Approaches for Monitoring PKG1 Oxidative Activation

cGMP-dependent protein kinase, also known as protein kinase G (PKG), is activated independently of cGMP by a novel thiol-reactive mechanism involving the formation of an intermolecular disulfide. This oxidative modification within PKG is generally not detected by conventional Western immunoblot analysis due to the experimental conditions used. Here, we describe the proteomic approach that lead to PKG being identified as a kinase susceptible to oxidant-dependent disulfide dimer formation, these methods being applicable for the identification of other disulfide bound protein complexes. In addition a nonreducing Western immunoblot method for routinely measuring PKG oxidation in complex protein mixtures generated from cell lysates or tissue homogenates is also described.