Partial purification and characterization of acetylcholinesterase isozymes from adult bovine filarial parasite Setaria cervi.

Filariasis is a major health problem, affecting millions of people in tropical and sub-tropical regions of the world. The isolation and characterization of parasite-specific enzyme targets is essential for developing effective control measures against filariasis. Acetylcholinesterase (AchE, E.C. 3.1.1.7), an important enzyme of neuromuscular transmission is found in a number of helminths including filarial parasites and may be playing a role in host-parasite interactions. Earlier, we demonstrated the presence of two isozymes of AchE, different from the host enzyme in the human (Brugia malayi) and bovine (Setaria cervi) filarial parasites. In the present study, two isozymes of AchE (pAchE1 and pAchE2) were isolated from S. cervi adults and characterized biochemically and immunochemically. The AchE was partially purified on Con-A Sepharose column and then subjected to preparative polyacrylamide gel electrophoresis (PAGE) for separation of the isozymes. The AchE activity was localized by the staining of gel and the isozymes were isolated from the PAGE strips by electroelution. Both isozymes preferentially utilized acetylcholine iodide as substrate and were strongly inhibited by the true AchE inhibitor (BW284c51), suggesting that they were true AchE. The polyclonal antibodies produced against the isozymes showed significant cross-reactivity with B. malayi AchE, but not against the host enzyme. These findings suggested that both the isozymes were biochemically (in terms of their substrate specificity and inhibitor sensitivity) and immunochemically similar, but different from the host enzyme.

Succinate oxidase and fumarate reductase systems of filarial parasite Setaria digitata.

Activities of succinate oxidase, fumarate reductase (FR) and succinate dehydrogenase (SDH) under a set of defined conditions were determined in the mitochondrial isolate from Setaria digitata, the filarial parasite from the cattle Bos indicus. Presence of only two activities namely SDH and succinate--UQ reductase of the succinate oxidase system could be detected in S. digitata. In the absence of cytochromes, the 3rd enzyme of the complex namely cytochrome oxidase is absent and it is proposed that an alternative oxidase is responsible for completing the succinate oxidation expressed as succinate oxidase activity. Though SDH and FR catalyse reverse reactions, they responded differently to modulators such as oxaloacetate, aspartate, alanine, pyruvate and fumarate. The degree of response of the two activities against inhibitors of electron transport was also different. Interestingly fumarate caused only 50% inhibition of succinate oxidation, while the effect against FR was more convincing.

Partial characterization of affinity purified excretory-secretory protease from Setaria digitata.

Excretory-secretory protease of S. digitata released along with the microfilariae (mf) during hatching has been purified by affinity chromatography. No other activity could be detected in the affinity purified material. Homogeneity is checked by native PAGE. It has a pH optimum of 5.4 and a molecular weight of 70 kD. The purified material showed positivity against antibodies raised against ES material.