An-Najah Staff

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.

 We have purified and characterized the Ascaris suum γ-glutamylcysteine synthetase, the rate-limiting step in the glutathione biosynthesis. The purified enzyme exhibited a specific activity of 18 U (mg protein)−1. Estimation of the molecular mass of the native enzyme by FPLC on Superdex S-200 revealed the presence of two enzyme activity peaks corresponding to molecular masses of 100 and 70 kDa. The higher-molecular-mass component could be dissociated by repeated gel filtration into the 70-kDa protein which is the enzymatically active subunit. The apparent Km values of the A. suum enzyme for Image -aminobutyrate, Image -cysteine and Image -glutamate were 0.31, 0.41 and 0.94 mM, respectively. Image -Buthionine-S,R-sulfoximine and cystamine showed time-dependent irreversible inhibitory effects on the A. suum enzyme activity with Ki values of 0.05 and 1.11 μM, respectively. The Ki values for the corresponding enzyme from rat kidney with Image -buthionine-S,R-sulfoximine and cystamine were 7.19 and 22.2 μM, respectively. The time of half-inactivation of the enzyme at infinite concentration of Image -buthionine-S,R-sulfoximine, τ50, was determined to be 3.1 and 1.34 min, for the parasite and mammalian enzymes respectively. For cystamine, a τ50 value of 3.32 min for the A. suum γ-glutamylcysteine synthetase was determined, while a value of 2 min in case of rat kidney enzyme was found. The A. suum enzyme activity was competitively inhibited by glutathione with a Ki value of 0.11 mM. The higher sensitivity of the A. suum enzyme to Image -buthionine-S,R-sulfoximine compared to the rat kidney γ-glutamylcysteine synthetase recommends it as a target for chemotherapy.