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Class III Peroxidases

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Plant Proteomics

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1072))

Abstract

Class III peroxidases are heme-containing proteins of the secretory pathway with an extremely high number of isoenzymes, indicating the tremendous and important functions of this protein family. This chapter describes fractionation of the cell in subproteomes, their separation by polyacrylamide gel electrophoresis (PAGE) and visualization of peroxidase isoenzymes by heme and specific in-gel staining procedures. Soluble and membrane-bound peroxidases were separated by differential centrifugation. Aqueous polymer two-phase partitioning and discontinuous sucrose density gradient were applied to resolve peroxidase profiles of plasma membranes and tonoplast. Peroxidase isoenzymes of subproteomes were further separated by PAGE techniques such as native isoelectric focussing (IEF), high resolution clear native electrophoresis (hrCNE), and modified sodium dodecyl sulfate (modSDS)-PAGE. These techniques were used as stand-alone method or in combination for two-dimensional PAGE.

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Abbreviations

2D:

Two dimensional

AB:

Acrylamide/Bis

ACA:

Aminocaproic acid, 6-aminohexanoic acid

APS:

Ammonium persulfate

BCA:

Bicinchoninic acid

BLAST:

Basic Local Alignment Search Tool

BN-PAGE:

Blue native PAGE

BSA:

Bovine serum albumin

CHAPS:

3-[(3-Cholamidopropyl)dimethylammonio]-1-propanesulfonate

DAB:

3′3′-Diaminobenzidine

DMSO:

Dimethyl sulfoxide

DTT:

Dithiothreitol

EDTA:

Ethylenediaminetetraacetic acid

ESI:

Electrospray ionization

GB:

Gel buffer

Guaiacol:

2-Methoxyphenol

HEPES:

4-(2-Hydroxyethyl)-1-piperazineethanesulfonic acid

hrCNE:

High resolution clear native electrophoresis

ICM:

Intracellular membranes

IEF:

Isoelectric focussing electrophoresis

LB:

Loading buffer

MALDI:

Matrix-assisted laser desorption/ionization

modSDS-PAGE:

Modified SDS-PAGE

PAGE:

Polyacrylamide gel electrophoresis

PEG:

Poly-ethylene glycol

PM:

Plasma membrane

PMSF:

Phenylmethylsulfonyl fluoride

PVPP:

Polyvinylpolypyrrolidone

TEMED:

N,N,N,N′-Tetramethyl-ethylenediamine

TIFF:

Tag Image File Format

TMB:

Tetramethylbenzidine

Tris:

Tris(hydroxymethyl)aminomethane 2-amino-2-hydroxymethyl-propane-1,3-diol

References

  1. Duroux L, Welinder KG (2003) The peroxidase gene family in plants: a phylogenetic overview. J Mol Evol 57:397–407

    Article  PubMed  CAS  Google Scholar 

  2. Passardi F, Cosio C, Penel C et al (2005) Peroxidases have more functions than a Swiss army knife. Plant Cell Rep 24:255–265

    Article  PubMed  CAS  Google Scholar 

  3. Passardi F, Theiler G, Zamocky M et al (2007) PeroxiBase: the peroxidase database. Phytochemistry 68:1605–1611

    Article  PubMed  CAS  Google Scholar 

  4. Lüthje S, Meisrimler CN, Hopff D et al (2011) Phylogeny, topology, structure and functions of membrane-bound class III peroxidases in vascular plants. Phytochemistry 72:1124–1135

    Article  PubMed  Google Scholar 

  5. Almagro L, Gómez Ros LV, Belchi-Navarro S et al (2009) Class III peroxidases in plant defence reactions. J Exp Bot 60:377–390

    Article  PubMed  CAS  Google Scholar 

  6. Welinder KG, Justesen AF, Kjærsgård IVH et al (2002) Structural diversity and transcription of class III peroxidases from Arabidopsis thaliana. Eur J Biochem 269:6063–6081

    Article  PubMed  CAS  Google Scholar 

  7. De Gara L (2004) Class III peroxidases and ascorbate metabolism in plants. Phytochem Rev 3:195–205

    Article  Google Scholar 

  8. Martín M, Casano LM, Zapata JM et al (2004) Role of thylakoid Ndh complex and peroxidase in the protection against photo-oxidative stress: fluorescence and enzyme activities in wild-type and ndhF-deficient tobacco. Physiol Plant 122:443–452

    Article  Google Scholar 

  9. Mika A, Buck F, Lüthje S (2008) Membrane-bound class III peroxidases: identification, biochemical properties and sequence analysis of isoenzymes purified from maize (Zea mays L.) roots. J Proteomics 71:412–424

    Article  PubMed  CAS  Google Scholar 

  10. Costa MMR, Hilliou F, Durate P et al (2008) Molecular cloning and characterization of a vacuolar class III peroxidase involved in the metabolism of anticancer alkaloids in Catharanthus roseus. Plant Physiol 146:403–417

    Article  PubMed  CAS  Google Scholar 

  11. Albertsson PA (1971) Partition of cell particles and macromolecules, 2nd edn. Wiley, New York

    Google Scholar 

  12. Dolowy K (1984) Bioelectrochemistry of cell surfaces. Prog Surface Sci 15:245–368

    Article  CAS  Google Scholar 

  13. Widell S, Lundborg T, Larsson C (1982) Plasma membranes from oats prepared by partition in an aqueous polymer two-phase system. Plant Physiol 70:1429–1435

    Article  PubMed  CAS  Google Scholar 

  14. Yoshida S, Uemura M, Niki T et al (1983) Partition of membrane particles in aqueous two-polymer phase system and its practical use for purification of plasma membranes from plants. Plant Physiol 72:105–114

    Article  PubMed  CAS  Google Scholar 

  15. Larsson C (1985) Plasma membranes. In: Linskens HF, Jackson JF (eds) Modern methods of plant analysis, cell components, new series, vol 1. Springer-Verlag, Berlin, pp 85–104

    Google Scholar 

  16. Yamazaki T, Kawamura Y, Uemura M (2009) Extracellular freezing-induced mechanical stress and surface area regulation on the plasma membrane in cold-acclimated plant cells. Plant Signal Behav 4:231–233

    Article  PubMed  CAS  Google Scholar 

  17. Minami A, Fujiwara M, Furuto A et al (2009) Alterations in detergent-resistant plasma membrane microdomains in Arabidopsis thaliana during cold acclimation. Plant Cell Physiol 50:341–359

    Article  PubMed  CAS  Google Scholar 

  18. Meisrimler CN, Planchon S, Renaut J et al (2011) Alteration of plasma membrane-bound redox systems of iron deficient pea roots by chitosan. J Proteomics 74:437–449

    Article  Google Scholar 

  19. Takahashi D, Kawamura Y, Yamashita T et al (2011) Detergent-resistant plasma membrane proteome in oat and rye: similarities and dissimilarities between two monocotyledoneous plants. J Proteome Res 11:1654–1665

    Article  Google Scholar 

  20. Lüthje S, Van Gestelen P, Córdoba-Pedregosa MC et al (1998) Quinojnes in plant plasma membranes—a missing link? Protoplasma 205:43–51

    Article  Google Scholar 

  21. Mika A, Boenisch MJ, Hopff D et al (2010) Membrane-bound guaiacol peroxidases from maize (Zea mays L.) roots are regulated by methyl jasmonate, salicylic acid, and pathogen elicitors. J Exp Bot 61:831–841

    Article  PubMed  CAS  Google Scholar 

  22. Bérczi A, Larsson C, Widell S et al (1989) On the presence of inside-out plasma membrane vesicles and vanadate-inhibited K+/Mg2+-ATPase in microsomal fractions from wheat and maize roots. Physiol Plant 77:12–19

    Article  Google Scholar 

  23. Alexandersson E, Gustavsson N, Bernfur K et al (2008) Purification and proteomic analysis of plant plasma membranes. Methods Mol Biol 432:161–173

    Article  PubMed  CAS  Google Scholar 

  24. Aidemark M, Andersson CJ, Rasmusson AG et al (2009) Regulation of callose synthase activity in situ in alamethicin-permeabilized Arabidopsis and tobacco suspension cells. BMC Plant Biol 9:27

    Article  PubMed  Google Scholar 

  25. Bérczi A, Lüthje S, Asard H (2001) b-Type cytochromes in plasma memrbranes of Phaseolus vulgaris hypocotyls, Arabidopsis thaliana leaves, and Zea mays roots. Protoplasma 217:50–55

    Article  PubMed  Google Scholar 

  26. Ratajczak R, Hinz G, Robinson DG (1999) Localization of pyrophosphatase in membranes of cauliflower inflorescence cells. Planta 208:205–211

    Article  PubMed  CAS  Google Scholar 

  27. Oberbeck K, Drucker M, Robinson DG (1994) V-ATPase and pyrophosphatase in endomembranes of maize roots. J Exp Bot 45:235–244

    Article  CAS  Google Scholar 

  28. Whiteman SA, Nühse TS, Ashford D et al (2008) A proteomic and phosphoproteomic analysis of Oryza sativa plasma membrane and vacuolar membrane. Plant J 56:146–156

    Article  PubMed  CAS  Google Scholar 

  29. Bérzci A, Asard H (2003) Soluble proteins, an often overlooked contaminant in plasma membrane preparations. Trends Plant Sci 8:250–251

    Article  Google Scholar 

  30. Costa MMR, Hilliou F, Durate P et al (2008) Molecular cloning and characterization of a vacuolar class III peroxidase involved in the metabolism of anticancer alkaloids in Catharanthus roseus. Plant Physiol 146:403–417

    Article  PubMed  CAS  Google Scholar 

  31. Mika A, Lüthje S (2003) Properties of guaiacol peroxidase activities isolated from corn root plasma membranes. Plant Physiol 132:1489–1498

    Article  PubMed  CAS  Google Scholar 

  32. Kukavica BM, Veljović-Jovanovic SD, Menckhoff L et al (2012) Cell wall bound cationic and anionic class III isoperoxidases of pea root—biochemical characterisation and function in root growth. J Exp Bot 63:4631–4645

    Article  PubMed  CAS  Google Scholar 

  33. Wittig I, Karas M, Schägger H (2007) High resolution clear native electrophoresis for in-gel functional analysis and fluorescence studies of membrane protein complexes. Mol Cell Proteomics 6:1215–1225

    Article  PubMed  CAS  Google Scholar 

  34. Thomas PE, Ryan D, Levin W (1976) An improved staining procedure for the detection of the peroxidase activity of cytochrome P-450 on sodium dodecyl sulfate polyacrylamide gels. Anal Biochem 75:168–176

    Article  PubMed  CAS  Google Scholar 

  35. Miller DJ, Nicholas DJD (1984) 3′,5,5′-Tetramethylbenzidine/H2O2 staining is not specific for heme proteins separated by gel electrophoresis. Anal Biochem 140:577–580

    Article  PubMed  CAS  Google Scholar 

  36. Herzog V, Fahimi HD (1973) A new sensitive colorimetric assay for peroxidase using 3,3′-diaminobenzidine as hydrogen donor. Anal Biochem 55:554–562

    Article  PubMed  CAS  Google Scholar 

  37. Guilbault GC, Kramer DN (1964) 4-Methoxy-α-naphthol as a spectrophotometric reagent substrate for measuring peroxidatic activity. Anal Chem 36:2494–2496

    Article  CAS  Google Scholar 

  38. Bergmeyer HU, Gawehn K, Grassl M (1974) Enzymes as biochemical reagents. In: Bergmeyer HU (ed) Methods of enzymatic analysis, 2nd ed, vol 1. Academic, New York, NY, pp 495–496

    Google Scholar 

  39. Führs H, Götze S, Specht A et al (2009) Characterization of leaf apoplastic peroxidases and metabolites in Vigna uniguiculata in response to toxic manganese supply and silicon. J Exp Bot 60:1663–1678

    Article  PubMed  Google Scholar 

  40. Lüthje S, Hopff D, Schmitt A et al (2009) Hunting for low abundant redox proteins in plant plasma membranes. J Proteomics 73:475–483

    Article  Google Scholar 

  41. Wilm M, Shevchenko A, Houthaeve T et al (1996) Femtomole sequencing of proteins from polyacrylamide gels by nano-electrospray mass spectrometry. Nature 379:466–469

    Article  PubMed  CAS  Google Scholar 

  42. Bradford MM (1976) A rapid and sensitive method for quantification of microgram quantities of protein dye binding. Anal Biochem 72:248–254

    Article  PubMed  CAS  Google Scholar 

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Acknowledgments

This work was supported by the Deutsche Forschungsgemeinschaft (DFG Lu 668/4-4) and the University of Hamburg (Young Researcher Initiative grant to C.N. Meisrimler and PhD student grant to D. Hopff).

Author information

Authors and Affiliations

  1. Biocentre Klein Flottbek, University of Hamburg, Hamburg, Germany

    Sabine Lüthje, David Hopff, Tim Schütze, Jenny Köppe & Katrin Heino

  2. Leibniz Institute of Plant Genetics and Crop Plant Research, Gatersleben, Germany

    Claudia-Nicole Meisrimler

Authors
  1. Sabine Lüthje
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  2. Claudia-Nicole Meisrimler
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  3. David Hopff
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  4. Tim Schütze
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  5. Jenny Köppe
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  6. Katrin Heino
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Editor information

Editors and Affiliations

  1. Dept of Biochem & Molecular Bio, University of Cordoba, Cordoba, Spain

    Jesus V. Jorrin-Novo

  2. University of Tsukuba National Institute of Crop Science, Tsukuba, Japan

    Setsuko Komatsu

  3. Molecular Systems Biology, University of Vienna, Vienna, Austria

    Wolfram Weckwerth

  4. Dept. Molecular Systems Biology, University of Vienna, Vienna, Austria

    Stefanie Wienkoop

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Lüthje, S., Meisrimler, CN., Hopff, D., Schütze, T., Köppe, J., Heino, K. (2014). Class III Peroxidases. In: Jorrin-Novo, J., Komatsu, S., Weckwerth, W., Wienkoop, S. (eds) Plant Proteomics. Methods in Molecular Biology, vol 1072. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-631-3_48

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  • DOI: https://doi.org/10.1007/978-1-62703-631-3_48

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  • Publisher Name: Humana Press, Totowa, NJ

  • Print ISBN: 978-1-62703-630-6

  • Online ISBN: 978-1-62703-631-3

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