Intracellular localization, isolation and characterization of two distinct varieties of superoxide dismutase from Neurospora crassa

Abstract

N. crassa contains 2 distinct superoxide dismutases. Most of the activity is associated with the cytosolic fraction and is the Cu/Zn-containing form of the protein. Mitochondria isolated from N. crassa showed 2 distinct superoxide dismutases: a cyanide-sensitive Cu/Zn-containing protein and a cyanide-insensitive form which probably contains Mn. Localization experiments, using selective marker enzymes and digitonin fractionation, indicated that the cyanide-sensitive form is localized in the intermembrane space, whereas the cyanide-insensitive form is confined to the mitochondrial matrix space. The cytosolic Cu/Zn-containing superoxide dismutase was isolated in high yields and extensively characterized by using EPR spectroscopy, isoelectric focusing and analytical ultracentrifugation. EPR spectroscopy was used to monitor changes in the Cu environment of the native protein after the addition of a number of potential inhibitors and after high-pH treatment. Both of the cyanide-sensitive Cu/Zn-containing enzymes (cytosolic and mitochondrial) appeared to have identical properties which in turn were different from the cyanide-insensitive enzyme. It is probable that the cyanide-insensitive enzyme was not previously detected, owing to its low amount (less than 10% of the total activity), greater lability than the cyanide-sensitive enzyme and the necessity of obtaining a mitochondrial-enriched fraction before its isolation.