Pyruvate kinase is protected by glutathione-dependent redox balance in human red blood cells exposed to reactive oxygen species.
To determine the antioxidant role of glutathione (GSH) in human red blood cells (RBCs), we investigated the effect of disrupting GSH homeostasis on the oxidative modification of thiol-dependent enzymes by exposure to tert-butyl hydroperoxide (BHP). When hemolysate was incubated with BHP, significant decreases in enzyme activity were observed. However, the inactivation did not occur in intact RBC suspensions that were exposed to BHP. In this study, we used two independent treatments aimed at decreasing the level of reduced form of GSH, pre-incubation with a glutathione reductase inhibitor or glucose-free medium to examine the influences of preventing GSH-dependent antioxidant and reactivation activity on thiol-dependent enzyme. Pyruvate kinase (PK) activity clearly decreased along with depletion of GSH compared to other glycolytic enzyme activities by BHP exposure in RBCs. The addition of GSH, but not glucose, before BHP exposure completely prevented the inactivation of PK in hemolysate; however, partial reactivation of inactivated PK was observed by post-addition of both GSH and glutaredoxin at an early stage during BHP exposure. Moreover, hydroxyl radicals but not hydrogen peroxide inactivated PK. These results suggest that PK is highly susceptible to radicals and that GSH is essential to protect PK activity by not only directly scavenging radicals but also by systematically reactivating oxidized enzyme in human RBCs.
Ogasawara
Yuki
Y
Department of Environmental Biology, Meiji Pharmaceutical University, 2-522-1 Noshio, Kiyose, Tokyo 204-8588,Japan. yo@my-pharm.ac.jp
Funakoshi
Masayo
M
Ishii
Kazuyuki
K
D000818:Animals; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004912:Erythrocytes / Q000378:metabolism*; D054477:Glutaredoxins / Q000378:metabolism; D005978:Glutathione / Q000502:physiology*; D006706:Homeostasis; D006801:Humans; D017665:Hydroxyl Radical / Q000378:metabolism; D066298:In Vitro Techniques; D018482:Muscle, Skeletal / Q000187:drug effects / Q000378:metabolism; D010084:Oxidation-Reduction; D011770:Pyruvate Kinase / Q000037:antagonists & inhibitors / Q000097:blood*; D011817:Rabbits; D017382:Reactive Oxygen Species / Q000633:toxicity*; D020122:tert-Butylhydroperoxide / Q000494:pharmacology
D004791:Enzyme Inhibitors; D054477:Glutaredoxins; D017382:Reactive Oxygen Species; D017665:Hydroxyl Radical; D020122:tert-Butylhydroperoxide; D011770:Pyruvate Kinase; D005978:Glutathione