Assay, purification, properties and mechanism of action of gamma-glutamylcysteine synthetase from the liver of the rat and Xenopus laevis.
1. An improved radioassay for glutathione synthetase and gamma-glutamylcysteine synthetase was developed. 2. Xenopus laevis liver gamma-glutamylcysteine synthetase was purified 324-fold by saline-bicarbonate extraction, protamine sulphate precipitation, CM-cellulose and DEAE-cellulose column chromatography, and gel filtration. 3. Rat liver gamma-glutamylcysteine synthetase was purified 11400-fold by a procedure similar to that employed for the Xenopus laevis enzyme. 4. Rat liver gamma-glutamylcysteine synthetase activity was inhibited by GSH and activated by glycine. These effects, which were not found in the enzyme from Xenopus laevis, may have a regulatory significance. 5. Isotope-exchange experiments revealed fundamental differences in the partial reactions catalysed by the rat and Xenopus laevis synthetases. The enzyme from Xenopus laevis appears to follow a Bi Bi Uni Uni Ping Pong mechanism, with glutamyl-enzyme as intermediate before the addition of cysteine and the release of gamma-glutamylcysteine. The results for the rat liver enzyme are consistent with a Tri Tri sequential mechanism.
D000818:Animals; D001001:Anura; D001639:Bicarbonates; D002250:Carbon Radioisotopes; D002458:Cell Fractionation; D011232:Chemical Precipitation; D002848:Chromatography, DEAE-Cellulose; D002850:Chromatography, Gel; D003545:Cysteine; D005973:Glutamine; D005978:Glutathione; D005998:Glycine; D007474:Ion Exchange; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D010453:Peptide Synthases / Q000302:isolation & purification*; D011479:Protamines; D051381:Rats; D012965:Sodium Chloride; D013462:Sulfur Radioisotopes; D014981:Xenopus
D001639:Bicarbonates; D002250:Carbon Radioisotopes; D011479:Protamines; D013462:Sulfur Radioisotopes; D005973:Glutamine; D012965:Sodium Chloride; D010453:Peptide Synthases; D005978:Glutathione; D003545:Cysteine; D005998:Glycine