• We are sorry, but NCBI web applications do not support your browser and may not function properly. More information
Logo of plntphysLink to Publisher's site
Plant Physiol. May 1992; 99(1): 317–323.
PMCID: PMC1080442

Broad Bean Leaf Polyphenol Oxidase Is a 60-Kilodalton Protein Susceptible to Proteolytic Cleavage

Abstract

Polyphenol oxidase (PPO) in leaves is generally considered to be a 45-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 end 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.

Full text

Full text is available as a scanned copy of the original print version. Get a printable copy (PDF file) of the complete article (1.6M), or click on a page image below to browse page by page. Links to PubMed are also available for Selected References.

Images in this article

Selected References

These references are in PubMed. This may not be the complete list of references from this article.
  • Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. [PubMed]
  • Flurkey WH. In Vitro Biosynthesis of Vicia faba Polyphenoloxidase. Plant Physiol. 1985 Oct;79(2):564–567. [PMC free article] [PubMed]
  • Flurkey WH. Polyphenoloxidase in higher plants: immunological detection and analysis of in vitro translation products. Plant Physiol. 1986 Jun;81(2):614–618. [PMC free article] [PubMed]
  • Flurkey WH. Polypeptide composition and amino-terminal sequence of broad bean polyphenoloxidase. Plant Physiol. 1989 Oct;91(2):481–483. [PMC free article] [PubMed]
  • Golbeck JH, Cammarata KV. Spinach Thylakoid Polyphenol Oxidase : ISOLATION, ACTIVATION, AND PROPERTIES OF THE NATIVE CHLOROPLAST ENZYME. Plant Physiol. 1981 May;67(5):977–984. [PMC free article] [PubMed]
  • Kupper U, Niedermann DM, Travaglini G, Lerch K. Isolation and characterization of the tyrosinase gene from Neurospora crassa. J Biol Chem. 1989 Oct 15;264(29):17250–17258. [PubMed]
  • Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. [PubMed]
  • Ploug M, Jensen AL, Barkholt V. Determination of amino acid compositions and NH2-terminal sequences of peptides electroblotted onto PVDF membranes from tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis: application to peptide mapping of human complement component C3. Anal Biochem. 1989 Aug 15;181(1):33–39. [PubMed]
  • Rezaian MA, Krake LR. Nucleic acid extraction and virus detection in grapevine. J Virol Methods. 1987 Sep;17(3-4):277–285. [PubMed]
  • Schägger H, von Jagow G. Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa. Anal Biochem. 1987 Nov 1;166(2):368–379. [PubMed]

Articles from Plant Physiology are provided here courtesy of American Society of Plant Biologists

Formats:

Related citations in PubMed

See reviews...See all...

Cited by other articles in PMC

See all...

Links

Recent Activity

Your browsing activity is empty.

Activity recording is turned off.

Turn recording back on

See more...