Click on image to view larger version.
Figure 2. Mechanisms of glutathiolation of actin in vitro. A) ESI-mass spectra of rabbit skeletal muscle incubated with GSNO (a), GSSG (b), or NOC-22 (c). Purified rabbit skeletal muscle actin (7 µM) was incubated with 1 mM of the indicated reagents, desalted, and analyzed by ESI/MS. B) Western blots of actin incubated with the indicated concentrations of GSNO and GSSG using anti-PSSG antibodies. C) Immunoblot analysis of glutathiolated actin. Purified actin was incubated with NOC-22, and after desalting, nitrosylated actin was incubated without (+NOC-22) or with GSH (NOC-22+GSH). As a control, naive actin was incubated with GSH. Samples were treated with 50 mM NEM before electrophoresis and Western blotting with the anti-PSSG Ab.
