions of six human brains. Duration and conditions of the postmortem period did not affect enzyme activity. Specific activity ranged between 103 and 377 nmoles/min/mg protein at 25 degrees C and it was 10-fold higher than that found in leukocytes. Apart from exclusively white matter regions (corpus callosum and centrum ovale), there was a moderate regional distribution (2.5-fold variation), with highest values in the inferior olive and hypothalamus, and lowest in the cerebellum and lenticular nucleus. With alpha-ketoglutarate (alpha-KG), NADH, or NH4+ as variable substrate, the apparent Km values in human brain were Km alpha-KG = 1.9 X 10(-3) M, KmNADH = 0.21 X 10(-3) M, and KmNH4+ = 28 X 10(-3) M, and in leukocytes they were Km alpha-KG = 1.7 X 10(-3) M, KmNADH = 0.24 X 10(-3) M, and KmNH4+ = 28 X 10(-3) M. The effects of cofactors, inhibitor, and pH were similar in brain and leukocyte GDH.     

Carbon catabolism in continuous cultures and bacteroids of Rhizobium leguminosarum MNF 3841

Bacteroids of R. leguminosarum MNF3841 isolated from pea nodules using Percoll gradients had activities of TCA cycle enzymes up to 6-fold higher than those measured in free-living cells grown on fumarate or sucrose. Activities of sugar catabolic enzymes on the other hand were 2–14-fold lower in isolated bacteroids than in sucrose-grown free-living cells. In continuous culture, cells of strain MNF3841 grown on sucrose under P _i limitation had 2–3-fold higher activities of invertase, glucose-6-phosphate dehydrogenase, the Entner-Doudoroff enzymes and 6-phosphogluconate dehydrogenase, than cells grown on fumarate. With one exception O₂ limited cultures had similar activities of the carbon catabolic enzymes to P _i-limited cultures grown in the same substrate. Glucose-6-phosphate dehydrogenase in O₂-limited cells grown of fumarate was 50% lower than in P _i-limited cells. Co-utilization of fumarate and sucrose occurred with chemostat cultures supplied with both under a variety of conditions.Abbreviations E-D Entner-Doudoroff - EMP Embden-Meyerhof-Parnas - PEPCK phosphoenolpyruvate carboxy kinase - HEPES N-[2-hydroxyethyl]piperazine-N-[2-ethanesulphonic acid]     

The Effect of Light and Exogenously Supplied Ammonium Ions on Glutamate Dehydrogenase Activity and Isoforms in Young Mustard (Sinapis alba L.) Seedlings

Glutamate dehydrogenase (GDH, E.C. 1.4.1.3) of mustard cotyledons was investigated during the first 4 days of seedling development. The enzyme was found to be composed of seven catalytically active isoforms (each with a molecular mass of 270 kDa) which exhibited a charge heterogeneity when investigated by isoelectric focusing. Antibodies against the purified isoform 7, raised in rabbits, cross-reacted with each of the isoforms in Western blotting experiments. In addition, each of the isoforms was composed of four immunopositive reacting polypeptides with 19, 21, 23 and 25 kDa. During development of the seedlings, a shift in the isoform pattern towards the more acidic forms was found which was more pronounced when the seedlings were supplied with 15 mM NH₄Cl. The time course of changes in total GDH level can be correlated with the time course of disappearance of storage proteins. Both parameters are negatively regulated by light possibly via the photoreceptor, phytochrome. There are some indications that GDH in young mustard cotyledons mainly acts in the deaminating direction.     

Study of a fructose-negative mutant of Rhizobium meliloti

Abstract Rhizobium meliloti grows on fructose as sole carbon source. Following nitrosoguanidine mutagenesis, a mutant of R. meliloti M5N1 was isolated as unable to grow on fructose. Enzyme assays with cell-free extracts showed it to lack significative phosphoglucose isomerase activity. Other enzymes were present at low levels. Both fructose and fructose 6-phosphate were accumulated within this mutant. The in vitro inhibition of fructokinase by fructose 6-phosphate was show. Symbiotic properties remained unaffected in the mutant strain.     

Enhancement of Extracellular Glutamate Scavenge System in Injured Motoneurons

Abstract: An increase in glutamine synthetase (GS) mRNA expression after peripheral motor nerve injury was demonstrated by differential display PCR using single arbitrary primer coupled with in situ hybridization screening called in situ display. Differential display PCR was carried out to compare differences in mRNA expression between axotomized (6 h after the transection) and normal hypoglossal nuclei in mice. Several gene fragments were increased after nerve injury; one was identified as GS. Subsequent emulsion autoradiography of hybridization tissue sections revealed that the increase in GS mRNA was observed in injured motoneurons. As GS is a key enzyme participating in the metabolism of the major excitatory neurotransmitter glutamate, we examined the significance of increased GS expression on glutamate-uptake kinetics. GS-transfected human embryonic kidney cells showed an up-regulation in glutamate-uptake kinetics. Therefore, newly expressed GS together with an increased expression of the neuronal glutamate transporter EAAC1 in the injured motoneurons accelerates glutamate uptake. The present results may suggest that the glutamate-uptake system involving the neuronal glutamate transporter and GS in injured neurons is enhanced so as to provide resistance against neurotoxic glutamate accumulation during the early process of nerve regeneration.     

黄色短杆菌GDK-9谷氨酸脱氢酶基因的克隆、序列分析及表达

利用PCR技术从黄色短杆菌GDK-9的基因组DNA中扩增出谷氨酸脱氢酶基因(gdh)片段(EC.1.4.1.4), 连到pUCm-T载体上测序。核酸序列分析结果表明, 该片段全长1927 bp, 包含一个ORF, 推测此ORF区编码一条448个氨基酸的多肽, 分子量约为48 kD。与已报道的gdh序列相似性为99.55%, 其中1190位碱基(C→A)突变导致了编码氨基酸的变化(Thr→Asn), 其它的碱基变化不影响编码的氨基酸。将gdh基因克隆入穿梭质粒pXMJ19中, 并转化E. coli XL-Blue和Brevibacterium flavum GDK-9, 经IPTG诱导后, SDS-PAGE电泳结果显示, 在预计位置出现明显的诱导蛋白条带, 分子量约为48.7 kD。谷氨酸发酵实验表明, 尽管谷氨酸脱氢酶GDH能明显提高胞内的谷氨酸含量, 但其不影响谷氨酸的分泌。     

Glutamate dehydrogenase regulation in callus cultures of Nicotiana plumbaginifolia: effect of glucose feeding and carbon source starvation on the isoenzymatic pattern

Abstract. In callus cultures of Nicotiana plumbaginifolia , the activity of glutamate dehydrogenase was repressed by glucose, whereas, on the contrary, carbon and energy source deprivation induced a remarkable increase in specific activity. Definition of these two opposite types of response was made possible by the use of glycerol as a non-repressing carbon source: in this condition, glutamate dehydrogenase activity reached an intermediate level, which was similar to the derepressed values of activity obtainable when cultures were allowed to exhaust the glucose supply in the medium. Isoelectric focusing analysis revealed the existence of three different isoenzymatic patterns which could be correlated to the three different levels of specific activity: repressed (glucose), induced (carbon starvation) and intermediate (glycerol). Repression affected mainly the four more cathodic bands which were predominant in non-repressed conditions. The possible catabolic role of these isoenzymes is discussed.     

Glutamate dehydrogenase in the first leaf of wheat

Glutamate dehydrogenase extracted from wheat leaves ( Triticum aestivum L. cv. Capitole) taken at two different physiological stages was analysed by electrophoretic and immune-chemical techniques. Two NAD-dependent antigens were identified which bear the balk of the glutamate dehydrogenase activity in the two extracts. The first enzyme was found in much larger amounts in young than in senescent leaves and the reverse situation was observed for the second antigen. The possible relationships between this antigenic polymorphism and the heterogeneity detected by isoelectric focusing from the two extracts were investigated. A charge heterogeneity (isoelectric points about 5.7 and 4.8) was found for the first antigen in both extracts. The second antigen appeared homogeneous (isoelectric point about 5.7) at least in senescent leaves. The last result indicates that two quite different antigens appear in the same isoelectric focusing zone.     

Glucose and Synaptosomal Glutamate Metabolism: Studies with [15N]Glutamate

The metabolism of [15N]glutamate was studied with gas chromatography-mass spectrometry in rat brain synaptosomes incubated with and without glucose. [15N]Glutamate was taken up rapidly by the preparation, reaching a steady-state level in less than 5 min. 15N was incorporated predominantly into aspartate and, to a much lesser extent, into gamma-aminobutyrate. The amount of [15N]ammonia formed was very small, and the enrichment of 15N in alanine and glutamine was below the level of detection. Omission of glucose substantially increased the rate and amount of [15N]aspartate generated. It is proposed that in synaptosomes (a) the predominant route of glutamate nitrogen disposal is through the aspartate aminotransferase reaction; (b) the aspartate aminotransferase pathway generates 2-oxoglutarate, which then serves as the metabolic fuel needed to produce ATP; (c) utilization of glutamate via transamination to aspartate is greatly accelerated when flux through the tricarboxylic acid cycle is diminished by the omission of glucose; (d) the metabolism of glutamate via glutamate dehydrogenase in intact synaptosomes is slow, most likely reflecting restriction of enzyme activity by some unknown factor(s), which suggests that the glutamate dehydrogenase reaction may not be near equilibrium in neurons; and (e) the activities of alanine aminotransferase and glutamine synthetase in synaptosomes are very low.     

Aspartate Aminotransferase and Glutamate Dehydrogenase Activities in the Squid Giant Nerve

Abstract: The present study sought to investigate the presence and distribution of some enzymatic activities involved in the metabolism of glutamate in the giant nerve fiber of the tropical squid Sepioteuthis sepioidea . Specific activities of aspartate aminotransferase and glutamate dehydrogenase were evaluated in homogenates of the isolated giant fiber, extruded axoplasm, and axoplasm-free giant nerve fiber sheaths. The activities of both enzymes were present in the tissue. The specific activity of aspartate aminotransferase was similar in axoplasm and sheaths. However, the specific activity of glutamate dehydrogenase was an order of magnitude higher in the sheaths. This finding is discussed in the framework of the hypothesis that proposes that a differential distribution of the enzymes of the glutamatergic system between the axonal and neuroglial compartments forms part of a system of communication between these cells whose neuronal signal may be glutamate.     

Glutamate dehydrogenase from Pisum sativum L.

A 2–8-fold increase in the activity of glutamate dehydrogenase (GDH), accompanied by an alteration of the GDH isoenzyme pattern, was observed in detached pea shoots floated on tap water (preincubated shoots). Sugars supressed the process, whereas NH ₊ ⁴ and various metabolites as well as inhibitors of energy metabolism and protein synthesis were ineffective. The subcellular distribution pattern revealed evidence that the GDH isoenzymes are exclusively located in the mitochondrial matrix. The alterations in GDH activity occurring in preincubated shoots are restricted to the mitochondria.An experimental device suitable for studying the GDH function in isolated intact mitochondria has been established. Using [¹⁴C] citrate as the carbon source and hydrogen donor, the mitochondria synthesized considerable amounts of glutamate upon addition of NH ₊ ⁴ . The rates of glutamate formation in dependency of increasing NH ₊ ⁴ levels follow simple Michaelis-Menten kinetics. Half-saturation concentrations of NH ₊ ⁴ of 3.6±1.2 mM; 1.9±0.06 mM and 1.6±0.1 mM were calculated for the mitochondria isolated from pea shoots, roots, and preincubated shoots, respectively. The results are discussed in relation to the possible role of GDH in NH_+/4 assimilation at elevated intracellular NH_+/4 levels.Abbreviations GDH Glutamate dehydrogenase - MDH malate dehydrogenase - GOT aspartate aminotransferase - SDH succinate dehydrogenase - HEPES 4-(2-hydroxyethyl)-1-piperazineethan-sulfonic acid - BSA bovine serum albumin - TPP thiamine pyrophosphate - DNP 2,4-dinitrophenol - CCCP carbonyl cyanide m-chlorophenylhydrazone - DCPIP 2,6-dichlorophenolindophenol Dedicated to Professor Dr. Maximilian Steiner on the occasion of his 75th birthday