Copper,zinc superoxide dismutase is primarily a cytosolic protein in human cells.

Abstract

The intracellular lalization of human copper,zinc superoxide dismutase (Cu,Zn-SOD; superoxide:superoxide oxidoreductase, EC 1.15.1.1) was evaluated by using EM immunocytochemistry and both Isolated human cell lines and human tissues. Eight monoclonal antibodies raised against either native or recombinant human Cu,Zn-SOD and two polyclonal antibodies raised against either native or recombinant human Cu,Zn-SOD were used. Fixation with 2% paraformaldehyde/0.2% glutaraldehyde was found necessary to preserve normal distribution of the protein. Monoclonal antibodies were less effective than polydonal antibodies in recognizing the antigen after adequate fixation oftissue. Cu,Zn-SOD was found widely distributed in the cell cytosol and in the cell nucleus, consistent with it being a soluble cytosolic protein.Mitochondria and secretory compartments did not label for this protein. In human cells, peroxisomes showed a labeling density slightly less than that of cytoplasm.The superoxide dismutases (SODs; superoxide:superoxide oxidoreductase, EC 1.15.1.1) are a family of enzymes commonly characterized by the metals that they contain and by their function to dismute 2-, thus providing essential protection of biological tissues against uncontrolled reactions with oxygen-based radicals. The copper,zinc form of SOD (Cu,Zn-SOD) is a dimer having a molecular mass of 32,000 Da. It has been identified as a soluble enzyme widely distributed in the cytoplasm of all mammalian cells (1). Previous EM immunocytochemical localization of this protein has been done on rat liver hepatocytes. The enzyme was found to be excluded from many membrane-bound compartments, such as nuclear envelope, endoplasmic reticulum, Golgi elements, secretory vesicles, and mitochondria (2). Quantitative immunocytochemistry done on rat Cu,Zn-SOD identified it to be widely distributed throughout the cytoplasm and nucleus; the cytoplasmic matrix had a concentration of 1.36 mg/ml, a concentration --50%o higher than that of the nuclear matrix (3). Lysosomes contained the highest concentration (5.81 mg/ml). Rat hepatocyte peroxisomes were found to contain the Cu,Zn-SOD in relatively low concentrations (0.27 mg/ml) (3).Keller et al. (4) recently evaluated the intracellular localization of Cu,Zn-SOD in human fibroblasts and hepatoma cells with four monoclonal antibodies (mAbs) raised against recombinant human (rh) Cu,Zn-SOD. Immunolocalization was done by using immunofluorescence (4, 5). The enzyme was reported to be localized only in punctate regions of the cells that were identified as peroxisomes because of colocalization of catalase to the same sites with a dual-immunofluorescence method. These authors speculated that the use of mAbs raised against a recombinant protein insured the reliability of their studies localizing SOD only to peroxisomes. MATERIALS AND METHODS mAbs. Four mAbs raised against rh Cu,Zn-SOD were obtained from Robert A. Hallewell (Chiron) and were designated CZSOD F2, CZSOD A3, CZSOD A6, and CZSOD A7, as reported by Keller et al. (4). A mAb ...

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