Assay, purification, properties and mechanism of action of γ-glutamylcysteine synthetase from the liver of the rat and Xenopus laevis

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Abstract
1. An improved radioassay for glutathione synthetase and γ-glutamylcysteine synthetase was developed. 2. Xenopus laevis liver γ-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 γ-glutamylcysteine synthetase was purified 11400-fold by a procedure similar to that employed for the Xenopus laevis enzyme. 4. Rat liver γ-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 γ-glutamylcysteine. The results for the rat liver enzyme are consistent with a Tri Tri sequential mechanism.