FIGURE 5.

H₂O₂ exposure and Cys³¹⁸–Cys³²⁶ disulfide bond formation impacts the AMP-dependent activation of bGP. A, bGP was incubated with 250 μm H₂O₂ for 30 min at 37 °C. The oxidized enzyme was then incubated with AMP or phosphorylase kinase prior to activity measurement. Phos. Ser, phosphorylated serine. Results are expressed as the percentage of the control. Data represent mean values of three independent experiments ± S.D., ***, p < 0.001 when compared with control; ###, p < 0.001 when two non-control groups are compared. B, analysis of the binding of AMP to reduced and oxidized bGP was performed by incubating the treated or untreated enzyme with 5 μm fluorescent mant-AMP. To ascertain that the fluorescence was due to binding of mant-AMP to the enzyme, increasing concentrations of AMP were added. The addition of increasing concentrations of AMP resulted in a loss of fluorescence. A. U., arbitrary units.