Dtt

The close association that has been observed between GSH-dependent, diamide-induced PKCa inactivation and PKCa S-glutathiolation indicates that PKCa S-glutathiolation is the cause of the inactivation.4 PKCa S-glutathiolation can be detected by nonreducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) as the diamide-induced and DTT-reversible covalent incorporation of [35S]GSH into the isozyme.4 This is a broadly applicable approach that can be used to investigate whether any PKC isozyme or other purified protein of interest is subject to S-glutathiolation. In some cases, S-glutathiolation of a protein may produce a shift in its migration position in nonreducing SDS-PAGE; this is true of recombinant human PKCa, which shifts from an apparent molecular weight of 82,000 to 88,000.4 If a measurable shift in apparent molecular weight is detected for an 35S-glutathiolated protein by nonreducing SDS-PAGE, then nonreducing Western analysis can be used as a nonradioactive method for routine monitoring of the S-glutathiolation modification. However, because a comparable shift in the migration position could, in principle, be produced by intramolecular disulfide bridge formation, it is important to first establish through [35S]GSH labeling

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