Free radical-induced protein modification and inhibition of Ca2+-ATPase of cardiac sarcoplasmic reticulum.
The effect of oxidative stress on the Ca2+-ATPase activity, lipid peroxidation and protein modification of cardiac sarcoplasmic reticulum (SR) membranes was investigated. Isolated SR vesicles were exposed to FeSO4/EDTA (0.2 micromol Fe2+ per mg of protein) at 37 degrees C for 1 h in the presence or absence of antioxidants. FeSO4/EDTA decreased the maximum velocity of Ca2+-ATPase reaction without a change of affinity for Ca2+ or Hill coefficient. Treatment with radical-generating system led also to conjugated diene formation, loss of sulfhydryl groups, changes in tryptophan and bityrosine fluorescences and to production of lysine conjugates with lipid peroxidation end-products. Lipid antioxidants butylated hydroxytoluene (BHT) and stobadine partially prevented inhibition of Ca2+-ATPase and decrease in tryptophan fluorescence, while the loss of -SH groups and formation of bityrosines or lysine conjugates were completely prevented. Glutathione also partially protected Ca2+-ATPase activity and decreased formation of bityrosine, but it was not able to prevent oxidative modification of tryptophan and lysine. These findings suggest that combination of amino acid modifications, rather than oxidation of amino acids of one kind, is responsible for inhibition of SR Ca2+-ATPase activity.
Kaplan
Peter
P
Comenius University, Jessenius Faculty of Medicine, Department of Biochemistry, Martin, Slovak Republic. kaplan@jfmed.uniba.sk
Babusikova
Eva
E
Lehotsky
Jan
J
Dobrota
Dusan
D
D000251:Adenosine Triphosphatases / Q000737:chemistry; D000596:Amino Acids / Q000737:chemistry; D000818:Animals; D000975:Antioxidants / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism; D000252:Calcium-Transporting ATPases / Q000737:chemistry* / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004492:Edetic Acid / Q000494:pharmacology; D005609:Free Radicals; D007700:Kinetics; D050356:Lipid Metabolism; D015227:Lipid Peroxidation; D008239:Lysine / Q000737:chemistry; D009206:Myocardium / Q000378:metabolism*; D018384:Oxidative Stress; D010100:Oxygen / Q000378:metabolism; D051381:Rats; D012519:Sarcoplasmic Reticulum / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D013997:Time Factors; D014364:Tryptophan / Q000737:chemistry; D014443:Tyrosine / Q000031:analogs & derivatives* / Q000737:chemistry
D000596:Amino Acids; D000975:Antioxidants; D005609:Free Radicals; D014443:Tyrosine; D014364:Tryptophan; D004492:Edetic Acid; C007543:dityrosine; D000251:Adenosine Triphosphatases; D000252:Calcium-Transporting ATPases; D008239:Lysine; D010100:Oxygen; D002118:Calcium