Abstract
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.
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Acknowledgments
We would like to acknowledge support from the Medical Research Council, the British Heart Foundation, the Leducq Foundation, and the Department of Health via the NIHR cBRC award to Guy’s and St Thomas’ NHS Foundation Trust. Also JR Burgoyne is supported by a Sir Henry Wellcome postdoctoral fellowship from The Wellcome Trust (sponsor reference 085483/Z/08/Z).
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Burgoyne, J.R., Eaton, P. (2013). Approaches for Monitoring PKG1α Oxidative Activation. In: Krieg, T., Lukowski, R. (eds) Guanylate Cyclase and Cyclic GMP. Methods in Molecular Biology, vol 1020. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-459-3_10
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DOI: https://doi.org/10.1007/978-1-62703-459-3_10
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