Binding of cytosolic proteins to myofibrils in ischemic rat hearts.
Myofibrillar proteins (MPs) were extracted from isolated and perfused rat hearts subjected to different periods of ischemia to investigate the occurrence of protein degradation and/or the association of cytosolic proteins with the myofibrillar pellet. A 23-kD band was detected by SDS-PAGE of MPs after 5 minutes of ischemia, with its density gradually increasing to a plateau after 20 minutes. Longer periods of ischemia were associated with the appearance of a 39-kD band. Irrespective of the duration of ischemia, both these bands persisted during reperfusion. A partial proteolytic degradation of troponin T (TnT) and troponin I (TnI) has been claimed to be responsible for the generation of these peptides. However, the N-terminal sequence of the 39-kD band was identical to that of GAPDH, whereas Edman sequencing after pepsin digestion showed that the 23 kD is alpha B-crystallin. The binding of the two cytosolic proteins to myofibrils was confirmed by immunofluorescence analysis on cryosections of ischemic hearts. In vitro studies showed that acidosis was sufficient to induce the binding of alpha B-crystallin, whereas the inhibition of ATP depletion prevented the binding of GAPDH. Thiol oxidation is unlikely to promote GAPDH binding, since perfusion with iodoacetate under aerobic conditions or treatment of homogenates with N-ethylmaleimide or diamide failed to induce GAPDH association with the myofibrils. These changes of the myofibrillar proteins could be considered as intracellular markers of the evolution of the ischemic damage. In addition, the binding of the 23-kD peptide might be involved in alterations of contractility.
Barbato
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R
Dipartimento di Chimica Biologica e Centro per lo Studio delle Biomembrane, Università di Padova, Italy.
Menabò
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Dainese
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P
Carafoli
E
E
Schiaffino
S
S
Di Lisa
F
F
D000595:Amino Acid Sequence; D000818:Animals; D003459:Crystallins / Q000378:metabolism; D003600:Cytosol / Q000737:chemistry*; D004591:Electrophoresis, Polyacrylamide Gel; D005455:Fluorescent Antibody Technique; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000235:genetics / Q000378:metabolism; D015151:Immunoblotting; D066298:In Vitro Techniques; D008297:Male; D008969:Molecular Sequence Data; D009124:Muscle Proteins / Q000378:metabolism*; D017202:Myocardial Ischemia / Q000378:metabolism*; D015425:Myocardial Reperfusion; D009206:Myocardium / Q000378:metabolism*; D009210:Myofibrils / Q000378:metabolism*; D011485:Protein Binding; D051381:Rats; D017208:Rats, Wistar
D003459:Crystallins; D009124:Muscle Proteins; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases