Purchase this article with an account.
A. L. Kloosterboer, J. R. Ribich, L. A. Levin; Identification of Protein Targets of Novel Neuroprotective Sulfhydryl Reducing Drugs. Invest. Ophthalmol. Vis. Sci. 2009;50(13):3190.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
Previous work in our laboratory has shown elevated intracellular superoxide is a critical event in the death of retinal ganglion cells (RGCs) after axotomy. The sulfhydryl-reducing compounds tris(2-carboxyethyl) phosphine (TCEP) and bis(3-propionic acid methyl ester) phenylphosphine borane complex (PB1) rescue RGCs from apoptosis. We used a novel two-dimensional gel electophoresis method to detect changes in oxidation state of protein sulfyhydryls after treatment with PB1 in a transformed retinal ganglion cell-like cell line (RGC-5).
Cultured RGC-5 cells were treated with 10 µM PB1 for 8 hours following which cellular and mitochondrial proteins were separated on a 4-20% polyacrylamide gradient gel. The lane was excised, reduced with 25 mM dithiothreitol, rinsed, and alkylated with 100 mM iodoacetamide. The excised slice was placed horizontally on a second gel, electrophoresed into the second dimension, and stained with Pierce® color silver stain or Sypro® Ruby.
Several proteins were found in consistent locations below the diagonal when prepared from control cells, but not cells treated with PB1, indicating that they contained disulfides that were reduced by PB1.
A relatively small number of cellular and mitochondrial proteins are targets for the novel reducing agent PB1, and one or more may be critical for its activity as a neuroprotective agent.
This PDF is available to Subscribers Only