Hyperoxia elevates Cu,Zn-superoxide dismutase of endothelial cells as detected by a sensitive ELISA.
An enzyme-linked immunosorbent assay (ELISA) was developed for the measurement of bovine Cu,Zn-SOD. Accuracy of the ELISA and specificity of the antibody for cell-free extracts was established by: (1) measurement of antigen levels of bovine endothelial cell extracts reconstituted with pure antigen, and (2) immunoblotting with affinity purified antibody. The ELISA was highly sensitive and 0.05-0.10 ng of pure antigen could be accurately detected, which allowed the measurement of Cu,Zn-SOD in as few as 250 endothelial cells. With utilization of the ELISA for detection, DEAE-cellulose chromatography patterns of endothelial cell Cu,Zn-SOD overlapped those of pure bovine erythrocyte Cu,Zn-SOD. Exposure of cells in culture to 80% O2 for 48 h increased the relative abundance of the Cu,Zn-SOD as measured by the ELISA by 1.8-fold. Thus, endothelial cells in culture respond to hyperoxia by enhanced production of Cu,Zn-SOD protein. The ELISA developed in this study may be useful for assessing other factors that regulate cellular production of Cu,Zn-SOD.
Das
S K
SK
Department of Medicine, New England Medical Center, Boston, Mass.
Fanburg
B L
BL
D000818:Animals; D000906:Antibodies / Q000302:isolation & purification; D000936:Antigen-Antibody Complex; D002417:Cattle; D002846:Chromatography, Affinity; D003429:Cross Reactions; D004591:Electrophoresis, Polyacrylamide Gel; D004730:Endothelium, Vascular / Q000187:drug effects / Q000201:enzymology*; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D004912:Erythrocytes / Q000201:enzymology; D005260:Female; D007527:Isoenzymes / Q000032:analysis / Q000276:immunology / Q000378:metabolism*; D007700:Kinetics; D010100:Oxygen / Q000494:pharmacology*; D011651:Pulmonary Artery; D011817:Rabbits / Q000276:immunology; D013482:Superoxide Dismutase / Q000032:analysis / Q000276:immunology / Q000378:metabolism*
D000906:Antibodies; D000936:Antigen-Antibody Complex; D007527:Isoenzymes; D013482:Superoxide Dismutase; D010100:Oxygen