PROPERTIES OF THE GLUTAMINE-SYNTHETASE ISOFORMS ISOLATED FROM SUNFLOWER LEAVES

Glutamine synthetase (GS) from sunflower leaves was very unstable in v itro. The decay of GS activity was prevented by the presence of thiols and protease inhibitors in the extraction buffer. Properties of cytos olic (GS(1)) and chloroplastic (GS(2)) isoforms were studied after sep aration by ion-exchange chromatography. Transferase assay pH optima we re 7.0 for GS(1), and 7.5 for GS(2). Affinities of GS(2) for L-glutami ne and hydroxylamine were somewhat higher than those of GS(1). The act ivity of both isoforms was absolutely dependent on Mn2+, but GS(2) req uired a much lower Mn2+ concentration. GS(1) exhibited maximum activit y at 47 degrees C, with an activation energy of 37 kJ mol(-1) and a Q( 10) of 1.63. Maximum activity of GS(2) was shown at 43 degrees C, with an activation energy of 43 kJ mol(-1) and a Q(10) of 1.76. GS(2) was much more heat-labile than GS(1). The presence of Mn2+-ADP greatly pro tected the isoenzyme against thermal denaturation. Both isoforms were inactivated by p-hydroxymercuribenzoate and subsequently reactivated b y a short preincubation with dithioerythritol. Nevertheless, GS(1) sho wed a higher sensitivity to thiol reagents.