Detection, Quantitation, Purification, and Identification of Cardiac Proteins S-thiolated During Ischemia and Reperfusion

J Biol Chem. 2002 Mar 22;277(12):9806-11. doi: 10.1074/jbc.M111454200. Epub 2002 Jan 2.

Abstract

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.

MeSH terms

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

Substances

  • HSP27 Heat-Shock Proteins
  • Heat-Shock Proteins
  • Hspb1 protein, rat
  • Isoenzymes
  • Myeloma Proteins
  • Myoglobin
  • Neoplasm Proteins
  • Sulfhydryl Compounds
  • multiple myeloma M-proteins
  • Sepharose
  • Streptavidin
  • Glyceraldehyde 3-Phosphate Dehydrogenase (NADP+)
  • Protein Kinase C
  • Protein Kinase C-alpha
  • Nucleoside-Diphosphate Kinase
  • Protein Tyrosine Phosphatase, Non-Receptor Type 1
  • Protein Tyrosine Phosphatases
  • Ptpn1 protein, rat
  • Aconitate Hydratase
  • Triose-Phosphate Isomerase
  • Cysteine
  • Dithiothreitol