Superoxide dismutases in mitochondria from Helianthls tuberosus and Neurospora crassa.

Superoxide dismutase has been studied in a wide range of organisms (Fridovich, 1975). Early observations suggested that superoxide dismutases isolated from eukaryotes (McCord & Fridovich, 1969; Misra & Fridovich, 1972; Beauchamp & Fridovich, 1973) showed little diversity in their properties, having mol.wts. near 32000 and containing two copper and two zinc atoms per molecule. The activity of these enzymes was inhibited by cyanide. In contrast, two different cyanide-insensitive enzymes could be isolated from prokaryotes, e.g. Escherichia coli, which contained either two manganese atoms or one iron atom per molecule (Keele et al., 1970; Yost & Fridovich, 1973). From these original observations it would appear that there was a distinction between thesuperoxide dismutases in eukaryotes and prokaryotes. However, more recent observations suggest that this may not be correct (Puget & Michelson, 1974; Lumsden & Hall, 1975). Weisiger & Fridovich (19736) have reported that mitochondria isolated from chicken liver contained both manganese and cupro-zinc superoxide dismutases. The manganese enzyme was located in the matrix space and the cupro-zinc enzyme in the intermembrane space. A similar spatial distribution of these two enzymes has been reported in mitochondria isolated from rat liver by Peeters-Joris et al. (1975). Misra & Fridovich (1972) found that soluble extracts of Neurospora crassa contained only the cupro-zinc enzyme; however, the isolation procedure used by these workers involved extraction with an ethanol/chloroform mixture, which is known to inactivate the manganese enzyme (Weisiger & Fridovich, 1973~). The distribution of superoxide dismutase within the mitochondrion is of interest with respect to the endosymbiotic theory of mitochondrial evolution (Fridovich, 1975; Lumsden & Hall, 1975). Tyler (1975) reported that superoxide ions, formed during respiratory-chain activity, inhibited NADH oxidation in heart muscle submitochondrial particles and concluded that mitochondrial superoxide dismutase protected the NADH dehydrogenase against the harmful effects of superoxide ions. Superoxide dismutase may have a similar role in plant and fungal mitochondria, although there have been no reports of superoxide dismutase distribution and cyanide sensitivity in plant mitochondria. Misra & Fridovich (1972) reported a cupro-zinc superoxide dismutase in soluble extracts of N . crassu hyphae, but did not isolate mitochondria from this organism. In the present work we have studied the localization of superoxide dismutase in mitochondria isolated from Jerusalem artichoke (Helianthus tuberosus) and N. crassa. We provide evidence that in both these types of mitochondria the matrix contains a cyanide-insensitive enzyme (probably manganese dismutase) and the intermembrane space contains a cyanide-sensitive enzyme (cupro-zinc superoxide dismutase). Mitochondria were isolated from tubers of Jerusalem artichoke by the method of Palmer & Arron (1976) and from hyphae of N. crassa, which had been grown in submerged culture for 24h, by a modification of the method of Hall & Baltscheffsky (1968). The inner and outer membranes of the mitochondria were separated by osmotic swelling and contraction and then differential centrifugation as described by Moreau & Lance (1972). The different types of superoxide dismutase were separated and assayed on polyacrylamide gels by the method of Beauchamp & Fridovich (1971). The mitochondria from Jerusalem artichoke gave gels with three bands of superoxide dismutase activity (Fig. l), one of which was insensitive to cyanide and the remainder were sensitive. The cyanide-insensitive enzyme activity appeared to be confined to the matrix, whereas the cyanide-sensitive enzyme activities appeared to be