シイタケ子実体のスーパーオキシドジスムターゼの精製と諸性質 [in Japanese] Purification and Some Properties of Superoxide Dismutase from Pilei of Lentinus edodes Fruiting Bodies [in Japanese]
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Crude extract of Lentinus edodes(L. edodes)contained more than three kinds of superoxide dismutases(SODs)which were distinguished electrophoretically. All of these were found to be insensitive to H2O2 and cyanide. A centrifugal study of the crude extract in 0.25M sucrose solution revealed that most of the SOD activities are located in the cytoplasmic fraction. We purified one of these superoxide dismutases from pilei of L. edodes to homogeneity by ammonium sulfate fractionation, DE-32 ion-exchange, Sephadex G-100 gel filtration and Butyl-toyopearl hydrophobic chromatographies. The purified enzyme showed that a single protein band coincided with the single SOD activity band in native polyacrylamide gel electrophoresis(PAGE). The purified SOD showed a single band in PAGE in the presence of sodium dodecyl sulfate(SDS-PAGE)and its subunit molecular weight was estimated to 23±0.5kDa. The subunit molecular weight of SOD was also estimated by LC-MS analysis to be 22,184Da. Using the sedimentation equilibrium centrifugation method the molecular mass of native SOD was estimated to be 84,240Da. These observations suggest that the enzyme is a tetramer composed of subunits of equal size. Metal analysis of the native enzymes revealed 0.64g-atoms Mn per mole subunit in the preparations whose specific activity were 3500U/mg. Direct analysis of N-terminal amino acid of this enzyme by Edman degradation using a protein sequencer cannot detect any amino acid residue, but amino acid residue of N-terminal appeared as serine residue after the treatment of the enzyme with tetrafluoroacetic acid in a vapor phase at 60℃ for 10min. Therefore, the N-terminal amino acid was modified with acetyl group. The modification of N-terminal amino acid is the first example of Mn-SOD. Some of other physiological and biochemical properties of the enzyme were also investigated.
学苑 -(854), 1-9, 2011-12