Important role of Ser443 in different thermal stability of human glutamate dehydrogenase isozymes

Molecular biological studies confirmed that two glutamate dehydrogenase isozymes (hGDH1 and hGDH2) of distinct genetic origin are expressed in human tissues. hGDH1 is heat-stable and expressed widely, whereas hGDH2 is heat-labile and specific for neural and testicular tissues. A selective deficiency of hGDH2 has been reported in patients with spinocerebellar ataxia. We have identified an amino acid residue involved in the different thermal stability of human GDH isozymes. At 45 degrees C (pH 7.0), heat inactivation proceeded faster for hGDH2 (half life=45 min) than for hGDH1 (half-life=310 min) in the absence of allosteric regulators. Both hGDH1 and hGDH2, however, showed much slower heat inactivation processes in the presence of 1 mM ADP or 3 mM L-Leu. Virtually most of the enzyme activity remained up to 100 min at 45 degrees C after treatment with ADP and L-Leu in combination. In contrast to ADP and L-Leu, the thermal stabilities of the hGDH isozymes were not affected by addition of substrates or coenzymes. In human GDH isozymes, the 443 site is Arg in hGDH1 and Ser in hGDH2. Replacement of Ser by Arg at the 443 site by cassette mutagenesis abolished the heat lability of hGDH2 with a similar half-life of hGDH1. The mutagenesis at several other sites (L415M, A456G, and H470R) having differences in amino acid sequence between the two GDH isozymes did not show any change in the thermal stability. These results suggest that the Ser443 residue plays an important role in the different thermal stability of human GDH isozymes.     

Glutamate dehydrogenase of lupin nodules: Purification and properties

Glutamate dehydrogenase, GDH (l-glutamate: NAD⁺ oxidoreductase (deaminating) EC 1.4.1.2) was purified from the plant fraction of lupin nodules and the purity of the preparation established by gel electrophoresis and electrofocusing. The purified enzyme existed as 4 charge isozymes with a MW of 270000. The subunit MW, as determined by dodecyl sulphate electrophoresis, was 45 000. On the basis of the results of the MW determinations a hexameric structure is proposed for lupin-nodule GDH. The pH optima for the enzyme were pH 8.2 for the amination reaction and pH 8.8 for the deamination reaction. GDH from lupin nodules showed a marked preference for NADH over NADPH in the amination reaction and used only NAD⁺ for the deamination reaction. Pyridoxal-5′-P and EDTA inhibited activity. The enzyme displayed Michaelis-Menten kinetics with respect to all substrates except NAD⁺. When NAD⁺ was the varied substrate, there was a deviation from Michaelis-Menten behaviour towards higher activity at high concentrations of NAD⁺.     

Purification and immunological properties of an NAD(H) dependent glutamate dehydrogenase from soybean cells (Glycine max L.)

Studies were carried out on glutamate dehydrogenase (GDH, EC 1.4.1.2) isolated from the SB1 and SB3 soybean (Glyciene max L. cv. Mandarin) cell cultures. The NAD(H) dependent enzyme from SB1 and SB3 cells was purified to homogeneity, and that from the SB3 cells studied in detail. It was shown to be activated by calcium. The molecular weight of the native enzyme was found to be 263 000 ± 12 000. The molecular weight of the subunits was shown to be 41 000 ± 2000, which indicates that the enzyme has a hexameric structure. Anti-GDH antibodies were produced in rabbits, to GDH purified to homogeneity from both cell cultures. Each antibody preparation reacted with the purified enzyme produced from either cell culture. Antibodies to GDH from SB3 cells were utilized to study the apparent induction of GDH, which occurs when these cells are grown in a medium with ammonium ions as the sole nitrogen source. The increase in GDH activity was shown to be due to de-novo protein synthesis. The anti-SB3-GDH antibody preparation was also tested for cross reactivity with crude GDH preparations from a number of plant sources, and purified GDH from a number of other organisms. The antibody was shown to cross react with a number of the GDH preparations.     

Purification and properties of three NAD(P)+ isozymes of L-glutamate dehydrogenase of Chlamydomonas reinhardtii.

Three isozymes of glutamate dehydrogenase (GDH) of Chlamydomonas reinhardtii, induced under different trophic and stress conditions, have been purified about 800-1000-fold to electrophoretic homogeneity. They are hexamers of Mr 266,000-269,000 as deduced from gel filtration and sedimentation coefficient data. GDH1 consisted of six identical subunits of 44 kDa each, whereas both GDH2 and GDH3 consisted of six similar-sized monomers (4 of 44 kDa and 2 of 46 kDa). Optimum pH for the three activities with each pyridine nucleotide was identical (8.5 with NADH; 7.7 with NADPH; and 9.0 with NAD+). The isozymes exhibited similar high optimum temperature values (60-62 degrees C) and isoelectric points (7.9-8.1). Activity was enhanced in vitro by Ca2+ ions and strongly inhibited by pyridoxal 5'-phosphate, KCN, o-phenanthroline and EDTA, and to a lesser extent by pHMB and methylacetimidate. In the aminating reaction the three isozymes were inhibited in a concentration-dependent process by both NADH and NADPH, with apparent Km values for NH4+ ranging from 13-53 mM; 0.36-1.85 mM for 2-oxoglutarate and 0.07-0.78 mM for NADH and NADPH. In the deaminating reaction apparent Km values ranged from 0.64-3.52 mM for L-glutamate and 0.20-0.32 for NAD+. In addition, the three isozymes exhibited a non-hyperbolic kinetics for NAD+ with negative cooperativity (n = 0.8).     

Characterization of nuclear glutamate dehydrogenase of chicken liver and brain

Glutamate dehydrogenase (GDH) enzyme is recently being reported to be present in the nucleus in addition to the mitochondria in a number of organisms. Here we investigated the distribution of GDH in liver and brain tissues of chicken. Polyclonal anti-GDH antibody against bovine GDH was raised by us, which was later shown to be immunereactive to chicken GDH. The nuclear and the mitochondrial extracts from liver and brain tissues of chicken were made as described. By quantitative immunoreactivity, it was revealed that the nuclear GDH expressed in comparable efficiencies in the liver and brain. However, the activity of the brain nuclear GDH was lower than the liver counterparts. The allosteric regulation pattern for the brain nuclear GDH was also different from the other corresponding fractions and it was speculated that the brain nuclear GDH was inactive. The liver and brain nuclear GDH were purified to homogeneity and comparison of specific activities of both the GDH ruled out the existence of any inhibitor in the brain nuclear GDH. It is hypothesized that the inactivation of the brain nuclear GDH in chicken could be due to some already known posttranslational modification. The present report throws light on the differential regulation pattern of GDH enzyme.     

AMP deaminase isozymes in human tissues

In human, there are four AMP deaminase (AMP aminohydrolase, EC 3.5.4.6) isozymes: E1, E2, M and L. Chromatographic, electrophoretic and immunological studies showed the existence of isozymes E1 and E2 in erythrocytes, isozyme M in muscle and isozyme L in liver and brain. The tissues such as heart, kidney and spleen contained isozymes E1, E2 and L. Isozymes E1, M and L were isolated as apparently homogeneous preparations. The three isozymes were all tetramers composed of identical subunits, but differing slightly in molecular weight; isozyme E1 showed a subunit molecular weight of 80 000, isozyme M 72 000 and isozyme L 68 000. They were immunologically different from one another. The antisera precipitated only the corresponding enzyme and did not precipitate any other isozyme. The three isozymes were also different in kinetic and regulatory properties. Isozyme E2 was very similar to isozyme E1 in immunological and kinetic properties, although isozyme E2 could be separated from isozyme E1 by phosphocellulose chromatography, and zonal electrophoresis.     

Tissue specificity of tobacco peroxidase isozymes and their induction by wounding and tobacco mosaic virus infection

Peroxidases (EC 1.11.1.7) have been implicated in the responses of plants to physical stress and to pathogens, as well as in a variety of cellular processes including cell wall biosynthesis. Tissue samples from leaf, root, pith, and callus of Nicotiana tabacum were assayed for specific peroxidase isozymes by analytical isoelectric focusing. Each tissue type was found to exhibit a unique isozyme fingerprint. Root tissue expressed all of the detectable peroxidase isozymes in the tobacco plant, whereas each of the other tissues examined expressed a different subset of these isozymes. In an effort to determine which peroxidase isozymes from Nicotiana tabacum are involved in cell wall biosynthesis or other normal cellular functions and which respond to stress, plants were subjected to either wounding or infection with tobacco mosaic virus. Wounding the plant triggered the expression of several cationic isozymes in the leaf and both cationic and anionic isozymes in pith tissue. Maximum enzyme activity was detected at 72 hours after wounding, and cycloheximide treatment prevented this induction. Infection of tobacco with tobacco mosaic virus induced two moderately anionic isozymes in the leaves in which virus was applied and also systemically induced in leaves which were not inoculated with virus.     

On the ontogeny and interactions of phosphofructokinase in mouse tissues

The distribution and interactions of phosphofructokinase isozymes with cellular structure have been studied in the major tissues of the mouse during development. The ontogenic patterns of isozymes which were obtained were consistent with those observed for other species and are interpreted in terms of the presence of three genes and three homotetrameric forms of the enzyme (A4, B4 and C4) in the tissues of the mouse. In addition, the data provides a clear indication that interactions between the enzyme and cellular structure are appreciable in all major tissues and at all stages of development, with all isozyme types exhibiting such interactions. The significance of the study of subcellular interactions of these isozymes in contributing to a comprehensive physiological rationale for this mammalian enzyme and its multiple forms is discussed.     

Stress-induced changes in glutamate dehydrogenase activity imply its role in adaptation to C and N metabolism in lupine embryos

The modifying effect of sucrose on glutamate dehydrogenase (GDH) activity and isoenzyme pattern was investigated in isolated embryos of lupine ( Lupinus luteus L.), cultured in vitro in a medium with sucrose (+S) or without sucrose (−S) and exposed to cadmium (Cd) and lead (Pb) stress. Sucrose starvation of lupine embryos led to a rapid increase in the specific activity of GDH, immunoreactive β-polypeptide and it was accompanied by appearance of new cathodal isoforms of enzyme. This suggests that isoenzymes induced in lupine embryos by sucrose starvation combine into GDH hexamers with the predominance of β-GDH subunits synthetized under GDH1 gene control. The addition of sucrose to the medium caused an opposite effect. Along with upregulation of catabolic activity of GDH by sucrose starvation, activity of proteolytic enzymes was also induced. These data can point to regulatory mechanism implying a sucrose dependent repression of the GDH1 gene according to the mechanism of catabolic repression. Treatment of embryos with Cd ²⁺ or Pb²⁺ resulted in ammonium accumulation in the tissues, accompanied by an increase in anabolic activity of GDH and activity of anodal isoenzymes, in both (+S) and (−S) embryos without new de novo synthesis of α subunit proteins. Thus, GDH isoenzyme profiles may reflect the physiological function of GDH, which appears to be an important link of metabolic adaptation in cells, aimed at using carbon sources other than sugar during carbohydrate starvation (catabolic activity of GDH) and protecting plant tissues against ammonium accumulated because of heavy metal stress (anabolic activity of GDH).     

泡沙参同工酶基因位点的遗传分析

利用聚丙烯酰胺凝胶电泳技术 ,对来自天然群体 (居群 )的泡沙参 (Adenophora potaninii Korsh.)及其人工杂交子代进行了 8种同工酶的电泳检测和谱带遗传分析 ,以确定编码这些酶系统的基因位点和等位基因。选用 4种不同的凝胶缓冲系统 ,对下列不同酶系统进行了酶谱的遗传分析 :天冬氨酸转氨酶 (AAT)、酯酶 (EST)、甲酸脱氢酶 (FDH)、谷氨酸脱氢酶 (GDH)、异柠檬酸脱氢酶 (IDH)、乳酸脱氢酶(LDH)、苹果酸酶 (ME)和超氧化物歧化酶 (SOD)。结果表明 ,这 8种酶系统至少由 1 8个基因位点编码 ,其中 1 2个位点为遗传稳定的等位酶位点 ,是可靠的遗传标记。酶谱的分离式样表明 ,EST为单聚体结构 ,AAT、FDH、IDH、SOD为二聚体结构 ,GDH为六聚体结构。最后对同工酶的器官和发育特异性以及同工酶基因位点的遗传分析进行了讨论     

Isozymes of glutamate dehydrogenase from oat leaves: Properties and light effect on synthesis

Ammonium-dependent induction of a GDH isozyme in oat leaveswas proportional to light intensity and inhibited by DCMU. Thestimulation of GDH synthesis in response to ammonia was partiallyrepressed by benzimidazole. The inducible (no. 1) and noninducible(no. 2) GDH isozymes wereseparated and purified by approximately54 and 24 fold respectively. The two isozymes were highly specificfor NAD and the rate of NADH oxidation was 7 to 9 times higherthan NAD reduction. Both isozymes showed similar Km values forsubstrates of the reductive amination reaction and pH optimafor NADH oxidation. The pH optima for NAD reduction were 9 and8.2 for isozymes1 and 2 respectively. The two isozymes had asimilar molecular weight, 2.2–2.4 x 10⁵ but differed intheir isoelectric point and temperature sensitivity. Resultssuggest that the GDH isozymes in oat leaves are two differententities but might possess a similar metabolic function. (Received January 6, 1976; )     

Critical role of the cysteine 323 residue in the catalytic activity of human glutamate dehydrogenase isozymes

The role of residue C323 in catalysis by human glutamate dehydrogenase isozymes (hGDH1 and hGDH2) was examined by substituting Arg, Gly, Leu, Met, or Tyr at C323 by cassette mutagenesis using synthetic human GDH isozyme genes. As a result, the Km of the enzyme for NADH and alpha-ketoglutarate increased up to 1.6-fold and 1.1-fold, respectively. It seems likely that C323 is not responsible for substrate-binding or coenzyme-binding. The efficiency (kcat/Km) of the mutant enzymes was only 11-14% of that of the wild-type isozymes, mainly due to a decrease in kcat values. There was a linear relationship between incorporation of [14C]p-chloromercuribenzoic acid and loss of enzyme activity that extrapolated to a stoichiometry of one mol of [14C] incorporated per mol of monomer for wild type hGDHs. No incorporation of [14C]p-chloromer-curibenzoic acid was observed with the C323 mutants. ADP and GTP had no effect on the binding of p-chloromercuribenzoic acid, suggesting that C323 is not directly involved in allosteric regulation. There were no differences between the two hGDH isozymes in sensitivities to mutagenesis at C323. Our results suggest that C323 plays an important role in catalysis by human GDH isozymes.     

Genetic Analysis of Isozyme Loci in Adenophora potaninii Korsh.

Enzyme polymorphism in Adenophora potaninii Korsh. was investigated using vertical slab polyacrylamide gel electrophoresis. Genetic analysis of the population samples and the progeny of intraspecific crosses allowed the verification of the isozyme loci from eight enzyme systems. The system studies included aspartate aminotransferase (AAT), esterase (EST). formate dehydrogenase (FDH), glutamate dehydrogenase (GDH), isocitrate dehydrogenase (IDH), lactate dehydrogenase (LDH), malic enzyme (ME) and superoxide dismutase (SOD). The results indicated that the eight enzyme systems are specified by at least 18 loci, 12 of which behaved as al|ozyme loci. Zymogram patterns showed that EST is monomeric and GDH is hexameric. AAT, FDH, IDH and SOD are apparently dimeric. The tissue and developmental variability are also discussed along with the genetic analysis of isozymes.     

Use of gel electrophoresis for the study of enzymatic activities of cold-adapted bacteria

Glutamate dehydrogenase (GDH) and lactate dehydrogenase (LDH) activity of 13 cold-adapted strains, isolated from cold soils and showing GDH and/or LDH activity in spectrophotometric assays, were revealed by the use of electrophoresis on a nondenaturing acrylamide gel (zymogram). Psychrophilic strains were grown at 4 degrees C and 10 degrees C and the psychrotolerant strains at 4 degrees, 20 degrees and 28 degrees C. Incubation with the specific substrate and staining were done at 4, 28 or 37 degrees C. In the most cold-adapted strains, LDH and GDH production was high at 4 degrees C. In psychrotrophic strains, enzyme production and activity were greater at 20 or 28 degrees C than at lower temperatures. LDH remained active up to 37 degrees C while GDH activity was more thermolabile. GDH activity was NAD-dependent in some psychrophilic strains. In other strains, it was dependent on NAD(P) only or on both NAD and NAD(P). Two bands were seen for GDH or LDH activity in some strains. This method, which does not require a dialysis step, can be used to study the influence of temperature on enzyme production and activity, and the co-factor dependence. It detects phenotypic differences between isozymes, providing data for systematics.     

Control of Some Enzymes of Nitrogen Metabolism during Senescence of Detached Barley (Hordeum vulgare L.) Leaves

The effects of chloramphenicol, cycloheximide and kinetin onthe changes in activity of glutamate dehydrogenase (GDH), glutamatepyruvate transaminase (GPT), glutamate oxaloacetate transaminase(GOT) and nitrite reductase were studied during the senescenceof detached barley leaves in the light and dark. The four enzymesseemed to be synthesized at least during the first hours ofsenescence. The rate of synthesis of GDH was clearly higherthan that of its degradation, thus continuously increasing duringsenescence. Chloramphenicol and kinetin delayed the enzyme degradationprocesses of senescence in the dark. However, chloramphenicolaccelerated senescence in the light. Kinetin had no significanteffect on the enzyme activities in the light. Cycloheximidetreatments produced lower enzyme levels than their respectivecontrols in both the light and dark, but the enzyme levels werehigher in cycloheximide treated leaves in the light than inthe controls in water in the dark. The results are discussedwith reference to the requirement for protein synthesis in thedifferent processes of senescence. (Received August 17, 1981; Accepted February 22, 1982)     

The Effect of Red and Far-red Light on Subsequent Enzyme Activity in Arena Coleoptiles

The effect of red light (660 nm), far-red light (730 nm) and dark treatment on the subsequent enzyme activity in homogenates of Avena coleoptiles was investigated. The activities of succinic dehydrogenase (SDH), lactic dehydro-genase (LDH) and glucose-6-P dehydrogenase (GDH) were investigated. The activity of SDH was greatest in material receiving continuous darkness. LDH and GDH activity was stimulated by both light treatments compared with the dark values. Little or no difference in enzyme activity was found using either a single 15 min flash of light or continuous light for 24 h. Admixtures of extracts from dark treated and light treated material in a 1:1 ratio gave unexpected levels of enzyme activity. In all cases such admixtures gave much less than the anticipated enzyme activity.     

Cellular and subcellular localisation of glutamine synthetase and glutamate dehydrogenase in grapes gives new insights on the regulation of carbon and nitrogen metabolism

The subcellular localisation of glutamine synthetase (GS) and glutamate dehydrogenase (GDH) in grapevine (Vitis vinifera L.) leaves and flowers was investigated using immunogold-labelling experiments. In mature leaf tissue or fully developed flowers, GS was visualised both in the cytosol and in the chloroplasts, a high proportion of the protein being present in the phloem companion cells. GDH was preferentially located in the mitochondria of the phloem companion cells in both leaves and flowers. This observation suggests that, in conjunction with GS, GDH plays a major role in controlling the translocation of organic carbon and nitrogen metabolites in both vegetative and reproductive organs. Significant amounts of GDH protein were also visualised in multivesicular bodies within the flower receptacle. Although the function of such organelles is still unknown, its is possible that the presence of GDH in such cellular structures is important for the recycling of carbon and nitrogen molecules in senescing tissues in which the enzyme is generally induced.     

草鱼同工酶发育遗传学研究——Ⅰ.不同组织器官的同工酶分析

采用垂直淀粉凝胶电泳及特异性组织化学染色技术,研究了草鱼成体脑、眼、心、肾、肌、肝等6种组织中的6种同工酶系统(LDH、MDH、GDH、ADH、LDH、EST)的分化表达谱式。结果表明,草鱼的同工酶系统具有明显的组织特异性。与绝大多数硬骨鱼类相比,草鱼的LDH、m-MDH和ADH同工酶具有特殊的表达谱式:m-MDH和ADH均由两个基因座位编码;肾脏在LDH-A_3B与LDH-A_2B_3之间多出1条LDH酶带(LDH-X)。本文还讨论了草鱼同工酶的遗传基础和亚基组成,以及本实验的某些结果与其他作者的结果不相符的原因。     

From pancreatic islets to central nervous system, the importance of glutamate dehydrogenase for the control of energy homeostasis

Glutamate dehydrogenase (GDH) is a mitochondrial enzyme linking the Krebs cycle to the multifunctional amino acid glutamate. Thereby, GDH plays a pivotal role between carbohydrate and protein metabolisms, controlling production and consumption of the messenger molecule glutamate in neuroendocrine cells. GDH activity is under the control of several regulators, conferring to this enzyme energy-sensor property. Indeed, GDH directly depends on the provision of the co-factor NADH/NAD⁺, rendering the enzyme sensitive to the redox status of the cell. Moreover, GDH is allosterically regulated by GTP and ADP. GDH is also regulated by ADP-ribosylation, mediated by a member of the energy-sensor family sirtuins, namely SIRT4. In the brain, GDH ensures the cycling of the neurotransmitter glutamate between neurons and astrocytes. GDH also controls ammonia metabolism and detoxification, mainly in the liver and kidney. In pancreatic β-cells, the importance of GDH as a key enzyme in the regulation of insulin secretion is now well established. Inhibition of GDH activity decreases insulin release, while activating mutations are associated with a hyperinsulinism syndrome. Although GDH enzyme catalyzes the same reaction in every tissue, its function regarding metabolic homeostasis varies greatly according to specific organs. In this review, we will discuss specificities of GDH regulation in neuroendocrine cells, in particular pancreatic islets and central nervous system.