Changes in the location of polyphenol oxidase in potato (Solanum tuberosum L.) tuber during cell death in response to impact injury: comparison with wound tissue.

In order to elucidate the nature of the response of potato to impact injury at the biochemical level, changes in the location of the enzyme responsible for the discoloration, polyphenol oxidase, were determined using immunogold location with an antibody specific for potato tuber polyphenol oxidase. Tissue printing revealed that the enzyme was distributed throughout the tuber. Following impact injury, both tissue printing and quantitative electron microscopy indicated that there was no increase in the level of the enzyme although there was subcellular redistribution of polyphenol oxidase. This redistribution was first apparent at 12 h after impact, as determined by the use of confocal immunolocation, and coincided with loss of membrane integrity. These changes were examined in parallel with a number of stress-related parameters in both impact and wound responses. Wounding was accompanied by active gene expression and protein synthesis, leading to metabolic activity and tissue repair. In contrast, the bruising response was characterised by a limited active response and vital-staining methods indicated that after 16 h the tissue undergoes cell death.

Purification of polyphenol oxidase free of the storage protein patatin from potato tuber.

Routine protein purification to homogeneity from potato tuber, as from other storage tissues and seeds, is often hindered due to the large amounts of storage protein present. In potato, patatin, the major storage protein of the tuber, often contaminates preparations. The present work describes the purification of polyphenol oxidase (PPO) from the potato tuber (Solanum tuberosum cv Cara) to homogeneity including the critical step of hydrophobic chromatography on Octyl-Sepharose which was sufficient to completely remove patatin. The purified PPO was found to be a doublet of M(r) 60,000 and 69,000 when analysed by SDS-PAGE with a Km 4.3 +/- 0.3 mM for L-dihydroxyphenylalanine. Both bands were found to have similar N-terminal corresponding to PPO isoforms when sequenced.