Immunocytochemical localization of copper,zinc superoxide dismutase in peroxisomes from watermelon (Citrullus vulgaris Schrad.) cotyledons.

In previous works using cell fractionation methods we demonstrated the presence of a Cu,Zn-containing superoxide dismutase in peroxisomes from watermelon cotyledons. In this work, this intracellular localization was evaluated by using western blot and EM immunocytochemical analysis with a polyclonal antibody against peroxisomal Cu,Zn-SOD II from watermelon cotyledons. In crude extracts from 6-day old cotyledons, analysis by western blot showed two cross-reactivity bands which belonged to the isozymes Cu,Zn-SOD I and Cu,Zn-SOD II. In peroxisomes purified by sucrose density-gradient centrifugation only one cross-reactivity band was found in the peroxisomal matrix which corresponded to the isozyme Cu,Zn-SOD II. When SOD activity was assayed in purified peroxisomes two isozymes were detected, Cu,Zn-SOD II in the matrix, and a Mn-SOD in the membrane fraction which was removed by sodium carbonate washing. EM immunocytochemistry of Cu,Zn-SOD on sections of 6-day old cotyledons, showed that gold label was mainly localized over plastids and also in peroxisomes and the cytosol, whereas mitochondria did not label for Cu,Zn-SOD.