An-Najah Staff

 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.