The purification and properties of the glutamine synthetase from the cytosol of Soya-bean root nodules

Abstract

The major portion of glutamine synthetase [EC-6.3.1.2] activity in root nodules of soy-bean [Glycine max] plants is associated with the cytosol rather than with Rhizobium joponicum bacteriods. Glutamine synthetase accounts for about 2% of the total soluble protein in the nodule cytosol. Glutamine synthetase from nodule cytosol was purified by a procedure involving fractionation with protamine sulfate, ammonium sulfate and polypropylene glycol, chromatography on DEAE-Bio-Gel A and Bio-Gel A-5 m and affinity chromatography on glutamateagarose columns. The purified preparation appeared to be homogeneous in the analytical ultracentrifuge. From sedimentation-equilibrium experiments a molecular weight of about 376,000 was determined for the native enzyme and 47,300 for the enzyme in guanidinium chloride. From these data and measurements of electron micrographs, it was concluded that glutamine synthetase from nodule cytosol consists of 8 subunits arranged in 2 sets of planar tetramers which form a cubical configuration with dimensions of about 10 nm (100 .ANG.) across each side. Glutamine synthetase from nodule cytosol has a higher glycine and proline content and a lower content of phenylalanine than the glutamine synthetase that has been prepared from pea seed. The cytosol enzyme contains 4 half-cystine molecules per subunit, which is in contrast with 2 reported for the enzyme from pea seed. Enzyme activity is strikingly influenced by the relative proportion of Mg2+ and Mn2+ in the assay medium. Activity is inhibited by feedback inhibitors and is influenced by energy charge.