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 o f the skin epithelium of common carp (Cyprinus carpio) were investigat ed and compared with peroxidase activity of blood cells. Skin as well as blood-borne peroxidases eluted from the Superdex column as a 135 kD a protein and both probably are tetrameric molecules. Skill peroxidase activity was characterized by a V-max of 51.5 +/- 1.3 U mg(-1) min(-1 ) and a K-M of 1.64 +/- 0.18 mM ortho-phenylenediamine (OPD), whereas blood-borne peroxidase was characterized by a 1,000 fold higher specif ic activity (V-max = 30.5 . 10(3) +/- 2.5 . 10(3) U mg(-1) min(-1)) an d a higher affinity (K-M = 0.875 +/- 0.003 mM OPD). Polyclonal antibod ies were raised against concanavalin-A purified skin peroxidase as wel l as blood-borne peroxidase. Immunocytochemical labelling showed that peroxidase is present in mucous cells and in mucus covering the skin a nd gill epithelia, as well as in erythrocytes and leucocytes. We concl ude that the mucous cells of the skin produce a biochemically distinct peroxidase that is released in the mucus and may contribute to the an timicrobial properties of the mucous layer covering the skin. After ex posure of the fish to cadmium the kinetic characteristics of the enzym e activity, as determined in skin homogenates, changed considerably. T he V-max increased significantly to 61.9 +/- 1.1 U mg(-1) min(-1), and the affinity for OPD increased to the value demonstrated for blood-bo rne peroxidase (K-M = 0.888 +/- 0.045 mM OPD). Increased peroxidase le vels after cadmium exposure were also demonstrated immunochemically in a dotblot assay. However, no significant changes were observed when t he circulatory system of the fish was perfused prior to sampling, indi cating that erythrocytes are a major contributor to the increased pero xidase activity in carp skin during cadmium exposure. This likely refl ects the increased vascularization of the connective tissue layer unde rlying the skin epithelium, which takes place when the fish are expose d to chronic stressors including cadmium. In the cadmium-exposed fish this effect prevented the biochemical detection of stresser-related ch anges in epithelial peroxidase reported earlier with cytochemical meth ods.