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Articles

Glutamine Synthetase of Germinating Peanuts ¹

PROPERTIES OF TWO CHROMATOGRAPHICALLY DISTINCT FORMS AND THEIR ACTIVITY TOWARD 4-METHYLENEGLUTAMIC ACID

Department of Biological Chemistry, The University of Michigan, Ann Arbor, Michigan 48109

Glutamine synthetase activity, extracted from an acetone powder of 7-day germinated peanuts (Arachis hypogaea L.), was precipitated by ammonium sulfate (40-60% saturation) and further purified by gel filtration and calcium phosphate gel treatment. When it was adsorbed to and subsequently eluted from a column of diethylaminoethyl-cellulose, two peaks of activity (designated glutamine synthetase 1 and 2) were obtained which were enriched 150- and 20-fold, respectively, over the initial extract. Glutamine synthetase 1 was present in ungerminated seeds and in the cotyledons during germination; glutamine synthetase 2 appeared during germination and was found largely in the developing plant. Compared with glutamine synthetase 2, glutamine synthetase 1 appeared to have a slightly smaller molecular weight and was more stable to heat and storage. The catalytic properties of the two forms were essentially the same. Whereas neither form catalyzed -glutamyltransferase activity with 4-methyleneglutamine, both glutamine synthetases 1 and 2 catalyzed an ATP- and NH₄⁺-dependent conversion of [¹⁴C]-4-methyleneglutamic acid to [¹⁴C]-4-methyleneglutamine, but the K_m value for 4-methyleneglutamic acid was 10-fold greater and the V_max only one-fourth that measured with L-glutamic acid. This is the first report of glutamine synthetase activity with 4-methyleneglutamic acid as substrate, although the level of this activity does not appear adequate to account for the rapid synthesis of 4-methyleneglutamine observed in germinating peanuts.

¹ Supported by grant 5901-0410-8-0164-0 from the Competitive Research Grants Office of the United States Department of Agriculture, Science and Education Administration, by United States Public Health Service Grant AM-03718 from the National Institutes of Health, and by a Faculty Research Grant from the H. H. Rackham School of Graduate Studies, The University of Michigan.