Two forms of glutamine synthetase in leaves of Cucurbita pepo

Abstract

It has been shown that the leaves of pumpkin ( Cucurbita pepo) contain two molecular forms of glutamine synthetase (GS), one occurring in the cytosol (GS 1)and the other in the chloroplasts (GS 2). The activities of both forms were greater when ammonium ion was infiltrated into the leaves and this was shown to be due to de novo synthesis. The two synthetases were purified by ammonium sulphate fractionation, ion exchange chromatography on DEAE-cellulose, selective adsorption on calcium phosphate gel, and preparative polyacrylamide gel electrophoresis. The MWs of GS 1 and GS 2, estimated by gel filtration on Sephacryl S-200, were 480 000 and 370 000 respectively. During polyacrylamide gel electrophoresis in the presence of SDS both GS 1 and GS 2 were dissociated into polypeptide chains with MWs of 58 000 and 50 000 respectively, suggesting that GS, 1 and GS 2 are octamers consisting of identical monomers. The synthetases showed noticeable differences in their amino acid composition. In GS 1 and GS 2 the proportions of α- helical segments were 34 and 17 % respectively. In the presence of Mg 2+, the pH optima for GS 1 and GS 2 were 7.25 and 7.75 respectively, and K m values toward l-glutamate were 13 and 46 mM respectively. From the experimental data it is inferred that GS 1 and GS 2 are isoenzymes.