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.
Sandalio
L M
LM
Departamento de Bioquímica, Biología Celular y Molecular de Plantas, Estación Experimental del Zaidín, CSIC, Granada, Spain. lmsanda@eez.csic.es
López-Huertas
E
E
Bueno
P
P
Del Río
L A
LA
D015153:Blotting, Western; D018548:Cotyledon / Q000201:enzymology; D004591:Electrophoresis, Polyacrylamide Gel; D005638:Fruit / Q000201:enzymology*; D007150:Immunohistochemistry / Q000379:methods*; D007527:Isoenzymes; D008830:Microbodies / Q000201:enzymology*; D010936:Plant Extracts / Q000737:chemistry; D013482:Superoxide Dismutase / Q000276:immunology* / Q000378:metabolism
D007527:Isoenzymes; D010936:Plant Extracts; D013482:Superoxide Dismutase