NADPH dependent activation of microsomal glutathione transferase 1.
Microsomal glutathione transferase 1 (MGST1) can become activated up to 30-fold by several mechanisms in vitro (e.g. covalent modification by reactive electrophiles such as N-ethylmaleimide (NEM)). Activation has also been observed in vivo during oxidative stress. It has been noted that an NADPH generating system (g.s.) can activate MGST1 (up to 2-fold) in microsomal incubations, but the mechanism was unclear. We show here that NADPH g.s treatment impaired N-ethylmaleimide activation, indicating a shared target (identified as cysteine-49 in the latter case). Furthermore, NADPH activation was prevented by sulfhydryl compounds (glutathione and dithiothreitol). A well established candidate for activation would be oxidative stress, however we could exclude that oxidation mediated by cytochrome P450 2E1 (or flavine monooxygenase) was responsible for activation under a defined set of experimental conditions since superoxide or hydrogen peroxide alone did not activate the enzyme (in microsomes prepared by our routine procedure). Actually, the ability of MGST1 to become activated by hydrogen peroxide is critically dependent on the microsome preparation method (which influences hydrogen peroxide decomposition rate as shown here), explaining variable results in the literature. NADPH g.s. dependent activation of MGST1 could instead be explained, at least partly, by a direct effect observed also with purified enzyme (up to 1.4-fold activation). This activation was inhibited by sulfhydryl compounds and thus displays the same characteristics as that of the microsomal system. Whereas NADPH, and also ATP, activated purified MGST1, several nucleotide analogues did not, demonstrating specificity. It is thus an intriguing possibility that MGST1 function could be modulated by ligands (as well as reactive oxygen species) during oxidative stress when sulfhydryls are depleted.
Rinaldi
Rosanna
R
Division of Biochemical Toxicology, Institute of Environmental Medicine, Karolinska Institutet, Nobels Vg 13, Box 210, S-171 77 Stockholm, Sweden.
Aniya
Yoko
Y
Svensson
Richard
R
Eliasson
Erik
E
Swedmark
Stellan
S
Shimoji
Miyuki
M
Morgenstern
Ralf
R
D000818:Animals; D004229:Dithiothreitol / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005033:Ethylmaleimide / Q000378:metabolism; D005978:Glutathione / Q000494:pharmacology; D005982:Glutathione Transferase / Q000096:biosynthesis*; D006861:Hydrogen Peroxide / Q000494:pharmacology; D008297:Male; D008862:Microsomes, Liver / Q000187:drug effects / Q000201:enzymology*; D009249:NADP / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D013481:Superoxides / Q000494:pharmacology
D004791:Enzyme Inhibitors; D013481:Superoxides; D009249:NADP; D006861:Hydrogen Peroxide; C122107:microsomal glutathione S-transferase-I; D005982:Glutathione Transferase; D005978:Glutathione; D005033:Ethylmaleimide; D004229:Dithiothreitol