Reaktivierung von Ammonium‐inaktivierter Glutaminsynthetase in Escherichia coli

It is known from previous work that the regulation of Glutaminesynthetase in Escherichia coli is effected by two mechanisms. First, the synthesis of GS is repressed by NH4+; and secondly, the activity of GS is regulated by inactivation and reactivation of the enzyme molecule. In the present paper we demonstrate and characterize the reactivation of inactivated GS in intact cells of E. coli. GS is inactivated after addition of NH4+ to the culture medium. Experiments with a cell free extract from E. coli have shown, that the inactivation is mediated by a specific “inactivating enzyme”, ATP, Mg2+, and Glutamine which is probably formed from the added NH4+. The reactivation is observed, when the inactivated cells are centrifuged from the NH4+ containing medium and then resuspended in an NH4+ free medium. In about 5–7 minutes the activity increases 5–30‐fold. Inhibitors of protein synthesis such as chloramphenicol and p‐fluoro‐phenylalanine do not affect the reactivation proces. Only a slight retardation of the reactivation is observed. A carbon source is required for reactivation. When inactivated cells are incubated in a medium containing only salts and buffer no reactivation is observed. Probably the carbon source is metabolized to form intermediates and energy, which are needed for glutamine catabolizing reactions or for prevention of glutamine synthesis. Reactivation does not take place in the presence of NH4+i.e. under inactivating conditions. However if inactivation is inhibited by fluoride, reactivation can be observed in the presence of NH4+. When inactivated cells are incubated with fluoride only, without a carbon source in the reactivation medium, a reactivation is found. Inactivation as well as reactivation are inhibited by thiol‐group reagents. From these results we conclude that: (a) Reactivation is independent of protein synthesis; (b) Reactivation does not require energy; (c) Probably thiol‐groups are involved in the reactivation process.