Expression of a hybrid Cu/Zn-type superoxide dismutase which has high affinity for heparin-like proteoglycans on vascular endothelial cells.
Since plasma levels of enzymes, such as superoxide dismutase (SOD), that scavenge reactive oxygen species are low, surface membranes of endothelial and parenchymal cells of various tissues are often exposed to oxidative stress. To dismutase superoxide radicals efficiently in and around vascular endothelial cells, we constructed a fusion gene encoding a hybrid SOD (HB-SOD) consisting of human Cu/Zn-SOD and a C-terminal basic peptide that binds to heparin-like proteoglycans. The fusion gene was expressed in yeast, and the resulting HB-SOD was highly purified. Upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis, HB-SOD revealed a protein band with an apparent molecular weight of 20,000. HB-SOD bound to endothelial cells of aortic segments by a mechanism which was inhibited by heparin but not by antithrombin III. When injected intravenously to rats, 125I-labeled HB-SOD rapidly disappeared from the circulation; the rate of disappearance was decreased by heparin. Less than 1% of the injected HB-SOD was found in the urine 20 min after administration at which time more than 70% of SOD was excreted in its intact form. Immunohistochemical studies revealed that HB-SOD predominantly bound to heparin-like proteoglycans on endothelial cells of the artery and other tissues. HB-SOD might permit studies on pathophysiological roles of superoxide radicals in and around vascular endothelial cells in vivo.
Inoue
M
M
Department of Biochemistry, Kumamoto University Medical School, Japan.
Watanabe
N
N
Matsuno
K
K
Sasaki
J
J
Tanaka
Y
Y
Hatanaka
H
H
Amachi
T
T
D000595:Amino Acid Sequence; D000818:Animals; D001011:Aorta; D001483:Base Sequence; D002417:Cattle; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D004247:DNA; D004591:Electrophoresis, Polyacrylamide Gel; D004730:Endothelium, Vascular / Q000378:metabolism*; D006493:Heparin / Q000031:analogs & derivatives* / Q000378:metabolism; D006801:Humans; D007150:Immunohistochemistry; D007700:Kinetics; D008297:Male; D008969:Molecular Sequence Data; D011509:Proteoglycans / Q000378:metabolism*; D051381:Rats; D011919:Rats, Inbred Strains; D013482:Superoxide Dismutase / Q000235:genetics* / Q000378:metabolism
D011509:Proteoglycans; C036090:heparin proteoglycan; D006493:Heparin; D004247:DNA; D013482:Superoxide Dismutase