Internalization of human extracellular-superoxide dismutase by bovine aortic endothelial cells.
The high heparin-affinity subtype C of the secretory enzyme extracellular-superoxide dismutase (EC-SOD) mainly exists on the outside of endothelial cell surface in the vasculature. Radioiodinated recombinant EC-SOD C(r-EC-SOD C) bound to cultured bovine aortic endothelial cells (BAE cells) at 4 degrees C with an association constant of 9.35 x 10(6) M-1 and maximum binding of 600 ng/dish (3109 ng/mg cellular protein). When incubated at 37 degrees C for 1 h, some 125I-r-EC-SOD C was no longer releasable by heparin treatment, suggesting that 125I-r-EC-SOD C was internalized by BAE cells. Since the internalization was inhibited in the presence of heparin in medium, this step was mediated by the binding to cell surface heparin sulfate proteoglycans. When cells containing internalized 125I-r-EC-SOD C were incubated in newly added medium at 37 degrees C for up to 1 h, 54% of radioactivity was recovered in new medium. However, 71% of the radioactive materials released to the medium, presumably 125I-r-EC-SOD C and its metabolic products, had lost heparin binding activity. Much of internalized 125I-r-EC-SOD C was degraded to low molecular weight peptides, because 54% of the radioactive products released to the medium were trichloroacetic acid-soluble and 59% of them were below 10 kDa. About one-fourth of radioactive materials were recycled 125I-r-EC-SOD judged from heparin-HPLC and Sephacryl S-200 column chromatography. In the presence of chloroquine, lysosomal protease inhibitor, the release of internalized 125I-r-EC-SOD C decreased to 59% compared with the control culture.(ABSTRACT TRUNCATED AT 250 WORDS)
Ohta
H
H
Department of Pharmaceutics, Gifu Pharmaceutical University, Japan.
Adachi
T
T
Hirano
K
K
D000818:Animals; D001011:Aorta; D001665:Binding Sites; D001692:Biological Transport; D002417:Cattle; D002462:Cell Membrane / Q000378:metabolism; D002478:Cells, Cultured; D002850:Chromatography, Gel; D004730:Endothelium, Vascular / Q000378:metabolism*; D019812:Heparan Sulfate Proteoglycans; D006497:Heparan Sulfate / Q000378:metabolism; D006801:Humans; D007457:Iodine Radioisotopes; D007527:Isoenzymes; D007700:Kinetics; D011509:Proteoglycans / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D013482:Superoxide Dismutase / Q000378:metabolism*
D019812:Heparan Sulfate Proteoglycans; D006497:Heparan Sulfate; D007457:Iodine Radioisotopes; D007527:Isoenzymes; D011509:Proteoglycans; D011994:Recombinant Proteins; D013482:Superoxide Dismutase