Structural and functional properties of glutamate dehydrogenases

Glutamate dehydrogenases are homooligomeric enzymes that catalyse the reversible oxidative deamination of glutamate to 2-oxoglutarate, thereby linking nitrogen metabolism and the tricarboxylic acid cycle. Four different classes of the enzyme are known in microbes that differ in size and cofactor specificity. Despite some common features, the kinetic properties of the enzymes from different species vary considerably. We consider the relationship between the sequences, those structures that are available and the enzyme kinetics in the context of the metabolic role of the enzyme

Structural and functional properties of glutamate dehydrogenases

Glutamate dehydrogenases are homooligomeric enzymes that catalyse the reversible oxidative deamination of glutamate to 2-oxoglutarate, thereby linking nitrogen metabolism and the tricarboxylic acid cycle. Four different classes of the enzyme are known in microbes that differ in size and cofactor specificity. Despite some common features, the kinetic properties of the enzymes from different species vary considerably. We consider the relationship between the sequences, those structures that are available and the enzyme kinetics in the context of the metabolic role of the enzyme

Effect of Ostertagia circumcincta excretory/secretory products on gastrin release in vitro

It has been suggested that parasite excretory/secretory (ES) products may be capable of direct stimulation of gastrin secretion and of contributing to the hypergastrinaemia typical of abomasal parasitism. Ostertagia circumcincta ES products were tested on an ovine antral mucosal preparation which had been developed for a pharmacological study of gastrin secretion in the sheep. Its responsiveness to chemical stimulation was established by stimulation with amino acids and amines: tryptophan (0.1–5 mM) and phenylalanine (10–100 mM) stimulated gastrin release (151–160 and 117–129%, respectively), whereas glycine (0.1–100 mM) was without effect; ammonium sulphate, but not sodium sulphate, stimulated gastrin release in concentrations from 1 mM (122%) to 50 mM (148%). ES products were prepared by incubation of exsheathed third-stage larvae (L3) or parasites recovered on Day 8 p.i. (L4), Day 12 p.i. (10% L4, 90% immature adults), Day 21 p.i. (5% L4, 30% immature adults, 65% adults), Day 22 p.i. (20% immature adults, 80% adults), Day 30 p.i. (adults) and Day 35 p.i. (adults), or a mixed-age parasite population. Worms were recovered from agar and incubated in either distilled water or Hank's balanced salt solution (HBSS) adjusted to pH 2.5, 3.5, 4.5, 5.0 or 7.4. HBSS pH 7.4 was also prepared with antibiotics, without glucose, and with antibiotics but without glucose. Survival of Day 21 and 35 worms and exsheathed L3 in water or in a series of HBSS adjusted to pH 2.5, 3.5, 4.5, 5.0 or 7.4 was assessed from the percentage of motile parasites. L3 slowly became immotile over several days except in HBSS pH 2.5, in which survival was reduced, whereas adult worms did not tolerate incubation at 37 °C in water or HBSS at pH 2.5, retained motility for about 2 days at pH 3.5, but survived well at pH 4.5 and above. Incubates prepared from all stages of O. circumcincta, both in media favourable and unfavourable for parasite survival, failed to stimulate consistently the secretion of gastrin by tissue from both parasite-naive and previously exposed sheep, whereas a considerable number of incubates were significantly inhibitory. The inhibitor may not be produced by the nematodes, but by contaminating abomasal or environmental microflora, as inhibitory activity was predominantly generated by prolonged incubation, it was less potent when glucose was omitted and was not present in media containing antibiotics. This study did not find evidence for a gastrin stimulant in O. circumcincta ES products, but did demonstrate the acid intolerance of adult worms and suggests that abomasal microbes may be capable of modulating the secretory activity of the host digestive tract

Abomasal bacteria produce an inhibitor of gastrin secretion in vitro

Previously, proliferating microflora transferred with abomasal nematodes, were suspected to be the source of the gastrin inhibitor in some parasite excretory/secretory products. Aerobic cultures in HBSS of abomasal fluid from uninfected sheep became inhibitory during the static growth phase, unless antibiotics were present. Basal gastrin secretion was reduced by up to 90%. Rumen fluid and incubates and medium in which Streptococcus bovis and ovine rumen Actinomycete spp. had been grown also contained the inhibitor. Unlike abomasal cultures, rumen fluid and incubates also reduced the measurement of gastrin standards. Rumen incubates were less potent after exposure to pH 2–3, suggesting that inactivation normally occurs in the unparasitised abomasum. Contaminating bacteria which generate the gastrin inhibitor in parasite ES products are probably rumen organisms which survive in the abomasum and proliferate during subsequent incubation. Significantly, rumen bacteria have been shown to be capable of affecting the secretory activity of the gastric mucosa

Variation in the form of iron in beef and lamb meat and losses of iron during cooking and storage

Iron levels were lower in semitendinosus muscle of beef and lamb than in longissimus or triceps brachii muscles, and were lower for muscles of male than female lambs, but were similar for muscles from bulls, heifers and mature cows. Soluble proportions of haem iron were higher for beef than lamb (66% vs. 56% of total iron, P < 0.0001), and percentages, as insoluble haem iron or insoluble and soluble non-haem iron, were lower in beef. For beef semitendinosus muscle, increases in cooking time and temperature led to losses of soluble iron and haem iron, with increases in insoluble and non-haem forms of iron, and also in the level of iron in the cooking juice. Losses of iron in drip from a free meat sample or a sample on a pad were over 90% soluble haem iron and were greater following freezing and thawing due mainly to higher volumes of drip

Abomasal contents of parasitised sheep contain an inhibitor of gastrin secretion in vitro

Serum gastrin concentrations are typically elevated in parasitised sheep; however, in some animals serum gastrin concentrations may fall abruptly despite a very high abomasal pH. Although proliferating abomasal bacteria in culture generate a potent inhibitor of in vitro gastrin secretion, this inhibitor has not been detected in abomasal contents of unparasitised sheep. In sheep parasitised by O. circumcincta, all abomasal fluid samples of pH 5 and above were inhibitory to gastrin release in vitro. Inhibitory activity and abomasal pH were correlated in two separate experiments; the model best fitting the data being sigmoidal in each case, with zero activity at pH 3.6 and 4.6, respectively. There was no clear evidence that the presence of a gastrin inhibitor in the abomasal contents reduced the serum gastrin concentration in parasitised sheep. Serum gastrin was correlated with abomasal pH (log10 serum gastrin concentrations conformed to log-linear sigmoidal models)

Gastrin secretion by ovine antral mucosa in vitro

The effect on gastrin and somatostatin release in sheep of stimulatory and inhibitory peptides and pharmacological agents was investigated using an in vitro preparation of ovine antral mucosa. Carbachol stimulated gastrin release in a dose-dependent manner but had no effect on somatostatin release. As atropine blocked the effect of carbachol, cholinergic agonists appear to stimulate gastrin secretion directly through muscarinic receptors on the G-cell and not by inhibition of somatostatin secretion. Both vasoactive-intestinal peptide (VIP) and gastric-inhibitory peptide (GIP) increased somatostatin release but did not inhibit basal gastrin secretion, although VIP was effective in reducing the gastrin response to Gastrin-releasing peptide (GRP). Porcine and human GRP were stimulatory to gastrin secretion in high doses but bombesin was without effect. The relative insensitivity to GRP (not of ovine origin) previously reported from intact sheep may be caused either by a high basal release of somatostatin or by the ovine GRP receptor or peptide differing from those of other mammalian species

Effect of excretory/secretory products of abomasal parasites on epithelial tight junctions

The presence of abomasal parasites is thought to be associated with an increase in the permeability of the gastric epithelium. Epithelial permeability is regulated by junctional complexes between adjacent cells. The most apical component of this junctional complex is the tight junction which functions as a paracellular diffusion barrier. Any disruption of tight junctions results in impaired barrier function and an associated increase in epithelial permeability. To investigate the effect of abomasal parasites on the integrity and barrier function of epithelia, Caco-2 cell monolayers were exposed to the excretory/ secretory products (ES) of adult Ostertagia (Teladorsagia) circumcincta and Haemonchus contortus. Changes in epithelial barrier function were monitored by measuring transepithelial electrical resistance (TEER) and tight junction integrity was visualised using immunofluorescence localisation of the tight junction-associated proteins, occludin and zonula occludens-1 (ZO-1), by confocal microsopy. Under control conditions, occludin and ZO-1 were localised to a continuous pericellular ring around individual cells when viewed from the apical surface. In cells exposed to ES for 24 h, staining of this pericellular ring was diminished in intensity and corresponded with an increase in the presence of punctuate, intracellular staining. Exposure to ES was also shown to interfere with tight junction integrity, which was detected as a decrease in TEER from 678 ± 10 Ωcm2 (control) to 526 ± 8 Ωcm2 (ES-treated) (n = 12) in 6 h. These alterations in TEER, along with intracellular changes in occludin and ZO-1 distribution, suggest that parasite ES disrupts tight junctions, leading to an increase in epithelial permeability which may be of importance in the pathology of abomasal parasitism

The tricarboxylic acid cycle in L3Teladorsagia circumcincta: metabolism of acetyl CoA to succinyl CoA

Nematodes, like other species, derive much of the energy for cellular processes from mitochondrial pathways including the TCA cycle. Previously, we have shown L3Teladorsagia circumcincta consume oxygen and so may utilise a full TCA cycle for aerobic energy metabolism. We have assessed the relative activity levels and substrate affinities of citrate synthase, aconitase, isocitrate dehydrogenase (both NAD+ and NADP+ specific) and α-ketoglutarate dehydrogenase in homogenates of L3T. circumcincta. All of these enzymes were present in homogenates. Compared with citrate synthase, low levels of enzyme activity and low catalytic efficiency was observed for NAD+ isocitrate dehydrogenase and especially α-ketoglutarate dehydrogenase. Therefore, it is likely that the activity of these to enzymes regulate overall metabolite flow through the TCA cycle, especially when [NAD+] limits enzyme activity. Of the enzymes tested, only citrate synthase had substrate affinities which were markedly different from values obtained from mammalian species. Overall, the results are consistent with the suggestion that a full TCA cycle exists within L3T. circumcincta. While there may subtle variations in enzyme properties, particularly for citrate synthase, the control points for the TCA cycle in L3T. circumcincta are probably similar to those in the tissues of their host species

The kinetics and regulation of phosphofructokinase from Teladorsagia circumcincta

Phosphofructokinase (PFK-1) activity was examined in L3 and adult Teladorsagia circumcincta, both of which exhibit oxygen consumption. Although activities were higher in the adult stage, the kinetic properties of the enzyme were similar in both life cycle stages. T. circumcincta PFK-1 was subject to allosteric inhibition by high ATP concentration, which increased both the Hill coefficient (from 1.4 ± 0.2 to 1.7 ± 0.2 in L3s and 2.0 ± 0.3 to 2.4 ± 0.4 in adults) and the K½ for fructose 6 phosphate (from 0.35 ± 0.02 to 0.75 ± 0.05 mM in L3s and 0.40 ± 0.03 to 0.65 ± 0.05 mM in adults). The inhibitory effects of high ATP concentration could be reversed by fructose 2,6 bisphosphate and AMP, but glucose 1,6 bisphosphate had no effect on activity. Similarly, phosphoenolpyruvate had no effect on activity, while citrate, isocitrate and malate exerted mild inhibitory effects, but only at concentrations exceeding 2 mM. The observed kinetic properties for T. circumcincta PFK-1 were very similar to those reported for purified Ascaris suum PFK-1, though slight differences in sensitivity to ATP concentration suggests there may be subtle variations at the active site. These results are consistent with the conservation of properties of PFK-1 amongst nematode species, despite between species variation in the ability to utilise oxygen