Accessibility of Myofilament Cysteines and Effects on ATPase Depend on the Activation State during Exposure to Oxidants
Figure 4
Fluorescent labels identify cysteines as accessible to solvent in the filament lattice.
A. Rat EDL myofibrils were treated with one of three cysteine-specific fluorescent reagents, TMR, BFM or IAF. The myofibril proteins were then denatured and separated on an SDS PAGE gel. The gel was then scanned twice, at excitation and emission wavelengths appropriate to TMR or IAF and BFM. The gel was next stained overnight with Sypro, and scanned at excitation and emission wavelengths appropriate to Sypro. B. TMR scan of a SDS-PAGE gel of myofibrils treated with 100 uM TMR for 5 minutes at pH 6.2, 7.0, or 8.0. The actin bands from the three lanes stained with Sypro are displayed below the TMR scan as a loading control. C. Left: SDS-PAGE gel of rat EDL myofibrils treated for 5 minutes with a range of TMR concentrations (0–1 mM) and scanned using the TMR excitation emission settings. Right: The same gel was then stained with Sypro and scanned at Sypro frequencies.
