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

Abstract

An improved radioassay for glutathione synthetase and γ glutamylcysteine synthetase was developed. 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. Rat liver γ glutamylcysteine synthetase was purified 11400 fold by a procedure similar to that employed for the Xenopus laevis enzyme. 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. Isotope exchange experiments revealed fundamental differences in the partial reactions catalysed by the rat and Xenopus laevis synthetase. 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.