Purification and properties of glutathione peroxidase

Abstract

Glutathione peroxidase catalyses the decomposition of a wide range of hydroperoxides. In the present work this enzyme was purified approximately 2,500-fold from pig's blood. -- The kinetics of glutathione peroxidase were shown to be rather complex. Using glutathione as substrate, a linear Lineweaver-Burk plot was obtained with an approximate Km value of 3 mM. -- Using cumene hydroperoxide as substrate kinetic analysis on the purified enzyme gave non-linear Lineweaver-Burk plots. -- A wide range of nucleotides inhibited the enzyme, pyridine nucleotides exerting the strongest inhibition. Also, inhibitory effectiveness increased with the number of phosphate groups in the nucleotide. Nucleotide inhibition was competitive with respect to GSH and of a mixed nature with respect to hydroperoxides. In addition, increased levels of hydroperoxides enhanced nucleotide inhibition. -- The inhibitory action of nucleotides on enzyme activity could be substantially decreased by x-ray, ethanol or trypsin treatment with a lesser decrease of catalytic activity. Conversely, heat, pCMB, or sodium lauryl sulfate preferentially abolished the catalytic function with a lesser effect on the nucleotide inhibition. -- It was concluded that nucleotides interact with the enzyme at a site other than the active center and hence that glutathione peroxidase is an allosteric enzyme.