Purification and Characterization of Glutamine Synthetase Isoenzymes from Leaves and Roots of Brassica napus (L.)

Abstract

Summary The glutamine synthetase enzymes from leaves (GS2) and roots (GSR) of Brassica napus L. have been purified to homogeneity by the application of a three-stage isolation procedure comprising anion-exchange chromatography, adsorption by hydroxylapatite and gel filtration on Sephacryl 5-300 HR. The isoforms of the enzyme show a differential distribution in leaf and root tissues. Elution profiles of hydroxylapatite chromatography showed a distinct behaviour for GS proteins found in leaves and roots. Denaturing SDS-PAGE and Western blot experiments revealed molecular masses of approximately 43.5 and 40.5 kD for GS2 and GSR subunits, respectively. Moreover, kinetic properties determined using crude extracts confirmed different physiological roles for both GS enzymes, especially pH-optima and apparent Km-values. The affinity for glutamate was observed to be six times higher for GSR compared with GS2. Northern analysis of leaf and root RNA revealed two distinct GS mRNAs: a 1.6kb GS transcript was present in leaves, while a 1.4 kb GS mRNA was detected in roots. These results provide evidence for the predominant existence of a plastidic glutamine synthetase in leaves, whereas the roots of Brassica napus contain mainly cytosolic GS isoenzymes.