Proteomic analysis of rat heart in ischemia and ischemia-reperfusion using fluorescence two-dimensional difference gel electrophoresis.
Ischemia-reperfusion injury is a major complication occurring in acute myocardial infarction, cardiopulmonary bypass surgery, and heart transplantation. The aim of this study was to identify proteins that were involved in ischemia-reperfusion injury using fluorescence two-dimensional difference gel electrophoresis. We compared the 100,000 x g precipitate fractions of normal, ischemic and ischemia-reperfused rat hearts and detected six spots which changed more than two-fold in expression level and two additional spots related to these spots. Using peptide mass fingerprinting by matrix-assisted laser desorption/ionization-time of flight mass spectrometry, we identified five of these spots as protein disulfide isomerase A3 (PDA3), one as 60 kDa heat shock protein (HSP60) and two as elongation factor Tu (EF-Tu). HSP60 was increased during ischemia and decreased to normal expression level after reperfusion. EF-Tu was increased in ischemia but not decreased by reperfusion. We also found that several protein spots of PDA3 shifted towards a higher isoelectric point in ischemia and ischemia-reperfusion. Our data strongly suggested that PDA3 underwent dephosphorylation during ischemia and reperfusion and serine 343 of PDA3 was one of the phosphorylation sites.
Sakai
Jun
J
Genomic Science Laboratories, Sumitomo Pharmaceuticals, 2-1 Takatsukasa 4-Chome, Takarazuka, Hyogo 665-0051, Japan. sakai@sumitomopharm.co.jp
Ishikawa
Hironori
H
Kojima
Shinichi
S
Satoh
Hideshi
H
Yamamoto
Setsuko
S
Kanaoka
Masaharu
M
D000818:Animals; D001665:Binding Sites; D002232:Carbocyanines / Q000737:chemistry; D018834:Chaperonin 60 / Q000378:metabolism; D019992:Databases as Topic; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods*; D005456:Fluorescent Dyes / Q000494:pharmacology; D007091:Image Processing, Computer-Assisted; D007511:Ischemia; D008297:Male; D008856:Microscopy, Fluorescence / Q000379:methods*; D009206:Myocardium / Q000378:metabolism*; D010444:Peptide Elongation Factor Tu / Q000378:metabolism; D010766:Phosphorylation; D019704:Protein Disulfide-Isomerases / Q000737:chemistry; D020543:Proteome; D051381:Rats; D017208:Rats, Wistar; D015427:Reperfusion Injury / Q000473:pathology*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013347:Subcellular Fractions
D002232:Carbocyanines; D018834:Chaperonin 60; D005456:Fluorescent Dyes; D020543:Proteome; C089187:cyanine dye 3; C085321:cyanine dye 5; D010444:Peptide Elongation Factor Tu; D019704:Protein Disulfide-Isomerases