Purification, molecular cloning, and some properties of a manganese-containing superoxide dismutase from Japanese flounder (Paralichthys olivaceus)

Manganese-superoxide dismutase (Mn-SOD) from Japanese flounder (Paralichthys olivaceus) hepatopancreas has been purified with high purification (781-fold) and recovery (10.8%). The molecular mass of the purified enzyme was estimated to be 26 kDa by SDS-PAGE under reducing conditions. In activity staining by native-PAGE, the Japanese flounder Mn-SOD gave three active bands and exhibited KCN-insensitive activity. In addition, the electrophoretic mobility of this enzyme was observed to be faster than that of Japanese flounder Cu,Zn-SOD. On the other hand, the N-terminal amino acid sequence of this Mn-SOD was determined to be 16 amino acid residues, and the sequence showed high homology to other Mn-SODs but not Japanese flounder Cu,Zn-SOD. Analysis of nucleotide and deduced amino acid sequences revealed that the Mn-SOD cDNA consisted of a 64 bp 5′-non-coding region, a 675 bp open reading frame encoding 225 amino acids, and a 465 bp 3′-non-coding region. The first 27 amino acids containing a mitochondria-targeting signal were highly conserved among other Mn-SODs.