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. 2011 Jun 10;286(33):29098–29107. doi: 10.1074/jbc.M111.240127

FIGURE 1.

FIGURE 1.

O2˙̄ generated from uncoupled eNOS induces eNOS protein radical formation. a, EPR spin trapping of O2˙̄ generated by eNOS. The reaction system contained 50 mm Tris-HCl buffer, pH 7.4, 1 mm NADPH, 0.5 mm Ca2+, 10 μg/ml CaM, 15 μg/ml purified eNOS, and 25 mm spin trap DEPMPO. The spectra were recorded at room temperature with a microwave frequency of 9.863 GHz, 20 milliwatts of microwave power, and 1.0 G modulation amplitude. b, immunoblotting analysis of protein radical formation. Lanes 1–6, O2˙̄ is generated from BH4-free eNOS. Lane 1, BH4-free eNOS; lane 2, +10 μm Arg; lane 3, +1 mm Arg; lane 4, +1 mm l-NAME; lane 5, +Mn-SOD; lane 6, +10 μm BH4. Lanes 7 and 8, O2˙̄ is generated from nNOS reductase domain. Lane 7, BH4-free eNOS + nNOS reductase domain (nNOSR); lane 8, +Mn-SOD.