Affinity purification, identification, and biochemical characterization of Gamma-glutamyl transpeptidase, a membrane anchored enzyme of Gigantocotyle explanatum.

Gamma-glutamyl transpeptidase is an enzyme that facilitates the transfer of glutamyl groups from glutamyl peptides to other peptides or water. Additionally, it also participates in important processes such as amino acid transport, cellular redox control, drug detoxification, apoptosis, and DNA fragmentation in a various organism. In the present study, GGT activity in Gigantocotyle explanatum was examined in order to characterize the enzyme in the helminth system. GGT is isolated using membrane solubilization and purified through affinity column chromatography (Con-A Sepharose column). Km and Vmax values, as well as the optimal pH, optimal temperature, and incubation period, are also determined using enzyme kinetics. The hetero-dimeric property of the enzyme is demonstrated by the purified GGT, which yielded two subunits of 65.5 and 55 kDa. The optimal pH and temperature are found to be 8.0 and 37 °C, respectively. While assessing the optimal incubation time of the enzyme, it was observed that the purified GGT not only retained its functional integrity up to 15 min but also reflected considerable thermostability at higher temperatures, by retaining 78% and 25% of its initial activities at 50 °C and 60 °C, respectively. One millimolar concentration of 6-Diazo-5-Oxo Nor-isoleucine (DON), a specific inhibitor of GGT, completely abolished GGT activity. These results suggest that GGT in these worms is a catalytically active enzyme with distinguishing characteristics that can be used for further study to comprehend its function in amphistome biology and in host-parasite relationships, especially since the potential therapeutic candidacy of the GGT enzyme has already been indicated in these groups of organisms.

In vitro effect of metrifonate on the indices of oxidative stress in Gigantocotyle explanatum.

Helminth infections in general and digenetic trematodes in particular cause a huge economic loss globally to our livestock. Gigantocotyle explanatum is a digenetic amphistome that infects the bile ducts of water buffalo and are highly prevalent in tropical and sub-tropical countries. In the present study, effects of an organophosphate compound, Metrifonate (MF) in three doses, viz., 9.4 × 10-5 M (Dose I), 14.4 × 10-5 M (Dose II), and 19.4 × 10-5 M (Dose III), have been studied in vitro, on the motility and on some enzymatic and non-enzymatic oxidative stress indices in G. explanatum. The worm's motility and their non-enzymatic oxidative stress biomarkers like lipid peroxides measured as thiobarbituric acid-reactive substances (TBARS) and reduced glutathione (GSH) were disrupted significantly in a dose-dependent manner. However, the enzymatic oxidative stress biomarkers like glutathione-S-transferase (GST) and superoxide dismutase (SOD) were affected by MF treatment in a biphasic manner. Exposure to Dose I significantly stimulated the activities of both GST and SOD, whereas exposure to Doses II and III resulted into significant inhibition in a dose-dependent manner. Our findings suggest that MF has potential to be a strong and effective anthelmintic, however, further studies in vitro as well as in vivo are needed to explore further these observations and understand the exact mode of MF action in G. explanatum and other trematodes of veterinary economic importance.

Paralytic effect of alcoholic extract of Allium sativum and Piper longum on liver amphistome, Gigantocotyle explanatum.

OBJECTIVE: To investigate the effects of alcoholic extract of Allium sativum and Piper longum on the muscular activity of a parasitic amphistome, Gigantocotyle explanatum. MATERIALS AND METHODS: Amphistomes were isometrically mounted to record the spontaneous muscular activity by using Chart 4 software program (Power Lab, AD Instruments, Australia) and to examine the effects of cumulative doses (100, 300, 1000, and 3000 µg/ml) of the plant extracts on the amplitude (g), frequency (per 10 min), and baseline tension (g) of the spontaneous muscular activity of the amphistome. RESULTS: Alcoholic extract of A. sativum produced significant reduction in the frequency and amplitude of contractile activity of the amphistome at 1000 and 3000 µg/ml bath concentrations. Complete paralysis of the amphistome was observed after 15 min of addition of 3000 µg/ml concentration. Alcoholic extract of P. longum also caused paralysis following 15-20 min exposure of the amphistome to 3000 µg/ml concentration. In both the cases the amphistomes did not recover from paralysis following 2-3 washes. CONCLUSION: The observations demonstrate the paralytic effect of alcoholic extract of A. sativum and P. longum on G. explanatum.

Characterization of the glutamate dehydrogenase activity of Gigantocotyle explanatum and Gastrothylax crumenifer (Trematoda: Digenea).

Glutamate dehydrogenase (GLDH) (EC 1.4.1.3) is a ubiquitous enzyme, which is present at the protein and carbohydrate metabolism crossroads. The enzyme activity was investigated in biliary and rumen amphistomes, Gigantocotyle explanatum and Gastrothylax crumenifer, respectively, infecting the Indian water buffalo Bubalus bubalis. The enzyme activity was consistently higher in G. explanatum as compared to G. crumenifer, where NAD(H) was utilized as coenzyme and the pH optima was recorded at 8. The K(m) and V(max) values for α-ketoglutarate were 2.1 mM and 9.09 units in G. explanatum, whereas 3.03 mM and 1.90 units in G. crumenifer, respectively. Among the allosteric modulator nucleotides, AMP, ADP, ATP, GMP, CMP and UMP, only AMP enhanced GLDH activity in G. crumenifer while ADP was stimulatory in G. explanatum. The amino acid leucine stimulated the GLDH activity in both the amphistomes while alanine was stimulatory only in G. crumenifer. Pronounced interspecific differences in response to different metabolic inhibitors like diethyldithiocarbamate, semicarbazide hydrochloride and mercurial ions were also observed. The osmotic stress alters the enzyme activity, particularly in hypertonic saline the GLDH activity increased significantly (p < 0.01) in G. explanatum, while insignificant effects were observed in rumen dwelling G. crumenifer. Histoenzymology revealed region/tissue specific distribution of GLDH with prominent staining in tissues like vitellaria, lymph system and tegument/subtegument, thus showing specific distribution of GLDH indicating differential metabolic state. Such intergeneric differences in GLDH activity could also be a consequence of occupying different microenvironments within the same host.