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By W. N. Aldridge (eds.)
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Extra resources for A Symposium on Mechanisms of Toxicity
8). The observation that glutamate decreases the rate at which methionine sulphoximine irreversibly inhibits the enzyme suggests that there are at least two steps in the inhibition process. Thus, glutamate appears to compete with methionine sulphoximine for attachment to the enzyme; attachment of methionine sulphoximine to the enzyme is followed by irreversible inhibition. The finding that ammonia (or hydroxylamine) plus glutamate protects against inhibition suggests that methionine sulphoximine can bind to the enzyme by attaching to both the glutamate and ammonia binding sites, essentially as a bifunctional reagent.
Furthermore, when the enzyme was preincubated with methionine sulphoximine in the presence of ATP and magnesium ions and then diluted and assayed, it was found to be inhibited. Many experiments in our laboratory have confirmed the observation that methionine sulphoximine in the presence of ATP and either manganese or magnesium ions irreversibly inactivates glutamine synthetase. There is an absolute requirement for ATP and one of these metal ions for irreversible inactivation. For example, 98-100% of the activity is inhibited when the enzyme is incubated at pH 7·2 and 37°Cfor 15 min with 5 x to-aM L-methionine-SR-sulphoximine, 0·01 M ATP, and 0·02 M magnesium chloride (or 0·002 M manganese chloride).
Of enzyme-synthesized and chemically synthesized methionine sulphoximine phosphate were found to be identical, as judged by paper chromatography in five solvent systems, paper electrophoresis at pH 2·8, 5·5, 7·5 and 9·0, relative stability at various values of pH and the rates of cleavage in 1 N at 100°C and by several phosphatases. Treatment of methionine sulphoximine phosphate with hydrosulphite gives a product with the properties of methionine sulphimine phosphate indicating that the phosphoryl moiety of methionine sulphoximine phosphate is attached to the sulphoximine nitrogen atom; possible structures of methionine sulphoximine phosphate are given in Fig.