Prokaryotic Expression of Gene Encoding Glutamate Dehydrogenase of Streptococcus suis Serotype 2 and Preparation of Polyclonal Antibodies against Its Expressed Products

[ Objective] To obtain detection antigen for diagnosis of Streptococcus suis infection. [ Method] The complete ORF of glutamate dehy- drogenase （GDH） gene was amplified from the genomic DNA of Streptococcus suis serotype 2 strain SC22 isolated in Sichuan Province by poly- merase chain reaction （PCR）. The resulting product was cloned into the prokaryotic expression vector pET-30a, which was then transformed into E. coil BL21 （DE3）. The identified positive transformants were screened for expression induced by IPTG. The expression products were subjected to SDS-PAGE and the recombinant protein was purified by nickel ion-agarose affinity chromatography. New Zealand rabbits were immunized with the purified recombinant GDH protein to prepare polyclonal antibodies. Titers of the anti-serum were determined by indirect ELISA and Western blot assay. [ Result] The recombinant GDH protein was effectively expressed in the host bacteria, and highly pure recombinant protein was obtained by nickel ion-agarose affinity chromatography. High-titer anti-serum against the recombinant protein was obtained. As evidenced by western blot as- say, the sera could react specifically with the lysates of all detected Streptococcus suis strains. In addition, the recombinant GDH protein could re- act specifically with serum samples collected from five pigs experimentally infected by strain SC22. [ Conclusion] The expressed GDH fusion protein has some common epitopes of natural GDH and can be used as detection antigen to develop ELISA and other diagnostic methods.

Effect of Exogenous Ammonium on GlutamineSynthetase, Glutamate Synthase, and Glutamate Dehydrogenase in the Root of Rice Seedling

Root biomass of rice seedlings was increased at lower concentration of exogenous NH 4 + , but it was decreased at higher concentration of exogenous NH 4 + . The level of free NH 4 + in the roots was accumulated gradually with the increase of NH 4 + concentration in the nutrient solution. The content of the soluble proteins was essentially constant at higher NH 4 + . The activities of glutamine synthetase (GS), NADH-dependent glutamate synthase (NADH-GOGAT), and NADH-dependent glutamate dehydrogenase (NADH-GDH) were risen with exogenous NH 4 + concentration at the lower NH 4 + concentration range. But the activities of GS and NADH-GOGAT were declined, and the level of NADH-GDH activity was kept constant under higher NH 4 + concentration. The GS/GDH ratio suggested that NH 4 + was assimilated by GS-GOGAT cycle under lower NH 4 + concentration, but NADH-GDH was more important for NH 4 + assimilation and detoxifying NH 4 + to the tissue cells at the higher NH 4 + level. According to the growth and the activity changes of these ammonium-assimilating enzymes of rice seedling roots, 10. 0 μg/mL NH 4 + -N in nutrient solution was more suitable to the rice growth.

Involvement of the ammonium assimilation mediated by glutamate dehydrogenase in response to heat stress in the scleractinian coral Pocillopora damicornis

Glutamate dehydrogenase(GDH)plays an important role in the ammonium assimilation and nitrogen metabolism by catalyzing the reversible oxidative deamination of L-glutamate toα-ketoglutarate.In the present study,the potential functions of GDH in response to heat stre ss were explored in the scleractinian coral Pocillopora damicornis(designated as PdGDH).The cDNA of PdGDH contained an open reading frame of 1611 bp encoding a polypeptide of 536 amino acids,which exhibited the highest sequence identity to GDH of Stylophora pistillata(96%identity),and the deduced PdGDH protein was predicted to contain one GdhA domain(from Val95 to Tyr525).The recombinant protein of PdGDH(rPdGDH)was expressed in Escherichia coli BL21(DE3)-Transetta,and its catalytic activity was measured under different temperatures,pH conditions and epigallocatechin-3-gallate(EGCG,a GDH inhibitor)concentrations.The purified rPdGDH only used reduced coenzyme nicotinamide adenine dinucleotide(NADH)as coenzyme,and its highe st activity was observed at 35℃and pH 7.5,re spectively.The rPdGDH activity was negatively correlated with the concentration of EGCG,and was inhibited by more than half(65%,P<0.05)at 10mol/L EGCG.No significant alteration of PdGDH mRNA expression was detected at 12 h after exposure to heat and ammonium(P>0.05).Furthermore,the activities of NADH-GDH in the scleractinian coral P.damicornis increased significantly at 12 h after the heat and ammonium stress,and the NADH-GDH activity in the heat stress group(32.66 U/mg,P<0.05)was significantly higher than that in the heat and ammonium stress group(11.26 U/mg).These results collectively suggested that PdGDH,as a homologue of glutamate dehydrogenase in the scleractinian coral P.damicornis,could respond to heat stress at the protein level,which would have ability to further promote ammonium assimilation to increase the heat acclimatization of the coral-Symbiodiniaceae symbiotic association.

Purification and characterization of a thermostable glutamate dehydrogenase from a thermophilic microorganism from Deception Island, Antarctica

Glutamate dehydrogenase （GDH） catalyzes the oxidative deamination of glutamate to a-ketoglutarate and ammonium ions. Currently the determination of ammonium and glutamate is carried out using a bovine GDH enzyme, which lacks optimal thermostability for long term storage at room temperature. From samples of Deception Island, Antarctica, we obtained the thermophilic bacteria PID 15 belonging to the Bacillus genera with high GDH specific activity. This new enzyme exhibited NAD＋ dependent activity and no activity was observed when NADP＋ was used. This enzyme shows a specific activity of 4.7 U.mg-1 for the oxidative deamination reaction and 15.4 U·mg-1 for the reduction of a-ketoglutarate. This enzyme has an optimum temperature of 65℃ and pH of 8.5 for the oxidative deamination. For the reduction of a-ketoglutarate, the optimum temperature is 60℃, with a pH of 8.0. One of the most important characteristics of this enzyme is its ability to retain more than 60% of its activity when it is incubated for 8 h at 65℃. The enzyme is also able to retain full activity when it is incubated for 48 d at 4℃ and over 80% of its activity when it is incubated at 25℃. Characterization of its kinetics suggests that it primarily catalyzes the formation of α-ketoglutarate. This enzyme has an important biological role in the catabolism of glutamate and may have some interesting biotechnological applications based on its thermostable properties.

Optimization of Cowpea Dry Grain Yield through the Stimulation of the RNA Synthetic Activity of NADH-Glutamate Dehydrogenase

Several potentially practical biochemical processes in plant systems still remain hidden, especially the NADH-glutamate dehydrogenase (GDH) synthesis of nongenetic code-based RNA that optimizes crop nutritious yield by degrading superfluous genetic code-based RNA. In continued characterization of the biochemistry of cowpea grain yield, GDH was purified by electrophoresis from seeds of cowpea treated with solutions of stoichiometric mixes of mineral salts. The GDH was made to synthesize RNAs in the amination (α-KG/NADH/) and then in the deamination (L-Glu/NAD+) direction. The initial product RNAs were captured and sequenced. The grand challenge was to discover the specific molecular roles of the redox enzyme in the optimization of cowpea grain yields. In the amination direction, the GDH hexamers synthesized plus-RNA, but in the deamination direction, they synthesized minus-RNA. The plus-RNAs and minus-RNAs were homologous to about the same numbers of different mRNAs encoding the key enzymes that regulate photosynthesis;saccharide biochemistry and glycolysis;phenylpropanoid biosynthesis;nodulation nitrogen fixing processes;dehydrin drought and glutathione environmental stress resistance processes;purine, pyrimidine, DNA, RNA and essential amino acid biosynthesis;storage protein vicilin accumulation;isoflavone earliness of cowpea maturity;peroxidase synthesis of lignin and sequestration of CO2 to enrich soil organic carbon contents;triglyceride physiology in the biosynthesis of bioactive compounds that render cowpea resistant to insects and fungi;etc., all of which constitute the GDH chemical pathways for discrimination of biochemical, physiological, metabolic, genetic reactions;and optimization of cowpea dry grain yields. Each stoichiometric mix of mineral salts produced optimally yielding biochemical variant of purple hull cowpea;the K + K + K mix was spectacular because it increased the grain yield to 7598 kg from the 3644 kg·ha-1 in the control cowpea. Optimized nutritious staple crop yield buttresses food security. The synthesis of plus-RNA in amination and minus-RNA in deamination is an economic tactical plan in biochemistry for the selection of superfluous mRNAs that would be degraded to assure the survival of cowpea growing under unfavorable environmental conditions.

The Role of Glutamate Dehydrogenase Activity in Development of Neurodegenerative Disorders

The specific role of Glutamate dehydrogenase (GLDH) in the brain is not yet clear, but it is an important enzyme in protein degradation as well as a metabolism regulator of glutamate as a neurotransmitter. The enzyme probably provides crucial protection for postsynaptic membranes against the neurotoxic effects of glutamate neurotransmitters. In men, GLDH activity declines almost evenly through the ages;in women, it declines faster in the first five decades. In the years of menopause, GLDH activity declines slower. The diminished GLDH activities in leukocytes and in the brain vary considerably, but they are parallel with the progress of neurodegenerative diseases. The GLDH activity is partly deficient in the brain, particularly in the leukocytes of patients with heterogeneous neurological disorders and degeneration of multiple neuronal systems. We found a statistically significant difference of GLDH activity in the cerebrospinal fluid in patients with neurological diseases and unexpected in patients with degenerative and inflammatory disorders. The decrease in GLDH activity in the cerebrospinal fluid of patients with neurodegenerative disorders may be one of the reasons for the neuro-excito-toxic glutamate effect. Defining the GLDH activity in leukocytes is at the moment the sole experimental method. The second one could be the measurement in cerebrospinal fluid. The results suggest a possibility to regulate glutamate level in human brain through activation of GLDH.

Research on Glutamate Dehydrogenase Activity in Sugar Beet (Beta vulgaris L) under Different Nitrogen Levels

The experiment of Glutamate Dehydrogenase (GDH) activity in various plant parts under different nitrogen levels in frame culture during the whole period of growth was carried out on campus of Northeast Agricltural University in 1993. The result showed that GDH activity in leaf blades under four nitrogen applied levels rose rapidly to the acme from the seedling to foliage rapid growth stage, then diminished rapidly to the lower level at the latter stage of foliage rapid growth. This level was kept to harvest. GDH activity in roots at each growth stage under all nitrogen levels exhibited little disparity and did not show ostensible regularity of changes. GDH activity in leaf blades was stimulated with nitrogen, however, it reduced with nitrogen fertilizer applying further. GDH activity in leaf blades was the biggest compared with crowns, petioles and roots, which suggested that it could represent the highest enzyme activityof the whole plant.

L-leucine stimulates glutamate dehydrogenase activity and glutamate synthesis by regulating mTORC1/SIRT4 pathway in pig liver

The liver is the most essential organ for the metabolism of ammonia, in where most of ammonia is removed by urea and glutamine synthesis. Regulated by leucine, glutamate dehydrogenase(GDH) catalyzes the reversible inter-conversion of glutamate to ammonia. To determine the mechanism of leucine regulating GDH, pigs weighing 20 ± 1 kg were infused for 80 min with ammonium chloride or alanine in the presence or absence of leucine. Primary pig hepatocytes were incubated with or without leucine. In the in vivo experiments with either ammonium or alanine as the nitrogen source, addition of leucine significantly inhibited ureagenesis and promoted the production of glutamate and glutamine in the perfused pig liver(P < 0.05). Similarly, leucine stimulated GDH activity and inhibited sirtuin4(SIRT4)gene expression(P < 0.01). Leucine could also activate mammalian target of rapamycin complex 1(m TORC1) signaling(P < 0.05), as evidenced by the increased phosphorylation levels of ribosomal protein S6 kinase 1(S6 K1) and ribosomal protein S6(S6). Interestingly, the leucine-induced m TORC1 pathway activation suitably correlated with increased GDH activity and decreased expression of SIRT4.Similar results were observed in primary cultured hepatocytes. Notably, leucine exerted no significant change in GDH activity in SIRT4-deficient hepatocytes(P > 0.05), while m TORC1 signaling was activated.Leucine exerted no significant changes in both GDH activity and SIRT4 gene expression in rapamycin treated hepatocytes(P > 0.05). In conclusion, L-leucine increases GDH activity and stimulates glutamate synthesis from different nitrogen sources by regulating m TORC1/SIRT4 pathway in the liver of pigs.

Differential Effect of Aluminium on Enzymes of Nitrogen Assimilation in Excised Bean Leaf Segments

Aluminium is a potent toxicant in acidic soils. The present study was taken up to analyze the effects of Al on enzymes of nitrogen assimilation in excised bean (Phaseolus vulgaris) leaf segments so as to gain an insight of the mechanism involved. Supply of 0.001 to 0.1 mM AlCl3 to excised bean leaf segments affected the in vivo nitrate reductase activity differently in the presence of various inorganic nitrogenous compounds, being inhibited with 5 mM ammonium nitrate and 10 mM ammonium chloride but enhanced with 10 mM potassium nitrate. Al effect with 50 mM KNO3 varied with time, showing an increased activity at shorter duration, but decreased at longer duration. Al effect on in vivo NRA was dependent upon the nitrate concentration, thus, inhibiting it at 0, 1 and 50 mM KNO3, while increasing at 2 and 10 mM. Further, saturating and non-saturating effects were observed in the absence and presence of Al. Al supply influenced the in vitro NRA also, being increased at 10 mM, but decreased at 50 mM KNO3. Supply of Al to excised leaf segments substantially inhibited the glutamate dehydrogenase activity in the absence as well as presence of 5 mM NH4NO3 but increased the glutamate synthase activity. Inhibition of specific glutamate dehydrogenase activity by Al supply was also observed. However, specific glutamate synthase activity was increased in the presence of NH4NO3 only. The experiments demonstrated that effect of supply of aluminium on in vivo nitrate reductase activity depended upon nitrogenous source as well as nitrate concentration and it exerted reciprocal regulation of glutamate dehydrogenase and glutamate synthase activities, which depended upon N supply too.