sepharose and Reperfusion-Injury

We have developed methods that allow detection, quantitation, purification, and identification of cardiac proteins S-thiolated during ischemia and reperfusion. Cysteine was biotinylated and loaded into isolated rat hearts. During oxidative stress, biotin-cysteine forms a disulfide bond with reactive protein cysteines, and these can be detected by probing Western blots with streptavidin-horseradish peroxidase. S-Thiolated proteins were purified using streptavidin-agarose. Thus, we demonstrated that reperfusion and diamide treatment increased S-thiolation of a number of cardiac proteins by 3- and 10-fold, respectively. Dithiothreitol treatment of homogenates fully abolished the signals detected. Fractionation studies indicated that the modified proteins are located within the cytosol, membrane, and myofilament/cytoskeletal compartments of the cardiac cells. This shows that biotin-cysteine gains rapid and efficient intracellular access and acts as a probe for reactive protein cysteines in all cellular locations. Using Western blotting of affinity-purified proteins we identified actin, glyceraldehyde-3-phosphate dehydrogenase, HSP27, protein-tyrosine phosphatase 1B, protein kinase Calpha, and the small G-protein ras as substrates for S-thiolation during reperfusion of the ischemic rat heart. MALDI-TOF mass fingerprint analysis of tryptic peptides independently confirmed actin and glyceraldehyde-3-phosphate dehydrogenase S-thiolation during reperfusion. This approach has also shown that triosephosphate isomerase, aconitate hydratase, M-protein, nucleoside diphosphate kinase B, and myoglobin are S-thiolated during post-ischemic reperfusion.

Topics: Aconitate Hydratase; Animals; Biotinylation; Blotting, Western; Chromatography, High Pressure Liquid; Cysteine; Cytoskeleton; Cytosol; Dithiothreitol; Electrophoresis, Polyacrylamide Gel; Glyceraldehyde 3-Phosphate Dehydrogenase (NADP+); Heat-Shock Proteins; HSP27 Heat-Shock Proteins; Ischemia; Isoenzymes; Models, Chemical; Myeloma Proteins; Myocardium; Myoglobin; Neoplasm Proteins; Nucleoside-Diphosphate Kinase; Oxidative Stress; Protein Binding; Protein Kinase C; Protein Kinase C-alpha; Protein Tyrosine Phosphatase, Non-Receptor Type 1; Protein Tyrosine Phosphatases; Rats; Reperfusion; Reperfusion Injury; Sepharose; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Streptavidin; Subcellular Fractions; Sulfhydryl Compounds; Triose-Phosphate Isomerase