Purification and Properties of Superoxide Dismutase from Drosophila melanogaster*

The major superoxide dismutase ((‘slow’’ electro- morph) of the fruit fly, Drosophila melanogaster, has been purified to homogeneity. “his enzyme contains 2 Cu2+ and 2 Zn2’/molecule. The ultraviolet absorption spectrum indicates a lack of tryptophan. This enzyme has a molecular weight of 32,000 and is composed of two subunits of equal size, which are joined by nonco-valent interactions. Cyanide at 1 and 3 m~ inhibits the activity of superoxide dismutase 92 and 1001, but 5 and 10 m~ azide caused 15 and 30% inhibition. The isoelec- tric point, assessed by isoelectric focusing, is 5.3. Amino acid analyses, as well as the spectral and catalytic properties, are reported. The f). melanogaster superoxide dismutase does not cross-react with antibodies to bovine erythrocyte Cu-Zn-containing superoxide dismutase nor to Escherichia coli manganese- and iron- containing superoxide dismutases. Superoxide catalytically OL, of the defense against (1-3). a of structural proteins

Superoxide dismutases, which catalytically scavenge O L , appear to be essential components of the biological defense against oxygen toxicity (1-3). These enzymes are neither a part of structural proteins nor involved in intermediate metabolism, providing a unique situation to be studied by population geneticists. Genetic polymorphism of superoxide dismutase has already been discovered in diverse living organisms including humans and the fruit fly, Drosophila (4-10). Little is known, however, about the structural basis of such polymorphisms.
Superoxide dismutases have been isolated from several organisms; thus far, only three grossly dissimilar kinds have been found. The structural and functional relationships of these three classes of superoxide dismutases have raised interesting and unresolved questions about their evolution. Copper-and zinc-containing superoxide dismutases have been isolated from various species (11-19) and considered to be characteristic of the cytosol of eukaryotic cells (20), but a similar enzyme has been found in a prokaryote, Photobacterium leioghathi (20). Manganese-containing superoxide dismutases have been isolated from several prokaryotes (21-23) and from the mitochondria of chicken liver (14) and of yeast eukaryotes would contain copper-zinc, while the mitochondria would contain manganese. However, the luminous fungus, Pleurotus olearius, has been shown to contain two superoxide dismutases, both of which contain manganese (28). Furthermore, substantial quantities of manganese enzyme have been found in the cytosol of chicken liver and of baboon liver (29). Superoxide dismutase isolated from the cytosol of unicellular red alga, Porphyridium cruentum, which is considered to be perhaps the most primitive eukaryote, contain manganese (30). However, blue-green algae, which are considered to be the most advanced prokaryotes, have an iron-containing superoxide dismutase (31, 32). Iron-containing enzymes have also been found in several bacteria (33-35). A survey of progressively more advanced plants has failed to find copperzinc superoxide dismutase in marine plants, but has found it in land plants such as mosses and ferns (36). Thus, the facts are not easily arranged into a coherent theory of descent.
There have been several reports indicating that superoxide dismutase protects against ionizing radiation damage to DNA, viruses, bacteria, mammalian cells in culture, and even whole animals (37-43). Since insects have been shown to be more resistant to ionizing radiation than mammals, Drosophila are reported to survive radiation exposure of 64,000 rads (44), and because a superoxide dismutase has not been isolated from an insect, it seemed important to purify and characterize this enzyme from Drosophila melanogaster. We here report the thorough purification and characterization of one of two electrophoretically detectable allozymes of superoxide dismutase from D. melanogaster with the expectation that this will relate to the radioresistance of the organism and will also bring us a step closer to disentangling the complex evolutionary history of these enzymes.

DISCUSSION
Cell-free extracts of the fruit fly, D. melanogaster, contain two superoxide dismutases which are separable on polyacrylamide gel electrophoresis. The major superoxide dismutase activity was inhibited by cyanide. This enzyme, like the bovine erythrocyte superoxide dismutase, survived an unusual purification step which included use of chloroform-ethanol to denature extraneous proteins. However, unlike other Cu-Zn superoxide dismutases, the Drosophila enzyme ("slow" electromorph) quickly lost its activity when salted out of an ethanol-rich phase with KzHPO,.
Isolation of the major superoxide dismutase of D. melanogaster revealed that the molecular properties of this enzyme appear to have been rigidly preserved during the evolution of eukaryotes. Thus, the enzyme is similar to the cytoplasmic enzymes of other eukaryotes (11)(12)(13)(14)(15)(16)(17)(18)(19) with respect to molecular weight, quaternary structure, metal prosthetic groups, and ultraviolet spectrum, but it does not cross-rsact with a rabbit antibody to the bovine erythrocyte enzyme. The D. melanogaster enzyme was stable to freezing and thawing and was homogeneous by the criteria of polyacrylamide gel electrophoresis and sodium dodecyl sulfate gel electrophoresis.
Since a 245-fold purification from the cell-free extract was homogeneous, and since the net recovery was 8%, we can estimate that this superoxide dismutase constituted 0.4% of the protein of the crude soluble extract. Drosophila superoxide dismutase is at least 1.5 times more active than the enzymes of all other species reported in the literature. Because superoxide dismutase has been implicated in protecting against ionizing radiation (37)(38)(39)(40)(41)(42)(43) and insects have been shown to be more radio-resistant than most animals (44), it seems reasonable to believe that the high concentrations of highly active superoxide dismutase detected in Drosophila could be contributing to the higher resistance of these flies to ionizing radiation. The evolutionary relationships among superoxide dismutases are obviously of great interest. The structural basis of genetic polymorphisms of this enzyme noticed in Drosophila (9) needs to be explored.