Purification and characterization of gamma-glutamylcysteine synthetase from Ascaris suum.
We have purified and characterized the Ascaris suum gamma-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 L-aminobutyrate, L-cysteine and L-glutamate were 0.31, 0.41 and 0.94 mM, respectively. D,L-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 microM, respectively. The Ki values for the corresponding enzyme from rat kidney with D,L-buthionine-S,R-sulfoximine and cystamine were 7.19 and 22.2 microM, respectively. The time of half-inactivation of the enzyme at infinite concentration of D,L-buthionine-S,R-sulfoximine, tau 50, was determined to be 3.1 and 1.34 min, for the parasite and mammalian enzymes respectively. For cystamine, a tau 50 value of 3.32 min for the A. suum gamma-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.(ABSTRACT TRUNCATED AT 250 WORDS)
Department of Biochemical Parasitology, Bernhard Nocht Institute for Tropical Medicine, Hamburg, Germany.
A S
AS
Hussein
R D
RD
Walter
D000818:Animals; D017165:Ascaris suum / Q000201:enzymology*; D019328:Buthionine Sulfoximine; D002850:Chromatography, Gel; D003538:Cystamine / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005721:Glutamate-Cysteine Ligase / Q000037:antagonists & inhibitors / Q000302:isolation & purification* / Q000378:metabolism; D005978:Glutathione / Q000096:biosynthesis; D015801:Helminth Proteins / Q000037:antagonists & inhibitors / Q000302:isolation & purification* / Q000378:metabolism; D007668:Kidney / Q000201:enzymology; D007700:Kinetics; D008717:Methionine Sulfoximine / Q000031:analogs & derivatives / Q000494:pharmacology; D008970:Molecular Weight; D051381:Rats; D013045:Species Specificity; D013379:Substrate Specificity
D004791:Enzyme Inhibitors; D015801:Helminth Proteins; D008717:Methionine Sulfoximine; D019328:Buthionine Sulfoximine; D005721:Glutamate-Cysteine Ligase; D005978:Glutathione; D003538:Cystamine