Figure 2.

mMical is a stereospecific monooxygenase that converts two conserved Met to Met sulfoxide residues in actin, which can be further reduced by MsrB. (A, B) Oxidation status of two conserved Met residues (Met46 and Met49 in rabbit muscle actin) was analyzed by mass spectrometry. The fraction of oxidized Met was calculated based on the ratio of Met sulfoxide residues to total Met and Met sulfoxide residues in the detected peptides of actin. (A) Actin was incubated with mMical1 (1 μM)/NADPH (200 μM) for 1 h and further treated with mMsrA (1 μM)/DTT (3 mM), mMsrB1 (10 μM)/DTT (3 mM), or mMsrB2 (1 μM)/DTT (3 mM) for 1 h (following buffer change with G-actin buffer). (B) Actin was incubated with mMical2 (1 μM)/NADPH (200 μM) for 3 h and further treated with mMsrA (1 μM)/DTT (3 mM) or mMsrB2 (1 μM)/DTT (3 mM) for 3 h following buffer change with free G-actin buffer. Other Met residues were not oxidized by Micals (three Met residues were not detected). Actin incubated with mMical1 (0.1, 0.5, 2 μM)/NADPH (200 μM) or H₂O₂ (1 mM) and the Mical (2 μM)/NADPH (200 μM)-treated actin incubated with mMsrA (1 μM) or mMsrB2 (1 μM) with DTT (3 mM) were subjected to (C) western blotting with the antibodies specific for the reduced form of actin. Membrane was also stained with Amido Black. Then, (D) the ratio of reduced to total actin was calculated based on the quantification of band density using ImageJ. All data were normalized to No treatment (control actin), and this experiment was independently repeated three times and statistically analyzed by Student's t-test (*: p < 0.05). Error bars represent SD. All experiments used 9.5 μM or 4.76 μM G-actin for further mass spectrometry and western blot analyses. See also Figure S2.