Thioredoxin regenerates proteins inactivated by oxidative stress in endothelial cells.
The thioredoxin/thioredoxin reductase system has been studied as regenerative machinery for proteins inactivated by oxidative stress in vitro and in cultured endothelial cells. Mammalian glyceraldehyde-3-phosphate dehydrogenase was used as the main model enzyme for monitoring the oxidative damage and the regeneration. Thioredoxin and its reductase purified from bovine liver were used as the regenerating system. The physiological concentrations (2-14 microM) of reduced thioredoxin, with 0.125 microM thioredoxin reductase and 0.25 mM NADPH, regenerated H2O2-inactivated glyceraldehyde-3-phosphate dehydrogenase and other mammalian enzymes almost completely within 20 min at 37 degrees C. Although the treatment of endothelial cells with 0.2-12 mM H2O2 for 5 min resulted in a marked decrease in the activity of glyceraldehyde-3-phosphate dehydrogenase, it had no effect on the activities of thioredoxin and thioredoxin reductase. Essentially all of the thioredoxin in endothelial cells at control state was in the reduced form and 70-85% remained in the reduced form even after the H2O2 treatment. The inactivated glyceraldehyde-3-phosphate dehydrogenase in a cell lysate prepared from the H2O2-treated endothelial cells was regenerated by incubating the lysate with 3 mM NADPH at 37 degrees C and the antiserum raised against bovine liver thioredoxin inhibited the regeneration. The inhibition of thioredoxin reductase activity by 13-cis-retinoic acid resulted in a decrease in the regeneration of glyceraldehyde-3-phosphate dehydrogenase in the H2O2-treated endothelial cells. The present findings provide evidence that thioredoxin is involved in the regeneration of proteins inactivated by oxidative stress in endothelial cells.
Fernando
M R
MR
Department of Biochemistry, Kyushu University School of Medicine, Japan.
Nanri
H
H
Yoshitake
S
S
Nagata-Kuno
K
K
Minakami
S
S
D000818:Animals; D002417:Cattle; D002478:Cells, Cultured; D004730:Endothelium, Vascular / Q000187:drug effects / Q000378:metabolism*; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000037:antagonists & inhibitors / Q000378:metabolism; D006861:Hydrogen Peroxide / Q000494:pharmacology; D015474:Isotretinoin / Q000494:pharmacology; D008099:Liver / Q000201:enzymology / Q000378:metabolism; D009132:Muscles / Q000201:enzymology; D010084:Oxidation-Reduction; D011506:Proteins / Q000378:metabolism*; D011817:Rabbits; D013880:Thioredoxin-Disulfide Reductase / Q000378:metabolism; D013879:Thioredoxins / Q000378:metabolism*
D011506:Proteins; D013879:Thioredoxins; D006861:Hydrogen Peroxide; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases; D013880:Thioredoxin-Disulfide Reductase; D015474:Isotretinoin