Several isoforms of superoxide dismutase (SOD) with a high isoelectric point (pI) have been identified by isoelectric focusing chromatography in protein extracts from Scots pine (Pinus sylvestris) needles. One of these isoforms, a CuZn-SOD with a pI of about 10 and thus denoted hipI-SOD, has been isolated and purified to apparent homogeneity. A cDNA encoding the hipI-SOD protein was cloned and sequenced. Northern hybridization of mRNA isolated from different organs and tissues showed that hipI-SOD has a markedly different pattern of expression compared with chloroplastic and cytosolic SOD. Furthermore, the transcript levels of hipI-SOD and cytosolic SOD were found to respond differently to mechanical wounding, treatment with oxidized glutathione, paraquat, and ozone. Immunogold electron microscopy localized the hipI-SOD in the plasma membrane of sieve cells and the Golgi apparatus of albuminous cells. Moreover, high protein density was also detected in extracellular spaces such as secondary cell wall thickenings of the xylem and sclerenchyma and in intercellular spaces of parenchyma cells.
Four new isoforms of superoxide dismutase (SOD; superoxide: superoxide oxidoreductase, EC 1.15.1.1.) were identified in extracellular washing fluid from Scots pine (Pinus sylvestris L.) needles. The isoforms had an apparent molecular mass of 33 kDa. No neutral carbohydrates were present in the enzymes. The enzymatic activities were inhibited by 3 mM NaCN. One of the putative extracellular SOD isoforms was purified and NH2-terminal-sequenced. The sequence contained the domain KAVAVL. The domains VEG and V(K/S)G, present in chloroplastic and cytosolic CuZn SODs of plants, respectively, were not detected. The enzyme was composed of two subunits of 17.8 kDa each. The isoelectric point was determined to be 6.5. The results suggest the existence of an extracellular SOD in Scots pine.
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