PURIFICATION AND PROPERTIES OF LUPIN NODULE GLUTAMINE-SYNTHETASE

Glutamine synthetase (GS) from the cytoplasm of L. luteus nodules was purified to apparent homogeneity using a final step of ADP-Sepharose affinity chromatogrpahy. Mercaptoethanol and divalent metals were essential to maintain the enzyme activity and keto compounds enhanced the stability during purification. From gel filtration a Mr for the native enzyme of 347,000 was determined with subunits of 41,500 indicated by SDS-PAGE. The pH optima for the biosynthetic and transferase activities were 7.9 and 6.5 respectively. Mg2+-activated GS was strongly inhibited by Mn2+ and Ca2+; Co2+, while also inhibitory, allowed an alternate, more active form of GS after addition of glutamate. Activity was also inhibited by possible feedback inhibitors. The apparent Km values for glutamate, NH4+ ATP, glutamine, NH2OH and ADP were 8.58 mM, 12.5 .mu.M, 0.22 mM, 48.6 mM, 3.37 mM and 59.7 nM respectively.