Purification and characterization of canine manganese superoxide dismutase and its immunohistochemical localization in canine heart compared with that of copper-zinc superoxide dismutase

Abstract

Several forms of superoxide dismutase (SOD; EC 1.15.1.1) are known to be major scavengers of superoxide radicals which are involved in various kinds of injuries including heart failure. In an attempt to elucidate the protective system of the canine heart against superoxide radical, the present study immunohistochemically examined the localizations of copper-zinc superoxide dismutase (Cu,Zn-SOD) and manganese superoxide dismutase (Mn-SOD) in the canine heart. Mn-SOD was purified homogeneously from canine liver by chromatography on a DE-52 column and a CM-52 column, followed by gel filtration on a Sephadex G-200 column: the purified enzyme consisted of two identical subunits of molecular weight 22,000. Polyclonal antibody was prepared against the purified Mn-SOD, and its specificity was confirmed by Western blotting. The Mn-SOD and Cu,Zn-SOD in canine hearts were examined by immunocytochemical staining using specific anti Mn-SOD serum and anti-canine Cu,Zn-SOD serum prepared previously. Cu,Zn-SOD was localized in vessel cells including the endothelium, whereas little Mn-SOD occurred in the vessel cells but some was located in the endothelium. The corrected Cu,Zn-SOD activity (714 ± 27 U/ml; mean ± SEM, n = 4) was similar to that of the corrected Mn-SOD activity (633 ± 42 U/ml; n = 4) in the canine heart. The present study shows that both forms of SOD are present in the endothelium. It is concluded that they may cooperatively scavenge superoxide radicals generated around the canine heart vessels, and Cu,Zn-SOD may be functional in secondary protection of the myocytes against more severe injury derived from superoxide radicals in the vessel cells.