PURIFICATION AND CHARACTERIZATION OF GAMMA-GLUTAMYLCYSTEINE SYNTHETASE FROM ASCARIS-SUUM

We have purified and characterized the Ascaris suum gamma-glutamylcyst eine synthetase, the rate-limiting step in the glutathione biosynthesi s. The purified enzyme exhibited a specific activity of 18 U (mg prote in)(-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-molecu lar-mass component could be dissociated by repeated gel filtration int o the 70-kDa protein which is the enzymatically active subunit. The ap parent K-m values of the A. suum enzyme for L-aminobutyrate, L-cystein e and L-glutamate were 0.31, 0.41 and 0.94 mM, respectively. D,L-Buthi onine-S,R-sulfoximine and cystamine showed time-dependent irreversible inhibitory effects on the A. suum enzyme activity with K-i values of 0.05 and 1.11 mu M, respectively. The K-i values for the corresponding enzyme from rat kidney with D,L-buthionine-S,R-sulfoximine and cystam ine were 7.19 and 22.2 mu M, respectively. The time of half-inactivati on of the enzyme at infinite concentration of D,L-buthionine-S,R-sulfo ximine, tau(50), was determined to be 3.1 and 1.34 min, for the parasi te and mammalian enzymes respectively. For cystamine, a tau(50) value of 3.32 min for the A. suum gamma-glutamylcysteine synthetase was dete rmined, while a value of 2 min in case of rat kidney enzyme was found. The A. suum enzyme activity was competitively inhibited by glutathion e with a K-i value of 0.11 mM. The higher sensitivity of the A. suum e nzyme to D,L-buthionine-S,R-sulfoximine compared to the rat kidney gam ma-glutamylcysteine synthetase recommends it as a target for chemother apy.