The amino-acid sequence of rabbit Cu-Zn superoxide dismutase

The primary structure of Cu-Zn superoxide dismutase from rabbit liver was investigated. The reduced and S-carboxymethylated enzyme was treated with cyanogen bromide, trypsin or Staphylococcus aureus proteinase V8. The resulting peptides were separated by high-performance liquid chromatography and sequenced by automated Edman degradation. With the exception of the N- and C-terminus the complete sequence was established by means of overlapping peptides. The N-terminus is blocked and thus not susceptible to Edman degradation. The amino-acid composition of the tryptic N-terminal peptide corresponds to that of the cytoplasmatic Cu-Zn superoxide dismutases of other mammals investigated. The chromatographic behaviour of these N-terminal peptides on a reversed phase C18 column is also identical, thus suggesting also for the rabbit Cu-Zn superoxide dismutase the N-terminal sequence Ac-Ala-Thr-Lys. The C-terminus was demonstrated to have the sequence -Ile-Ala-Pro by enzymatic degradation with carboxypeptidase Y. The complete amino-acid sequence of the rabbit Cu-Zn superoxide dismutase consists of 152 amino-acids and shows the expected homology to other Cu-Zn enzymes published so far. The aspartate and six histidine residues known to complex the metal ions are conserved at homologous positions. This also applies for the arginine residue near the C-terminus which is supposed to direct the anionic superoxide radical towards the active centre of the enzyme. The amino acid sequence of the rabbit Cu-Zn superoxide dismutase corresponds to those of other mammals in more than 80% of its amino-acid residues. From a total of 152 amino-acid residues the rabbit shares with rat 128, with mouse 130, with horse 127, with pig 126/127, with cattle 130 and with man 131 amino acids in homologous positions. However the Cu-Zn superoxide dismutases of closely related mammals like rats and mice differ in only five amino acid residues of their sequence. A phylogenetic closer relatedness between lagomorphs and rodents than between other orders of mammals, could not be derived from the sequence data given. Rather rodents and lagomorphs are to be considered as two evolutionary independent orders of mammals.