Mechanism of activation of rat liver microsomal glutathione transferase by noradrenaline and xanthine oxidase.
Activation of glutathione transferase activity in rat liver microsomes under a variety of conditions producing oxidative stress was investigated. Neither hydrogen peroxide (10 mM) (added or produced endogenously by glucose + glucose oxidase) nor duroquinone together with an NADPH-regenerating system (which generates the superoxide anion radical) had any significant effect on the glutathione transferase activity towards 1-chloro-2,4-dinitrobenzene. On the other hand, incubation of microsomes with 1 mM noradrenaline (which autooxidizes and generates superoxide anion radical) gave a 160% activation, as shown earlier (Aniya and Anders, J Biol Chem 264: 1998-2002, 1989). This was taken as an indication that microsomal glutathione transferase could be activated by oxidative stress. Here, we demonstrate that activation by this compound is due to covalent binding (presumably of the quinone formed during autooxidation). The xanthine/xanthine oxidase system, which generates the superoxide anion radical and hydrogen peroxide, increases microsomal glutathione transferase activity, but this activation was not dependent on the presence of xanthine. Western blots of microsomes treated with xanthine oxidase revealed that activation was due to proteolysis (presumably by contaminating proteases in the xanthine oxidase). In conclusion, there is no firm evidence that rat liver microsomal glutathione transferase is activated directly by reduced oxygen species in the microsomal system. The possibility remains that oxidative stress triggers secondary mechanisms such as generation of reactive intermediates and/or activation of proteolysis, which can in turn increase enzyme activity.
Lundqvist
G
G
Department of Toxicology, Karolinska Institutet, Stockholm, Sweden.
Morgenstern
R
R
D000818:Animals; D016227:Benzoquinones / Q000494:pharmacology; D004137:Dinitrochlorobenzene / Q000378:metabolism; D004229:Dithiothreitol / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects; D005982:Glutathione Transferase / Q000378:metabolism*; D008297:Male; D008862:Microsomes, Liver / Q000187:drug effects* / Q000201:enzymology; D009638:Norepinephrine / Q000494:pharmacology*; D010084:Oxidation-Reduction; D051381:Rats; D011919:Rats, Inbred Strains; D014969:Xanthine Oxidase / Q000494:pharmacology*
D016227:Benzoquinones; D004137:Dinitrochlorobenzene; D014969:Xanthine Oxidase; D005982:Glutathione Transferase; D004229:Dithiothreitol; C034257:duroquinone; D009638:Norepinephrine