Isolation, partial characterization and localization of integumental peroxidase, a stress-related enzyme in the skin of a teleostean fish (Cyprinus carpio L.)

Biochemical and immunological characteristics of peroxidase activity of the skin epithelium of common carp (Cyprinus carpio) were investigated and compared with peroxidase activity of blood cells. Skin as well as blood-borne peroxidases eluted from the Superdex column as a 135 kDa protein and both probably are tetrameric molecules. Skin peroxidase activity was characterized by a Vmax of 51.5 ± 1.3 U mg-1 min-1 and a KM of 1.64 ± 0.18 mM ortho-phenylenediamine (OPD), whereas blood-borne peroxidase was characterized by a 1,000 fold higher specific activity (Vmax = 30.5 ċ 103 ± 2.5 ċ 103 U mg-1 min-1) and a higher affinity (KM = 0.875 ± 0.003 mM OPD). Polyclonal antibodies were raised against concanavalin-A purified skin peroxidase as well as blood-borne peroxidase. Immunocytochemical labelling showed that peroxidase is present in mucous cells and in mucus covering the skin and gill epithelia, as well as in erythrocytes and leucocytes. We conclude that the mucous cells of the skin produce a biochemically distinct peroxidase that is released in the mucus and may contribute to the antimicrobial properties of the mucous layer covering the skin. After exposure of the fish to cadmium the kinetic characteristics of the enzyme activity, as determined in skin homogenates, changed considerably. The Vmax increased significantly to 61.9 ± 1.1 U mg-1 min-1, and the affinity for OPD increased to the value demonstrated for blood-borne peroxidase (KM = 0.888 ± 0.045 mM OPD). Increased peroxidase levels after cadmium exposure were also demonstrated immunochemically in a dotblot assay. However, no significant changes were observed when the circulatory system of the fish was perfused prior to sampling, indicating that erythrocytes are a major contributor to the increased peroxidase activity in carp skin during cadmium exposure. This likely reflects the increased vascularization of the connective tissue layer underlying the skin epithelium, which takes place when the fish are exposed to chronic stressors including cadmium. In the cadmium-exposed fish this effect prevented the biochemical detection of stressor-related changes in epithelial peroxidase reported earlier with cytochemical methods.