Purification of two superoxide dismutase isozymes and their subcellular localization in needles and roots of Norway spruce (Picea abies L.) trees

Abstract

Two isozymes of superoxide dismutase (SOD; EC 1.15.1.1) were purified from Norway spruce (Picea abies L.) needles to apparent electrophoretic homogeneity. Purification factors were 354 for SOD I and 265 for SOD II. The native molecular mass of both purified enzymes was approximately 33 kD, as determined by gel filtration. The subunit molecular weights, as estimated by sodium dodecyl sulfate polyacrylamide gel electrophoresis, were 20,000 for SOD I and 16,000 for SOD II in the presence of 2-mercaptoethanol, and 15,800 and 15,000, respectively, in its absence. These results indicate that the native enzymes were homodimers whose subunits contained intrachain disulfide bonds. Isoelectric points determined by nondenaturing isoelectric focusing were 4.5 and 5.5 for SOD I and II, respectively. NH2-terminal sequence analysis of the first 22 to 23 amino acids revealed 70 to 75% sequence identity with chloroplastic CuZn SODs from other plant species for SOD I, and 75% sequence identity with the cytosolic CuZn SOD from Scots pine for SOD II. SOD I was the major activity in needles and it was associated with chloroplasts. SOD II activity was dominant in roots.