Detection, quantitation, purification, and identification of cardiac proteins S-thiolated during ischemia and reperfusion.
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.
Eaton
Philip
P
Centre for Cardiovascular Biology and Medicine, The Rayne Institute, St Thomas' Hospital, King's College London, United Kingdom. philip.eaton@kcl.ac.uk
Byers
Helen L
HL
Leeds
Nicola
N
Ward
Malcolm A
MA
Shattock
Michael J
MJ
D000154:Aconitate Hydratase / Q000378:metabolism; D000818:Animals; D019921:Biotinylation; D015153:Blotting, Western; D002851:Chromatography, High Pressure Liquid; D003545:Cysteine / Q000737:chemistry; D003599:Cytoskeleton / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism; D004229:Dithiothreitol / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D024601:Glyceraldehyde 3-Phosphate Dehydrogenase (NADP+) / Q000378:metabolism; D055551:HSP27 Heat-Shock Proteins; D006360:Heat-Shock Proteins; D007511:Ischemia; D007527:Isoenzymes / Q000737:chemistry; D008956:Models, Chemical; D009194:Myeloma Proteins / Q000378:metabolism; D009206:Myocardium / Q000378:metabolism; D009211:Myoglobin / Q000378:metabolism; D009363:Neoplasm Proteins / Q000737:chemistry; D009701:Nucleoside-Diphosphate Kinase / Q000378:metabolism; D018384:Oxidative Stress; D011485:Protein Binding; D011493:Protein Kinase C / Q000737:chemistry; D051571:Protein Kinase C-alpha; D054562:Protein Tyrosine Phosphatase, Non-Receptor Type 1; D017027:Protein Tyrosine Phosphatases / Q000737:chemistry; D051381:Rats; D015424:Reperfusion; D015427:Reperfusion Injury; D012685:Sepharose / Q000737:chemistry; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D019809:Streptavidin / Q000737:chemistry; D013347:Subcellular Fractions / Q000378:metabolism; D013438:Sulfhydryl Compounds / Q000737:chemistry / Q000378:metabolism*; D014305:Triose-Phosphate Isomerase / Q000378:metabolism
D055551:HSP27 Heat-Shock Proteins; D006360:Heat-Shock Proteins; C486114:Hspb1 protein, rat; D007527:Isoenzymes; D009194:Myeloma Proteins; D009211:Myoglobin; D009363:Neoplasm Proteins; D013438:Sulfhydryl Compounds; C085016:multiple myeloma M-proteins; D012685:Sepharose; D019809:Streptavidin; D024601:Glyceraldehyde 3-Phosphate Dehydrogenase (NADP+); D011493:Protein Kinase C; D051571:Protein Kinase C-alpha; D009701:Nucleoside-Diphosphate Kinase; D054562:Protein Tyrosine Phosphatase, Non-Receptor Type 1; D017027:Protein Tyrosine Phosphatases; C516610:Ptpn1 protein, rat; D000154:Aconitate Hydratase; D014305:Triose-Phosphate Isomerase; D003545:Cysteine; D004229:Dithiothreitol