l-buthionine-S,R-sulphoximine

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The y-Glutamylcysteine Synthetase of Onchocerca Volvulus

Journal Title, Volume, Page: 
Molecular and Biochemical Parasitology Volume 111, Issue 2, 1 December 2000, Pages 243-251
Year of Publication: 
2000
Authors: 
Ayman Hussein
Department of Biochemistry, Imperial College of Science, Technology and Medicine, London SW7 2AY, UK
Current Affiliation: 
Faculty of Medicine & Health Sciences, Department of Biomedical Sciences, An-Najah National University, Nablus, Palestine
Kai Lüersen
Department of Biochemical Parasitology, Bernhard Nocht Institute for Tropical Medicine, Bernhard-Nocht-Strasse 74, D-20359 Hamburg, Germany
Sylke Müller
Department of Biochemical Parasitology, Bernhard Nocht Institute for Tropical Medicine, Bernhard-Nocht-Strasse 74, D-20359 Hamburg, Germany
Eva Liebau
Department of Biochemical Parasitology, Bernhard Nocht Institute for Tropical Medicine, Bernhard-Nocht-Strasse 74, D-20359 Hamburg, Germany
Rolf D. Walter
Department of Biochemical Parasitology, Bernhard Nocht Institute for Tropical Medicine, Bernhard-Nocht-Strasse 74, D-20359 Hamburg, Germany
Preferred Abstract (Original): 

The tripeptide glutathione (GSH) plays an important role in the maintenance of the intracellular thiol redox state and in detoxification processes. The intracellular GSH level depends on glutathione reductase as well as on GSH synthesis. The first and rate limiting step in the synthetic pathway is catalysed by γ-glutamylcysteine synthetase (γ-GCS). The γ-GCS was partially purified from the filarial parasite Onchocerca volvulus and preliminary steady state kinetics were performed. The Ki-value for Image -buthionine-S,R-sulphoximine (BSO), a specific inhibitor of γ-GCS, was determined to be 0.13 μM, which is 54-fold lower than the Ki-value for the mammalian enzyme. Filarial γ-GCS was also inhibited by cystamine with a Ki-value of 3.9 μM compared with 22.2 μM determined for the rat enzyme. Further, the cDNA and the gene of the O. volvulus γ-GCS were cloned and sequenced. The gene of 5762 bp is composed of 14 exons and 13 introns. Southern blot analysis indicates that the γ-GCS gene is present as a single-copy gene. In accordance with Northern blot analysis, the entire cDNA sequence encompasses 2377 bp. At its 5′ end a nematode-specific spliced leader 130 bp upstream of the first in frame methionine was identified. The cDNA encodes a polypeptide of 652 amino acids with 50 and 69% sequence identity to the human and the Caenorhabditis elegans counterparts, respectively. The filarial γ-GCS is proposed as a potential drug target.

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