Studu On The Oligomeric State Of Glutamine Synthetase In Plants

Glutamine Synthetase(GS)is a key enzyme in nitrogen assimilation and recycling in plants.GS are classified as the cytosolic isoform(GS1)and the chloroplastic isoform(GS2)depend on different subcellular localization in higher plants.The most studies of GS assembly and crystal structure used the proteins heterologously expressed by X-ray crystallography or electron cryomicroscopy.In this study,a modified blue native PAGE(BNE)was used to separate plants GS isoforms with the in-gel detection of GS activity,which can estimate the native molecular weight of GS holoenzymes succinctly;separated by the 2-D SDS-PAGE gel and analysed by the western-blot,combined with the identification of LC-MS/MS,the subunits composition of GS isoenzymes were identified;and on the basis of the holoenzymes' molecular weight and the subunits composition,the oligomeric state of GS isoenzymes were identified.The GS from eight plants were identified by using this method,The predominant active form of GS1 is a decamer in Arabidopsis thaliana,Nicotiana tabacun,Medicago truncatula,Pisum sativum,Phaseolus valgaris,Orya sativa and Zea mays;whereas the GS1 in Triticum aestivum is a dodecamer.The predominant active form of GS2 is a decamer in Nicotiana tabacun,Medicago truncatula,Pisum sativum,Phaseolus valgaris and Zea mays;whereas the GS1 in Arabidopsis thaliana,Orya sativa and Triticum aestivum is a hexamer.Given our experiment data of Mt GS1 and Zm GS1 are in accordance with the previous results using X-ray crystallography,our technique is an efficient alternative method to study the native GS oligomeric structure in plants.Moreover,the GS1 isoenzyme with highly expressed showed an additional hexamer in C4 plant like Zea mays;the GS2 isoenzyme with highly expressed showed an additional hexamer in leguminous plants such as Medicago truncatula,Pisum sativum and Phaseolus valgaris.It's suggest that the differential post-translational regulation results in the diversity oligomeric state of GS isoenzymes on species,and in which maybe a way of the expression regulation of GS isoforms.