| Abstract: | Peroxidase activity, assayed with 2 mM-H2O2 and suitable hydrogen donors (either p-phenyl-enediamine or diaminobenzidine), was demonstrated in homogenates of neural retina and pigment epithelium of both the dog and the cow. The enzyme is particle-associated in the native state, but is readily extractable by brief sonication or freeze-thawing. At optimum pH, which is between 4.0 and 4.5 for both sources, the specific activity is up to 40 times greater in pigment epithelial cells than in neural retina. Some catalase activity was detected in extracts from both bovine and canine neural retina, but catalase was essentially absent in pigment epithelium. Fractionation of bovine pigment epithelial cells showed that peroxidase activity is associated mainly with heavy organelles sedimenting at low centrifugal forces. Melanosomes, nuclei, melanolysosomes and plasma membranes were the principal organelles identified in these low speed sediments. It was not possible to separate them either by differential centrifugation or on discontinuous sucrose gradients. However, melanosomes were excluded as the only source of peroxidase activity by isolating separately the melanotic and amelanotic cell populations; equal peroxidase was found in both cell types. Since nuclei are not a likely source of this enzyme, it is suggested that most of the peroxidase activity in bovine pigment epithelial cells is localized in either the melanolysosomes, plasma membranes, or both. |
