An-Najah Staff

γ-Glutamyl transpeptidase, which is of central importance in the degradation of glutathione, was purified from Ascaris suum to apparent homogeneity. The enzyme was found to be a predominantly membrane-bound protein and was solubilized by Triton X-100. The purified enzyme, which exhibits a specific activity of 1009 U (mg protein)−1, showed a molecular mass of 70 kDa and was found to be composed of two non-identical subunits of molecular mass 43 and 30 kDa. Concerning the kinetic properties of the enzyme, the data presented in this study showed that various amino acids and dipeptides with L-configuration served as acceptors for the γ-glutamyl moieties of the enzyme reaction products and showed Km-values in the mM range. The apparent Km-value for the γ-glutamyl donor L-glutamyl-γ-7-amido-4-methylcoumarin of the enzyme was found to be 0.03 mM. L- and D-serine in combination with borate ions were competitive inhibitors of the enzyme activity with Ki-values of 0.30 and 0.61 mM, respectively. Acivicin was an irreversible inhibitor of the enzyme with a Ki-value of 0.42 mM and with a pseudo-first-order kinetics (kinact) of 0.18 min−1. In vitro treatment of the adult A. suum with acivicin resulted in a dose-dependent inhibition of the enzyme activity and an increase of the glutathione levels. These findings indicate the physiological role of the γ-glutamyl transpeptidase of this parasitic nematode in the catabolism of glutathione.

 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.