Summary The redox interconversion of
Escherichia coli glutathione reductase has been studied both
in situ, with permeabilized cells treated with different reductants, and
in vivo, with intact cells incubated with compounds known to alter their intracellular redox state.The enzyme from toulene-permeabilized cells was inactivated
in situ by NADPH, NADH, dithionite, dithiothreitol, or GSH. The enzyme remained, however, fully active upon incubation with the oxidized forms of such compounds. The inactivation was time-, temperature-, and concentration-dependent; a 50% inactivation was promoted by just 2 M NADPH, while 700 M NADH was required for a similar effect. The enzyme from permeabilized cells was completely protected against redox inactivation by GSSG, and to a lesser extent by dithiothreitol, GSH, and NAD(P)
+. The inactive enzyme was efficiently reactivated
in situ by physiological GSSG concentrations. A significant reactivation was promoted also by GSH, although at concentrations two orders of magnitude below its physiological concentrations. The glutathione reductase from intact
E. coli cells was inactivated
in vivo by incubation with DL-malate, DL-isocitrate, or higher L-lactate concentrations. The enzyme was protected against redox inactivation and fully reactivated by diamide in a concentration-dependent fashion. Diamide reactivation was not dependent on the synthesis of new protein, thus suggesting that the effect was really a true reactivation and not due to
de novo synthesis of active enzyme. The glutathione reductase activity increased significantly after incubation of intact cells with tert-butyl or cumene hydroperoxides, suggesting that the enzyme was partially inactive within such cells. In conclusion, the above results show that both
in situ and
in vivo the glutathione reductase of
Escherichia coli is subjected to a redox interconversion mechanism probably controlled by the intracellular NADPH and GSSG concentrations.
相似文献