Mixed-function oxidation of E. coli glutamine synthetase by ascorbate, oxygen, and iron has previously been shown to cause inactivation of the enzyme and enhanced susceptibility to proteolytic attack by a variety of proteases. One of these proteases, a high molecular weight nonlysosomal liver proteinase which does not degrade native glutamine synthetase at neutral pH, has been purified and characterized, and used to study the degradation of the oxidatively modified enzyme. In earlier studies only partial degradation of oxidatively inactivated glutamine synthetase was observed because only a fraction of the subunits had been rendered susceptible to proteolytic attack. Prolonged exposure to the ascorbate system results in more extensive susceptibility to degradation without causing a major conformational change. Study of the time course of exposure to the ascorbate system showed a lag before the increase in susceptibility to proteolysis, during which the enzyme was almost completely inactivated. It has been demonstrated that mixed-function oxidation causes a number of changes in the glutamine synthetase molecule and that these changes do not occur simultaneously. Moreover, different mixed-function oxidation systems may cause different effects on the protein and do not necessarily render the enzyme susceptible to degradation. Several mammalian enzymes have been shown, like glutamine synthetase, to be preferentially degraded after exposure to the ascorbate system but some apparently are not. Comparison of the results with the purified proteinase with those obtained by microinjection of control and oxidized 14C-labeled glutamine synthetase suggest that the increased proteolysis observed in vitro likely reflects susceptibility to degradation in in vivo.
