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Broad bean leaf polyphenol oxidase is a 60-kilodalton protein susceptible to proteolytic cleavage

Polyphenol oxidase (PPO) in leaves is generally considered to be a 46-kilodalton protein. PPO purified to homogeneity from broad bean (Vicia faba L.) leaves in the presence of protease inhibitors had an apparent molecular mass of 60 kD determined by denaturing sodium dodecyl sulfate (SDS) polyacryla...

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Bibliographic Details
Published in:Plant physiology (Bethesda) 1992-05, Vol.99 (1), p.317-323
Main Authors: ROBINSON, S. P, DRY, I. B
Format: Article
Language:English
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Summary:Polyphenol oxidase (PPO) in leaves is generally considered to be a 46-kilodalton protein. PPO purified to homogeneity from broad bean (Vicia faba L.) leaves in the presence of protease inhibitors had an apparent molecular mass of 60 kD determined by denaturing sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis. Under partially denaturing conditions, the protein had an apparent molecular mass of 43 kilodaltons, but this was shifted to 60 kilodaltons in the presence of sulfhydryl reductants, suggesting the presence of disulfide bonding. The purified protein was totally latent; i.e. PPO activity was only detected when assayed with sodium dodecyl sulfate. Treatment with proteases in the presence of 0.1% SDS inhibited enzyme activity, but in the absence of SDS the 60-kilodalton PPO was proteolytically cleaved with no loss of PPO activity. This yielded a 42-kilodalton peptide that had PPO activity and inactive peptides of 12 to 18 kilodaltons. Amino acid sequencing established that the 42-kilodalton protein was derived from the N-terminal and of the 60-kilodalton form of PPO. By comparison with the sequence of a cDNA clone for broad bean leaf PPO, the 18-kilodalton peptide was located at the C-terminal end of the 60-kilodalton protein. Northern analysis of mRNA from broad bean leaves probed with a cDNA clone of PPO identified a transcript of 2.2 kilobase pairs, which is more than sufficient to encode the 60-kilodalton protein. It is concluded that PPO is a 60-kilodalton protein in broad bean leaves but that it is susceptible to proteolysis during extraction, removing a peptide of 15 to 18 kilodaltons from the C-terminal end without decreasing enzyme activity.
ISSN:0032-0889
1532-2548
DOI:10.1104/pp.99.1.317