Cell Death by SecTRAPs: Thioredoxin Reductase as a Prooxidant Killer of Cells
Figure 5
Both SecTRAPs and TrxR1 are efficient in reducing juglone and thereby produce superoxide.
In (A) the formation of FAD-reduced disulfide charge transfer complex by NADPH (80 µM) in a SecTRAP preparation (truncated TrxR1, 16 µM subunit) and full-length TrxR1 (12 µM subunit) was analyzed with stopped-flow spectroscopy at 540 nm, showing similar kinetics for both enzymes. In (B) Michaelis-Menten kinetics for both full-length (filled symbols) and truncated (open symbols) TrxR1 using juglone as a substrate is demonstrated. In (C) it is shown that Trx1 and juglone compete for the reduction by full-length TrxR1 (filled bars) but that truncated TrxR1 (open bars) can only use juglone and not Trx1 as a substrate. This is illustrated from the initial NADPH consumption rate (0–200s) followed at 340 nm with or without Trx1 and insulin (upper panels). After 30 min of reaction, the number of exposed free thiols was determined (lower panels) in order to estimate to which extent the electrons from the NADPH oxidation were passed on to Trx1 and subsequently to insulin. The juglone concentration is indicated at the x-axes and each bar represents the mean±S.D. of three measurements. In (D), the reduction of juglone (5 µM) catalyzed by 10 nM SecTRAP (truncated TrxR1, left panel) or full-length TrxR1 (right panel) is shown following the consumption of NADPH (initial concentration 250 µM) by the decrease in absorbance at 340 nm (open symbols). Concomitantly, superoxide formation was detected at 480 nm with the adrenochrome method using 2 mM epinephrine (filled symbols). The formation of adrenochrome was completely inhibited by addition of 5 U SOD (circles), which also reduced the elevated NADPH consumption seen upon addition of only epinephrine (squares).