Molecular and biochemical characterisation of a Teladorsagia circumcincta glutamate dehydrogenase

A full length cDNA encoding glutamate dehydrogenase was cloned from Teladorsagia circumcincta (TcGDH). The TcGDH cDNA (1614 bp) encoded a 538 amino acid protein. The predicted amino acid sequence showed 96% and 93% similarity with Haemonchus contortus and Caenorhabditis elegans GDH, respectively. A soluble N-terminal 6xHis-tagged GDH protein was expressed in the recombinant Escherichia coli strain BL21 (DE3) pGroESL, purified and characterised. The recombinant TcGDH had similar kinetic properties to those of the enzyme in homogenates of T. circumcincta, including greater activity in the aminating than deaminating reaction. Addition of 1 mM ADP and ATP increased activity about 3-fold in the deaminating reaction, but had no effect in the reverse direction. TcGDH was a dual co-factor enzyme that operated both with NAD⁺ and NADP⁺, GDH activity was greater in the deaminating reaction with NADP⁺ as co-factor and more with NADH in the aminating reaction.     

Nucleotide allosteric regulation of the glutamate dehydrogenases of Teladorsagia circumcincta and Haemonchus contortus

The expression of glutamate dehydrogenase (GDH; EC 1.4.1.3) in L3 of the nematode Haemonchus contortus was confirmed by detecting GDH mRNA, contrary to earlier reports. The enzyme was active in both L3 and adult H. contortus homogenates either with NAD⁺/H or NADP⁺/H as co-factor. Although it was a dual co-factor GDH, activity was greater with NAD⁺/H than with NADP⁺/H. The rate of the aminating reaction (glutamate formation) was approximately three times higher than for the deaminating reaction (glutamate utilisation). GDH provides a pathway for ammonia assimilation, although the affinity for ammonia was low. Allosteric regulation by GTP, ATP and ADP of L3 and adult H. contortus and Teladorsagia circumcincta (Nematoda) GDH depended on the concentration of the regulators and the direction of the reaction. The effects of each nucleotide were qualitatively similar on the mammalian and parasite GDH, although the nematode enzymes were more responsive to activation by ADP and ATP and less inhibited by GTP under optimum assay condition. GTP inhibited deamination and low concentrations of ADP and ATP stimulated weakly. In the reverse direction, GTP was strongly inhibitory and ADP and ATP activated the enzyme.     

A study of the role of glutamate dehydrogenase in the nitrogen metabolism of Arabidopsis thaliana

Glutamate-dehydrogenase (GDH, EC 1.4.1.2) activity and isoenzyme patterns were investigated in Arabidopsis thaliana plantlets, and parallel studies were carried out on glutamine synthetase (GS, EC 6.3.1.2). Both NADH-GDH and NAD-GDH activities increased during plant development whereas GS activity declined. Leaves deprived of light showed a considerable enhancement of NADH-GDH activity. In roots, both GDH activities were induced by ammonia whereas in leaves nitrogen assimilation was less important. It was demonstrated that the increase in GDH activity was the result of de-novo protein synthesis. High nitrogen levels were first assimilated by NADH-GDH, while GS was actively involved in nitrogen metabolism only when the enzyme was stimulated by a supply of energy, generated by NAD-GDH or by feeding sucrose. When methionine sulfoximine, an inhibitor of GS, was added to the feeding solution, NADH-GDH activity remained unaffected in leaves whereas NAD-GDH was induced. In roots, however, there was a marked activation of GDH and no inactivation of GS. It was concluded that NADH-GDH was involved in the detoxification of high nitrogen levels while NAD-GDH was mainly responsible for the supply of energy to the cell during active assimilation. Glutamine synthetase, on the other hand was involved in the assimilation of physiological amounts of nitrogen. A study of the isoenzyme pattern of GDH indicated that a good correlation existed between the relative activity of the isoenzymes and the ratio of aminating to deaminating enzyme activities. The NADH-GDH activity corresponded to the more anodal isoenzymes while the NAD-GDH activity corresponded to the cathodal ones. The results indicate that the two genes involved in the formation of GDH control the expression of enzymes with different metabolic functions.Abbreviations GDH glutamate dehydrogenase - GS glutamine synthetase - MSO methionine sulfoximine     

A short-term divergent selection for resistance to Teladorsagia circumcincta in Romanov sheep using natural or artificial challenge

This experiment was conducted to assess the efficiency of selection on the basis of response to artificial challenges in order to breed sheep resistant to natural infection. A short-term divergent selection process was designed to estimate the genetic parameters of these two traits. Two flocks, including 100 Romanov ram lambs each, were challenged in 1990 when they were 6 months old. One flock received three artificial infections with 20 000 third-stage Teladorsagia circumcincta larvae, at intervals of 7 weeks. Faecal egg counts (FEC) were performed on Days 22, 25 and 28 post infection (p.i.) and the animals were drenched on Day 28 p.i. The other flock was grazed for 5 months on a pasture contaminated with the same species. Faecal samples were taken from the lambs at similar ages. About 5 rams with the lowest FEC and 5 with the highest FEC were selected in each flock and mated with unselected ewes. Their offspring (200 animals) were challenged in 1992, half in the same way as their sires, and the other half by the other method. Because of a drought in the summer of 1990, it was necessary to repeat part of the experiment, and in 1992 the 5 and 8 rams with the lowest and highest FEC, respectively, were selected from the offspring challenged on the pasture in 1992 and were mated with unselected ewes. Their progeny (about 80 animals) were challenged in 1994, half by natural infection, half by artificial infection. The mean FEC of the flock increased from the first to the third artificial infection. The natural infection was highly variable in different years, reflecting the difficulty of assessing resistance using this mode of challenge. Genetic parameters were estimated using animal models and REML solutions. The repeatabilities of the FEC following artificial and natural infection were 0.49 and 0.70 respectively within a period of one week, and 0.22 and 0.41 respectively for periods separated by intervals of 7 weeks; the heritabilities of the single egg count were 0.22 and 0.38 respectively. The genetic correlation was 0.87: the FEC recorded under natural or artificial infection appear to depend on the same genetic potential.     

Phenotypic analysis of host-parasite interactions in lambs infected with Teladorsagia circumcincta

Female Blackface lambs expected to exhibit genetic variability for resistance to gastrointestinal nematodes, were either exposed to continuous experimental infections of Teladorsagia circumcincta or were sham-dosed to monitor phenotypic responses to infection. As a measure of parasitism and host response, worm-eggs in faeces (faecal egg count, FEC) were counted over a 3-month period and worm burdens were ascertained at post-mortem. The host response to the infection was also measured by differential counts of white blood cells, anti-T. circumcincta IgA antibody levels and body weight. Results suggest that nematode abundance (mean number of parasites per host) and prevalence (proportion of infected animals) were maximal shortly after the beginning of infection (21 days p.i.) when virtually all the infected animals were shedding worm eggs. Increasing anti-T. circumcincta IgA antibody and eosinophil concentrations were associated with a reduction in total numbers of adult worms and an increase in the frequency of early L4s. The data also suggest that genetic selection for an enhanced anti-T. circumcincta IgA response might complement selection based on a reduced FEC as a strategy to select for resistance to gastrointestinal nematodes.     

The effect of corticosteroid treatment on local immune responses, intake and performance in lambs infected with Teladorsagia circumcincta

The nutritional cost of, and the sequential cellular changes associated with the developing immune response to the abomasal parasite Teladorsagia circumcincta were investigated using corticosteroid-induced immune-suppression. Six-month-old lambs with minimal nematode experience were either infected with 4000 L3 T. circumcincta per day (group IF), similarly infected and concurrently immune-suppressed with methylprednisolone acetate (group ISIF), immune-suppressed only (group IS) or remained as controls (group C). Food intake, faecal egg count (FEC) and antibody titres in plasma were recorded weekly, worm burden at necropsy on day 63 p.i. and body composition by X-ray computed tomography on days −2 and 62 p.i. Furthermore, sequential immunological changes at the site of parasite infestation in the abomasal mucosa were measured from serial biopsy tissue samples taken from additional animals that were fitted with an abomasal cannula and either infected with the same regime as IF animals above (group CnIF) or concurrently infected and immune-suppressed as above (group CnISIF). Corticosteroid treatment resulted in greater FECs (P < 0.01) and worm burdens (P < 0.01) in both ISIF and CnISIF compared with IF and CnIF sheep, respectively. Infection reduced feed intake by 17% between 14 and 28 days p.i. (P < 0.05) and efficiency of energy utilisation by 20% (P = 0.07) in IF animals but not in ISIF animals. Mast cells, globule leukocytes and IgA in tissue biopsy samples were elevated in CnIF from 42 days p.i., all of which were abrogated by corticosteroid treatment. The ability to regulate the worm population appeared to be associated with a rise in tissue IgA concentration and numbers of globule leucocytes (GL). The results support the hypothesis that a majority of the production losses that occur during infection of lambs with T. circumcincta in lambs are a consequence of the host immune response. These findings may have implications for regimes that promote the development of a strong host immune reaction to gastrointestinal parasites in lambs.     

Some properties of glutamate dehydrogenase,glutamine synthetase and glutamate synthase from Corynebacterium callunae

Characteristics of the three major ammonia assimilatory enzymes, glutamate dehydrogenase (GDH), glutamine synthetase (GS) and glutamate synthase (GOGAT) in Corynebacterium callunae (NCIB 10338) were examined. The GDH of C. callunae specifically required NADPH and NADP⁺ as coenzymes in the amination and deamination reactions, respectively. This enzyme showed a marked specificity for -ketoglutarate and glutamate as substrates. The optimum pH was 7.2 for NADPH-GDH activity (amination) and 9.0 for NADP⁺-GDH activity (deamination). The results showed that NADPH-GDH and NADP⁺-GDH activities were controlled primarily by product inhibition and that the feedback effectors alanine and valine played a minor role in the control of NADPH-GDH activity. The transferase activity of GS was dependent on Mn⁺² while the biosynthetic activity of the enzyme was dependent on Mg²⁺ as essential activators. The pH optima for transferase and biosynthetic activities were 8.0 and 7.0, respectively. In the transfer reaction, the K _m values were 15.2 mM for glutamine, 1.46 mM for hydroxylamine, 3.5×10^-3 mM for ADP and 1.03 mM for arsenate. Feedback inhibition by alanine, glycine and serine was also found to play an important role in controlling GS activity. In addition, the enzyme activity was sensitive to ATP. The transferase activity of the enzyme was responsive to ionic strength as well as the specific monovalent cation present. GOGAT of C. callunae utilized either NADPH or NADH as coenzymes, although the latter was less effective. The enzyme specifically required -ketoglutarate and glutamine as substrates. In cells grown in a medium with glutamate as the nitrogen source, the optimum pH was 7.6 for NADPH-GOGAT activity and 6.8 for NADH-GOGAT activity. Findings showed that NADPH-GOGAT and NADH-GOGAT activities were controlled by product inhibition caused by NADP⁺ and NAD⁺, respectively, and that ATP also had an important role in the control of NADPH-GOGAT activity. Both activities of GOGAT were found to be inhibited by azaserine.Abbreviations GDH glutamate dehydrogenase - GOGAT glutamate synthase - GS glutamine synthetase     

Heterologous expression of cDNAs encoding Chlorella sorokiniana NADP-specific glutamate dehydrogenase wild-type and mutant subunits in Escherichia coli cells and comparison of kinetic and thermal stability properties of their homohexamers

Full-length cDNAs encoding the - and -subunits and a truncated mutant subunit of the Chlorella sorokiniana NADP-GDH isozymes were constructed and expressed in Escherichia coli cells. The kinetic and thermal stability properties of the resultant homohexamers were examined. The electrophoretic mobility of the recombinant - and -subunits was identical to that of the native subunits as determined by immunoblotting. The homohexamers were purified by anion-exchange and gel-filtration chromatography. The - and -homohexamers that were synthesized in the bacterial cells were shown to have similar Michaelis constants for their substrates as previously shown after synthesis in C. sorokiniana cells (Bascomb and Schmidt, 1987). The homohexamer synthesized in the bacterium was allosteric with respect to NADPH but to a lesser degree than when isolated from the alga. The mutant homohexamer was composed of subunits that were truncated by 40 amino acids at their N-termini. This mutant isozyme was kinetically similar to the larger, anabolic -homohexamer, but it did not display the allosteric response to NADPH shown by the -homohexamer. The three isozymes had significant thermal tolerance and were stable at 50 °C. The temperature optimum for catalytic activity for the - and -homohexamers was 60 °C, and 65 °C for the 40N homohexamer. This study demonstrated that most of the kinetic properties of the Chlorella sorokiniana NADP-GDH isozymes were retained after their synthesis in a heterologous system, and that the distinctive N-terminal domains of these isozymes have dramatic effects on their biochemical characteristics.     

The glutamate dehydrogenase structural gene of Escherichin coli

Summary The glutamate dehydrogenase structural gene, gdhA, was mapped at 38.6 min on the genetic map and at 1860 kb on the physical map. A detailed map of this region is presented.     

The effect of neutral salt anions on the oxidative deamination activity of plant glutamate dehydrogenase

Increasing concentrations of anions of the Hofmeister series decrease the activity of highly purified glutamate dehydrogenase (EC 1.4.1.2.) from Pisum sativum L. The extent of the inactivation, as estimated by the ion concentration which causes a 50% transformation of the native form to the low activity form of the enzyme (approximately halfmaximal activity), follows the ranking Cl^–––3 ^– –. Sulfate has a slightly activating effect. At salt concentrations higher than 1 M (with SCN^– higher than 200 mM), the activity decreases to a value from 3–6% of the initial activity and remains then stable over a wide range of higher anion concentrations. From kinetic investigations it is seen that the treatment of the enzyme with anions decreases the affinity for the cosubstrate NAD⁺ and the substrate L-glutamate (K _M-values increased) and also increases the dissociation constant for NAD⁺. The salt induced inactivation is reversible by dilution. From a mathematical treatment of the kinetic data of the inactivation, it is seen that increasing concentrations of the anions exert cooperative effects on the inactivation process.     

Enzymes of the ornithine-glutamate-proline pathway in the sheep abomasal nematode parasites Haemonchus contortus and Teladorsagia circumcincta

A fully functional ornithine–glutamate–proline pathway was detected in L3 and adult Haemonchus contortus and Teladorsagia circumcincta, making the parasites capable of interconversion of these amino acids. Ornithine aminotransferase (OAT) (E.C. 2.6.1.13) was a reversible pyridoxal-5-phosphate (PLP)-dependent enzyme with an optimum pH 8.5. Hydroxylamine completely inhibited OAT activity in both parasites. For all five enzymes, substrate affinity was similar for each species and life cycle stage, the notable exceptions being the nearly 10-fold lower affinity for Δ¹-pyrroline-5-carboxylate (P5C) of P5C reductase (E.C. 1.5.1.2) in adult T. circumcincta and about half for P5C for L3 H. contortus P5C dehydrogenase (E.C. 1.5.1.12). P5C synthase (E.C. 1.2.1.41) activity was similar with either NADPH or NADH as co-factor. Proline oxidase (E.C. 1.5.99.8) was a co-factor independent enzyme with an optimal pH 8.5. Despite similarities to those in the host, enzymes of this pathway may still be useful as control targets if they differ antigenically, as a supply of proline is necessary for cuticle formation.     

The consequences of short-term grazing of bioactive forages on established adult and incoming larvae populations of Teladorsagia circumcincta in lambs

The objective of this study was to investigate the consequences of short-term grazing on bioactive forages on (i) the viability and fecundity of established adult Teladorsagia circumcincta population and (ii) the establishment and development of incoming T. circumcincta infective larvae. Forty-eight, parasite naive, 3-month old, grazing lambs were artificially infected with 8000 infective larvae of T. circumcincta on day 1 of the experiment. On day 21 p.i., lambs were allocated to one of three bioactive forage grazing treatments; chicory (Cichorium intybus), sulla (Hedysarum coronarium), lotus (Lotus pedunculatus), and the control grass/clover (Lolium perenne/Trifolium repens) forage. On day 28 of the experiment a second dose of 8000 T. circumcincta infective larvae was administered to the lambs to investigate the effects of forages on the ability of infective larvae to establish within the host. All animals were slaughtered for worm recovery on day 35, while liveweight gain, feacal egg counts (FEC) and total worm egg output were monitored regularly throughout the experiment. Although FEC or total egg output were similar among the groups, adult worm burdens at slaughter were significantly affected (P<0.05) by forage treatment during the 2 week grazing period. Lambs grazing chicory had the lowest adult worm burdens and significantly lower numbers of male worms compared to those grazing on grass/clover (P<0.01), while the lambs grazing on sulla or lotus had similar adult populations to grass/clover fed animals. The results from the worm recoveries of the second dose (immature worm burdens) were affected by physiologically and/or immunologically mediated mechanisms, which reduced larval establishment in all treatments. Nevertheless, immature worm burdens at slaughter were similar between chicory, sulla and grass/clover group, while the immature worm recoveries from the lotus group were significantly higher (P<0.05) compared to those from lambs grazing grass/clover. Overall, the results of the present study support the view that chicory can be a promising candidate species in pasture management practices to control T. circumcincta burdens.     

Arginine metabolism in the sheep abomasal nematode parasites Haemonchus contortus and Teladorsagia circumcincta

The ornithine urea cycle, polyamine synthesis, nitric oxide synthesis and metabolism of arginine to putrescine have been investigated in L3 and adult Haemonchus contortus and Teladorsagia circumcincta. Neither parasite had a detectable arginine deiminase/dihydrolase pathway nor a functional ornithine urea cycle. Nitric oxide synthase was present in central and peripheral nerves, but was not detected in whole parasite homogenates. Both arginase (E.C. 3.5.3.1) and agmatinase (E.C. 3.5.3.11) activities were present in both species. Arginase did not require added Mn²⁺ and had an optimal pH of 8.5. Polyamine metabolism differed in the two species and from that in mammals. Ornithine decarboxylase (E.C. 4.1.1.17) was present in both parasites, but no arginine decarboxylase (E.C. 4.1.1.19) activity was detected in T. circumcincta. The flexibility of synthesis of putrescine in H. contortus may make this pathway less useful as a target for parasite control than in T. circumcincta, in which only the ornithine decarboxylase pathway was detected.     

Purification and properties of the NADP-dependent glutamate dehydrogenase from Dictyostelium discoideum

Summary NADP-dependent glutamate dehydrogenase from Dictyostelium discoideum was purified 9300 fold with a yield of 4.6%. The enzyme is a hexamer of apparent molecular weight 294 kDa on Sephacryl S400 and a subunit molecular weight of 52 kDa as determined by SDS gel electrophoresis. The apparent K_mS for -ketoglutarate, NADPH and NH _inf4 ^sup+ are 1.2 mM, 9.7 µM and 2.2 mM respectively, and the purified enzyme has a broad pH optimum with a peak at pH 7.75. GTP has a slight stimulatory effect (22% at 83 µM) as does ADP (11% at 1 mM), and AMP is slightly inhibitory (9% at 1 mM) whereas adenosine, ATP and cAMP have little or no effect. Neither the Zn²⁺ chelating compound 1,10-phenanthroline nor EDTA have any effect on the enzyme while p-hydroxymercuribenzoic acid inhibits enzyme activity (50% at 80 µM) yet N-ethylmaleimide does not.In addition, the NADP-GDH activity varies little during the various stages of morphogenesis.Abbreviations EDTA Ethylenediamine Tetraacetic Acid - Tris Tris(hydroxymethyl)aminomethane - Bis-tris bis(2-hydroxyethyl)imino-tris(hydroxymethyl)methane - TRITON X-100 iso-octylphenoxypoly-ethoxyethanol - pHMB p-Hydroxymercuribenzoic acid     

Purification and characterization of the NADP-dependent glutamate dehydrogenase from Bacillus fastidiosus

Ammonia assimilation in Bacillus fastidiosus proceeds via the NADP-dependent glutamate dehydrogenase. The enzyme, purified to homogeneity, is composed of identical subunits with a molecular weight of about 48 000 dalton. Presumably the enzyme is a hexamer. The enzyme is specific for NADP (H). The pH optima for the amination and deamination reactions are 7.7 and 8.6, respectively. The temperature optimum is 60°C. Furthermore, temperature stability and apparent K_m values for substrates of both the amination and deamination reactions were determined. Several metabolites were tested for their effect on the enzyme activity. Only malate and fumarate showed some inhibitory effect.Abbreviation GDH glutamate dehydrogenase     

Methylglyoxal as substrate and inhibitor of human aldehyde dehydrogenase: Comparison of kinetic properties among the three isozymes

Methylglyoxal was demonstrated to be a substrate for the isozymes E1, E2 and E3 of human aldehyde dehydrogenase. Pyruvate was the product from the oxidation of methylglyoxal by the three isozymes. At pH 7.4 and 25^oC, the major and minor components of the E3 isozyme catalyzed the reaction with V_max of 1.1 and 0.8 μmol NADH min⁻¹ mg⁻¹ protein, respectively, compared to 0.067 and 0.060 μmol NADH min⁻¹ mg⁻¹ protein for the E1 and E2 isozymes, respectively. The E2 isozyme had a K_m for methylglyoxal of 8.6 μM, the lowest compared to 46 μM for E1 and 586 and 552 μM for the major and minor components of the E3 isozyme, respectively. Both components of the E3 isozyme showed substrate inhibition by methylglyoxal, with K_i values of 2.0 mM for the major component and 12 mM for the minor component at pH 9.0. Substrate inhibition by methylglyoxal was not observed with the E1 and E2 isozymes. Methylglyoxal strongly inhibited the glycolaldehyde activity of the E1 and E2 isozymes. Mixed-type models of inhibition were employed as an approach to calculate the inhibition constants, 44 and 10.6 μM for E1 and E2 isozymes, respectively.     

Interaction between glutamate dehydrogenase (GDH) and L-leucine catabolic enzymes: intersecting metabolic pathways

Branched-chain amino acids (BCAAs) catabolism follows sequential reactions and their metabolites intersect with other metabolic pathways. The initial enzymes in BCAA metabolism, the mitochondrial branched-chain aminotransferase (BCATm), which deaminates the BCAAs to branched-chain α-keto acids (BCKAs); and the branched-chain α-keto acid dehydrogenase enzyme complex (BCKDC), which oxidatively decarboxylates the BCKAs, are organized in a supramolecular complex termed metabolon. Glutamate dehydrogenase (GDH1) is found in the metabolon in rat tissues. Bovine GDH1 binds to the pyridoxamine 5′-phosphate (PMP)-form of human BCATm (PMP-BCATm) but not to pyridoxal 5′-phosphate (PLP)-BCATm in vitro. This protein interaction facilitates reamination of the α-ketoglutarate (αKG) product of the GDH1 oxidative deamination reaction. Human GDH1 appears to act like bovine GDH1 but human GDH2 does not show the same enhancement of BCKDC enzyme activities. Another metabolic enzyme is also found in the metabolon is pyruvate carboxylase (PC). Kinetic results suggest that PC binds to the E1 decarboxylase of BCKDC but does not effect BCAA catabolism. The protein interaction of BCATm and GDH1 promotes regeneration of PLP-BCATm which then binds to BCKDC resulting in channeling of the BCKA products from BCATm first half reaction to E1 and promoting BCAA oxidation and net nitrogen transfer from BCAAs. The cycling of nitrogen through glutamate via the actions of BCATm and GDH1 releases free ammonia. Formation of ammonia may be important for astrocyte glutamine synthesis in the central nervous system. In peripheral tissue association of BCATm and GDH1 would promote BCAA oxidation at physiologically relevant BCAA concentrations.     

Novel nitrogen sources for growth of Bacillus fastidiosus and their effect on the activity of NADP-dependent glutamate dehydrogenase

Abstract Although Bacillus fastidiosus assimilates ammonium formed internally during growth on urate, allantoin or allantoate via NADP-dependent glutamate dehydrogenase (NADP-GDH), growth on exogenous ammonium as nitrogen source has not been observed. Growth on ammonium, urea and ureidoglycolate, intermediates of the urate degradative pathway, was found to occur if the mineral growth medium containing glycerol as a carbon source was supplemented with both allantoin (0.5 mM) and brain heart infusion (BHI, 0.1%, w/v) or yeast extract. Neither allantoin nor BHI supported growth alone or in combination unless ammonium was present. NADP-GDH activity appeared to be regulated only by the extracellular concentration of allantoin or allantoate. Enzyme activity was not influenced by other nitrogen sources or the intracellular ammonium concentration.