NaCl对水稻谷氨酸合酶和谷氨酸脱氢酶的胁迫作用

在ＮａＣｌ的胁迫下,水稻幼苗根和叶的谷氨酸合酶和谷氨酸脱氢酶的活性随着营养液中的ＮａＣｌ浓度的升高而降低;游离ＮＨ４＾＋在叶中积累,在根中未见明显变化。与根相比,叶对ＮａＣｌ的胁迫作用更为敏感。叶的ＮＡＤＨ－ＧＯＧＡＴ和ＮＡＤＨ－ＧＤＨ活性在ＮａＣｌ胁迫降低的程度明显大于根。无论是否有ＮａＣｌ存在,根的ＮＡＤＨ－ＧＤＨ活性明显高于叶。ＧＳ／ＧＤＨ比值分析提示,对对照下,根中的ＮＨ４＾存在,根的ＮＡ     

Inhibitory effects of Cimicifuga heracleifolia extract on glutamate formation and glutamate dehydrogenase activity in cultured islets

Hyperinsulinism-hyperammonemia syndrome is due either to hyperactivity of GDH or impaired inhibition of GDH by GTP. We have investigated the effect of Cimicifuga heracleifolia extract on the activities of glutamate dehydrogenase (GDH) in cultured rat islets. When the extract was present in the culture medium for 24 h prior to cell harvest, the Vmax of GDH was decreased by 45% with no significant change in Km. In addition, the concentration of alpha-ketoglutarate increased by approximately 39%, and glutamate decreased by 48%. Perfusion of islets with C. heracleifolia extract reduced insulin release by up to 47%. Although the relation between GDH activity and insulin release remains to be clarified, our results suggest that C. heracleifolia extract regulates insulin release by altering GDH activity in primary cultured islets and that this natural compound may be used to modulate GDH activity in patients with hyperinsulinism-hyperammonemia syndrome.     

Lactic and alpha-glycerophosphate dehydrogenases in insects

Different tissues, especially muscles, from insects belonging to various groups were extracted and studied for their lactic dehydrogenase (LDH) and α-glycerophosphate dehydrogenase (GDH I) activities from the comparative point of view. In all cases assays of flight muscle extracts showed extremely low values of LDH activity whereas the GDH activities were surprisingly high. The activities in leg muscles were generally lower. In some insects, however, a very active LDH was found; in these cases the GDH activity seemed to be decreased. GDH I was rather active in the fat bodies. The presence of particulate glycerophosphate dehydrogenase (GDH II) was also demonstrated in insect muscles. These results were interpreted as indicating a system by which there is accomplished immediate and direct breakdown of metabolites to supply large amounts of energy during flight and other activities without the accumulation of intermediate substances.     

Glutamate dehydrogenase activity in soybean,and the effect on it of amino acids and growth substances

Summary Glutamate dehydrogenase (GDH) is largely particulate in soybean, and its specific activity is much higher in roots than in leaflets. The specific activity of GDH in soybean callus is considerably lowered by glycine and leucine and is markedly increased by glutamate, tyrosine, phenylalanine, and especially serine. Increasing concentrations of indoleacetic acid increase the specific activity of GDH in callus far more than they increase either fresh weight or specific activity of malate dehydrogenase. Increasing kinetin also causes an increase in the specific activity of GDH, but mainly at low indoleacetic acid concentrations.     

Human placental glucose dehydrogenase: IEF polymorphism in two Italian populations and enzyme activity in the six common phenotypes

Glucose dehydrogenase (hexose-6-phosphate dehydrogenase) has been assayed qualitatively and quantitatively in more than 600 human placentae collected in two Italian populations. The gene frequencies for GDH1, GDH2 and GDH3 were, respectively, 0.66, 0.21 and 0.12 in Continental Italy and 0.65, 0.23 and 0.12 in Sardinia. Among the six common phenotypes there was no difference in catalytic activity.     

Influence of hexaconazole, carbofuran and ethion on soil microflora and dehydrogenase activities in soil and intact cell

Total microbial count was highly affected (up to 61% at 1000 micrograms level) in presence of hexaconazole and persisted up to 21 days. Bacteria were more susceptible than actinomycetes. Carbofuran and ethion were moderately toxic to soil microflora. Inhibitory effects of all the three pesticides gradually decreased after 21 days as was evident by increase in total microbial count except in carbofuran. GDH activity in soil was also affected initially (up to 14 days) by all the three pesticides (60.3% in hexaconazole at 1000 micrograms level) and inhibition gradually decreased to zero except in carbofuran (15-20% toxicity persisted up to 35 days). GDH and LDH activity in presence of hexaconazole was strongly affected in intact cells of some standard culture of bacteria like Rhizobium sp. (host Dolichos sp., 32.1 and 72.5%), Bacillus subtilis Cohn (86.75 and 76.5%), Azotobacter sp. (36.9 and 55.4%) and B. sphaericus (67.6% GDH) respectively. Carbofuran inhibited the enzyme activity in B. subtilis (55.55 and 35.3%) and to some extent in B. sphaericus. Ethion moderately inhibited LDH activity in Rhodococcus sp. AK1 (17.1 and 33.3%), Rhizobium (27.6% LDH), E. coli HB 101 (34.2% LDH) as evidenced by formazan formation. From the result, it might be concluded that among the above three pesticides tested hexaconazole strongly inhibited the dehydrogenase system in bacteria including nitrogen fixing bacteria of soil and thus may affect soil fertility. It was concluded that hexaconazole was more toxic than ethion to dehydrogenase enzymes.     

Differences in the half-lives of some mitochondrial rat liver enzymes may derive partially from hepatocyte heterogeneity

The different turnover rates of rat liver mitochondrial enzymes make autophagy unlikely to be the main mechanism for degradation of mitochondria. Although alternatives have been presented, hepatocyte heterogeneity has not been considered. Lighter hepatocytes isolated in a discontinuous Percoll gradient contain more glutamate dehydrogenase (GDH) (half-life 1 day) and a more active autophagic system than heavier hepatocytes. The latter contain more carbamoyl phosphate synthase (CPS) and ornithine carbamoyl transferase (OTC) (half-lives 8 days) but less lysosomal activity. As expected, isolated autophagic vacuoles contain, relative to the mitochondrial content, 3-times less OTC and CPS than GDH, probably reflecting a faster lysosomal engulfment of mitochondria in the light hepatocytes (which contain more GDH). These data may explain some of the half-life differences of the enzymes studied.     

Glutamic Acid Dehydrogenase Activity in Linseed as Influenced by Plant Age and Photoperiod

Glutamic acid dehydrogenase (GDH) activity was measured in linseed Linum usitatissimum ev. SH-1 at different growth intervals under normal and long photoperiodic conditions. In ungerminated seeds a low activity of GDH was recorded which increased rapidly during germination and thereafter decreased to a level when no GDH activity could be detected at 47 days of growth. An increase in its activity was recorded at 67, 87 and 107 days. Longer photoperiod (continuous light) increased the activity in roots markedly as compared to shorter photoperiods (normal days).     

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.     

Microquantitative analysis of the intra-acinar profiles of glutamate dehydrogenase in rat liver.

In adult male and female rat liver, the activity of NAD(+)-and NADP(+)-dependent glutamate dehydrogenase (GDH) was microquantitatively measured in tissue samples of 50-150 ng, microdissected continuously along the sinusoidal length. Total activity of GDH with NAD+ as co-factor was found to be higher by a ratio of about 1:2.3 than with NADP+. All intra-acinar enzyme profiles, irrespective of sex, showed an increasing gradient of GDH activity from the periportal beginning to the perivenous end. These findings are at variance with the immunohistochemical localization of GDH in rat liver. The microquantitative GDH profiles with higher perivenous values could indicate a more pronounced glutamine synthesis in Zone 3 of the liver acinus.     

Proposed Enzymes of Auxin Biosynthesis and Their Regulation II. Tryptophan Dehydrogenase Activity in Plants.

In pea, maize and tomato plants a hitherto undescribed L-tryptophan dehydrogenase activity (TDH) has been detected. This enzyme catalyzes the reversible formation of indolepyruvic acid (IPyA) from L-tryptophan (L-trp). TDH and L-glutamate dehydrogenase (GDH), related enzymes in their mode of action, could be separated by gel chromatography. Enzymatic activity of TDH was sustained by both pyridine coenzymes NAD/NADP. With pea TDH the coenzyme NAD displays, at optimum pH 8.5 and at room temperature, only about 40-70 % of the activity of NADP. The amination of IPyA is catalysed more actively than the deamination of L-trp. L-trp/IPyA, L-glu/ketoglutarate, L-ala/pyruvate reacted as dehydrogenase substrates; L-phe/ phenylpyruvate, D-trp and D-phe did not react with pea enzyme extracts. A considerable similarity between the active centres of TDH and GDH has been found using inhibitors: absence of heavy metals, presence of a carbonyl group, indispensibility of bivalent ions for the enzyme activity. Pea TDH and GDH were distinctly inhibited by sodium azide. For the activity of TDH the presence of SH groups is less important than for GDH. The TDH activity in the investigated plants was lower than the GDH activity. The possible role of TDH in the regulation of the IPyA pool is discussed.Doc. RNDr. PhMr. M. Kutáček died on 28 November, 1989. The final form for print was prepared by dr. Ivana Machdckovd of the same Institute, who will also answer the reprint requests. Received June 6, 1990; accepted October 10, 1990     

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.     

Regulatory effects of 5'-deoxypyridoxal on glutamate dehydrogenase activity and insulin secretion in pancreatic islets

It has been known that glutamate, generated by glutamate dehydrogenase (GDH), acts as an intracellular messenger in insulin exocytosis in pancreatic beta cells. Here we demonstrate the correlation of GDH activity and insulin release in rat pancreatic islets perfused with 5'-deoxypyridoxal. Perfusion of islets with 5'-deoxypyridoxal, an effective inhibitor of GDH, reduced the islet GDH activity at concentration-dependent manner. Treatment of 5'-deoxypyridoxal up to 2 mM did not affect the cell viability. There was reduction in V(max) values on average about 60%, whereas no changes in K(m) values for substrates and coenzymes were observed. The concentration of GDH on the Western blot analysis and the level of GDH mRNA remained unchanged. The concentration of glutamate decreased by 52%, whereas the concentration of 2-oxoglutarate increased up to 2.3-fold in the presence of 5'-deoxypyridoxal. 5'-Deoxypyridoxal had no effects on inhibition by GTP and activation by ADP or L-leucine of islet GDH. In parallel with the inhibition of GDH activity, perfusion of islets with 5'-deoxypyridoxal reduced insulin release up to 2.5-fold. Although precise mechanism for correlation between GDH activity and insulin release remains to be studied further, our results suggest a possibility that the inhibitory effect of 5'-deoxypyridoxal on islet GDH activity may correlate with its effect on insulin release.     

Influence of three vesicular-arbuscular mycorrhizal fungi (Glomaceae) on the activity of specific enzymes in the root system of Cucumis sativus L.

The influence of three vesicular-arbuscular mycorrhizal (VAM) Glomus species on the activity of enzymes in the roots of Cucumis sativus was tested. Cucumber plants were grown in a split-root system, in which colonized and uncolonized roots of a single plant could be separated. The activity of the host root malate dehydrogenase (MDH), glucose 6-phosphate dehydrogenase (Gd), glutamate oxaloacetate transaminase (GOT) and glutamate dehydrogenase (GDH) was measured on a densitometer after separation of the host and fungal enzymes on polyacrylamide gels.The results showed that only minor changes in the activity of the host root enzymes occurred after VAM inoculation. Gd was stimulated by VAM and phosphorus, and one of the fungi decreased the activity of GDH in the host plant when both parts of the root system were colonized.     

Glutamate Dehydrogenase Activity in Roots: Distribution in a Seedling and Storage Root, and the Effects of Red and Far-red Illuminations

Pisum seedling and Pastinaca storage roots contained high glutanrate dehydrogenase (GDH) activity in areas of reported rapid growth and high phytoctrome content. A similar distribution was observed for malate dehydrogenase. Freeze-thawings of mitochondrial preparations from Pisum roots always resulted in increases of GDH specific activity; however, the observed increases were much larger with basal than apical sections. Both intact and freeze-thawed mitochondrial preparations from seedling roots exhibited increases in GDH activity with time after isolation. In intact mitochondrial preparations from roots of etiolated seedlings, an increase in malate dehydrogenase activity was observed similar to that of GDH activity; however, no increased malate dehydrogenase activity was noted in preparations from light-grown seedlings. Illuminating Pisum seedlings with far-red light slowly increased GDH activity in roots over a period of two weeks. Since these observed increases were not due to direct exposure of roots to light, other factors were likely involved.     

Comparison of human brain and liver glutamate dehydrogenase cDNAS

In order to investigate suggestions that more than one glutamate dehydrogenase (GDH) gene may be active in humans, seven human brain and seventeen human liver GDH cDNAs were isolated by probing with a 590 base cDNA from the coding region of human brain GDH. No sequence heterogeneity was revealed among any of the cDNAs by an oligonucleotide binding assay, nor did any cDNA appear to encode a hexapeptide contained in a published amino acid sequence of human liver GDH. Homologous regions of three liver and three brain cDNAs had identical sequences over more than 2 kb, including 3' nontranslated regions. This suggests that identical GDH mRNAs are present in human brain and human liver. Although only one gene appears to be expressed, human genomic DNA blots show a pattern of hybridization consistent with the existence of more than one GDH gene.     

The effect of nitrogen addition on N2-fixation and on glutamate dehydrogenase and glutamate synthase activities. in nodules and roots of soybeans inoculated with various strains of Rhizobium japonicum

The addition of nitrogen in the form of urea decreased the activitiesof glutamate dehydrogenase (GDH) and glutamate synthase (GOGAT)in root nodules of Glycine max, whereas the same addition greatlyenhanced root GDH activity. Division of nodules into a mitochondrialand bacteroid fraction indicated that the addition of nitrogenas urea, ammonia, or nitrate most greatly inhibits GDH activityin the mitochondrial fraction. Studies with plants having floralprimordia indicated that added nitrate inhibits nodular GDHmore than either ammonia or urea, while plants inoculated withan ineffective strain (non-nitrogen fixing) of Rhizobium japonicumshowed an increase in nodular GDH activity with nitrogen addition.GOGAT activity was greatly reduced after floral initiation.GDH, GOGAT, and nitrogenase activities in root nodules appearedto vary with the strain of Rhizobium japonicum used as inoculum.In general, strains which produced nodules with high GDH activityproduced bacteroids with low GOGAT activity and the strain whichproduced nodules with the lowest GDH activity produced bacteroidswith the highest GOGAT activity. (Received May 24, 1976; )     

Concerted modulation of alanine and glutamate metabolism in young Medicago truncatula seedlings under hypoxic stress

The modulation of primary nitrogen metabolism by hypoxic stress was studied in young Medicago truncatula seedlings. Hypoxic seedlings were characterized by the up-regulation of glutamate dehydrogenase 1 (GDH1) and mitochondrial alanine aminotransferase (mAlaAT), and down-regulation of glutamine synthetase 1b (GS1b), NADH-glutamate synthase (NADH-GOGAT), glutamate dehydrogenase 3 (GDH3), and isocitrate dehydrogenase (ICDH) gene expression. Hypoxic stress severely inhibited GS activity and stimulated NADH-GOGAT activity. GDH activity was lower in hypoxic seedlings than in the control, however, under either normoxia or hypoxia, the in vivo activity was directed towards glutamate deamination. (15)NH(4) labelling showed for the first time that the adaptive reaction of the plant to hypoxia consisted of a concerted modulation of nitrogen flux through the pathways of both alanine and glutamate synthesis. In hypoxic seedlings, newly synthesized (15)N-alanine increased and accumulated as the major amino acid, asparagine synthesis was inhibited, while (15)N-glutamate was synthesized at a similar rate to that in the control. A discrepancy between the up-regulation of GDH1 expression and the down-regulation of GDH activity by hypoxic stress highlighted for the first time the complex regulation of this enzyme by hypoxia. Higher rates of glycolysis and ethanol fermentation are known to cause the fast depletion of sugar stores and carbon stress. It is proposed that the expression of GDH1 was stimulated by hypoxia-induced carbon stress, while the enzyme protein might be involved during post-hypoxic stress contributing to the regeneration of 2-oxoglutarate via the GDH shunt.     

Application of an Electrochemical NAD+ Recycling System Involving a String-Like Carbon Fiber to an Enzyme Reactor

A string-like carbon fiber was found to be very suitable as a working electrode material for direct electrochemical oxidation of β-nicotinamide adenine dinucleotide reduced form (NADH), and direct use of it for an enzyme reactor was possible. The electrochemical NAD⁺ recycling system was applied to glucose dehydrogenase (GDH) and to the recombinant formate dehydrogenase (RFDH) reactors. The maximum oxidation current value increased to 3.9 mA in the case of the GDH reactor. The remaining GDH activity after the reaction for 10 h amounted to 57% of the initial level. The remaining NAD⁺ activity amounted to 78% of the initial level. The current efficiency was calculated to be 80%. Furthermore, RFDH, which was more stable than GDH, was applied to the system. The maximum current value reached 5.9 mA. The remaining RFDH activity after reaction for 10 h amounted to 81% of the initial level. The remaining NAD⁺ activity was 78% of the initial level. The current efficiency was calculated to be 73%. Based on these results, both the enzyme and NAD⁺ were found to be acceptably stable in the electrochemical NAD⁺ recycling system.