Purification and properties of glutamine synthetase from spinach leaves

Abstract

The chloroplastic glutamine synthetase of spinach leaves has been purified to homogeneity using affinity chromatography. This involves a tandem ;reactive blue A-agarose' and ;reactive red-A-agarose' as the final step in the procedure. This procedure results in a yield of 18 milligrams of pure glutamine synthetase per kilogram of starting material. The purity of our enzyme has been demonstrated on both one- and two-dimensional polyacrylamide gels.Purified glutamine synthetase has a molecular weight of 360,000 daltons and consists of eight 44,000 dalton subunits. The K(m) is 6.7 millimolar for glutamate, 1.8 millimolar for ATP (synthetase assay), and 37.6 millimolar for glutamine (transferase assay). The isoelectric point is 6.5 and the pH optima are 7.3 in the synthetase assay and 6.4 in the transferase assay. The irreversible, competitive inhibitors methionine sulfoxamine and phosphinothricin have K(i) values of 0.1 millimolar and 6.1 micromolar, respectively. Amino acid analysis has been carried out and the results compared with published analyses for other isoforms of glutamine synthetase.