Skip to main content
SpringerLink
Log in

Determination of Transglutaminase Activity in Plants

  • Protocol
  • First Online:
Polyamines

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1694))

Abstract

Transglutaminase (TGase:E.C. 2.3.2.13) catalyzes the acyl-transfer reaction between one or two primary amino groups of polyamines and protein-bound Gln residues giving rise to post-translational modifications. One increasing the positive charge on a proteins surface and the other results in the covalent crosslinking of proteins. Pioneering studies on TGase in plants started in the middle of the 1980’s but the methodology designed for use with animal extracts was not directly applicable to plant extracts. Here we describe radioactive and colorimetric methods adapted to study plant TGase, as well as protocols to analyze the involvement of TGase and polyamines in the functionality of cytoskeletal proteins.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution

Institutional subscriptions

References

  1. Folk JE, Park MH, Chung SI, Schrode J, Lester EP, Cooper HL (1980) Polyamines as physiological substrates for transglutaminases. J Biol Chem 255:3695–3700

    PubMed  CAS  Google Scholar 

  2. Beninati S, Martinet N, Folk JE (1988) High-performance liquid chromatographic method for the determination of ε-(γ-glutamyl)lysine and mono- and bis-γ-glutamyl derivatives of putrescine and spermidine. J Chromatogr A 443:329–335

    Article  CAS  Google Scholar 

  3. Siepaio MP, Meunier JCF (1995) Diamine oxidase and transglutaminase activities in white lupin seedlings with respect to cross-linking of proteins. J Agric Food Chem 43:1151–1156

    Article  CAS  Google Scholar 

  4. Lilley GR, Skill J, Griffin M, Bonner PL (1998) Detection of Ca2+-dependent transglutaminase activity in root and leaf tissue of monocotyledonous and dicotyledonous plants. Plant Physiol 117:1115–1123

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Slaughter TF, Achyuthan KE, Lai T, Greenberg CS (1992) A microtiter plate transglutaminase assay utilising 5-(Biotinamido) pentylamine as substrate. Anal Biochem 205:166–171

    Article  CAS  PubMed  Google Scholar 

  6. Del Duca S, Beninati S, Serafini-Fracassini D (1995) Polyamines in chloroplasts: identification of their glutamyl and acetyl derivatives. Biochem J 305:233–237

    Article  PubMed  PubMed Central  Google Scholar 

  7. Del Duca S, Tidu V, Bassi R, Serafini-Fracassini D, Esposito C (1994) Identification of transglutaminase activity and its substrates in isolated chloroplast of Helianthus tuberosus. Planta 193:283–289

    Article  Google Scholar 

  8. Della Mea M, Di Sandro A, Dondini L, Del Duca S, Vantini F, Bergamini C, Bassi R, Serafini-Fracassini D (2004) A Zea mays 39 kDa thylakoid transglutaminase catalyses light harvesting complex II by polyamines in a light-dependent way. Planta 219:754–764

    Article  CAS  PubMed  Google Scholar 

  9. Della Mea M, Serafini-Fracassini D, Del Duca S (2007) Programmed cell death: similarities and differences in animals and plants. A flower paradigm. Amino Acids 33:395–404

    Article  CAS  PubMed  Google Scholar 

  10. Lorand L, Graham RM (2003) Transglutaminases: crosslinking enzymes with pleiotropic functions. Nat Rev Mol Cell Biol 4:140–156

    Article  CAS  PubMed  Google Scholar 

  11. Aloisi I, Cai G, Serafini-Fracassini D, Del Duca S (2016a) Polyamines in pollen: from microsporogenesis to fertilization. Front Plant Sci 7:155

    PubMed  PubMed Central  Google Scholar 

  12. Aloisi I, Cai G, Serafini-Fracassini D, Del Duca S (2016b) Transglutaminase as polyamine mediator in plant growth and differentiation. Amino Acids 48:2467–2478

    Article  CAS  PubMed  Google Scholar 

  13. Signorini M, Beninati S, Bergamini C (1991) Identification of transglutaminase activity in the leaves of silver beet (Beta vulgaris L.) J Plant Physiol 137:547–552

    Article  CAS  Google Scholar 

  14. Trigwell SM, Lynch PT, Griffin M, Hargreaves AJ, Bonner PL (2004) An improved colorimetric assay for the measurement of transglutaminase (type II) –(gamma-gltamyl) lysine cross-linking activity. Anal Biochem 330:164–166

    Article  CAS  PubMed  Google Scholar 

  15. Bonner PL (2007) Protein purification: the basics. Taylor and Francis, Abingdon,UK

    Book  Google Scholar 

  16. Di Sandro A, Del Duca S, Verderio E, Hargreaves AJ, Scarpellini A, Cai G, Cresti M, Faleri C, Iorio RA, Hirose S, Furutani Y, Coutts IGC, Griffin M, Bonner PLR, Serafini-Fracassini D (2010) An extracellular transglutaminase is required for apple pollen tube growth. Biochem J 429:261–271

    Article  CAS  PubMed  Google Scholar 

  17. Brown RE, Jarvis KL, Hyland KJ (1989) Protein measurement using bicinchoninic acid: elimination of interfering substances. AnalBiochem 180:136–139

    CAS  Google Scholar 

  18. Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685

    Article  CAS  Google Scholar 

  19. Towbin H, Staehelin T, Gordon J (1979) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc.Natl.Acad.Sci.USA 76:4350–4354

    Article  CAS  PubMed  Google Scholar 

  20. Del Duca S, Serafini-Fracassini D, Bonner PL, Cresti M, Cai G (2009) Effects of post-translational modifications catalyzed by pollen transglutaminase on the functional properties of microtubules and actin filaments. Biochem J 418:651–664

    Article  PubMed  Google Scholar 

  21. Agustin C, Maria EC, Carlos AA (2013) A novel method for purification of polymerizable tubulin with a high content of the acetylated isotype. Biochem J 449:643–648

    Article  CAS  Google Scholar 

  22. Moore RC, Zhang M, Cassimeris L, Cyr RJ (1997) In vitro assembled plant microtubules exhibit a high state of dynamic instability. Cell Motil Cytoskeleton 38:278–286

    Article  CAS  PubMed  Google Scholar 

  23. Vallee RB (1982) A taxol-dependent procedure for the isolation of microtubules and microtubule-associated proteins (MAPs). J Cell Biol 92:435–442

    Article  CAS  PubMed  Google Scholar 

  24. Williams RC Jr, Lee JC (1982) Preparation of tubulin from brain. Methods Enzymol 85(Pt B):376–385

    Article  CAS  PubMed  Google Scholar 

  25. Carter NJ, Cross RA (2010) Microtubule motility assays. In: Kreitzer G, Jualin F, Espenel C (eds) Cel biology assays. Academic Press, Amsterdam, Boston

    Google Scholar 

  26. Meyer D, Rines DR, Kashina AS, Cole DG, Scholey JM (1998) Purification of novel kinesins from embryonic systems. Methods Enzymol 298:133–154

    Article  CAS  PubMed  Google Scholar 

  27. Cole DG, Scholey JM (1995) Purification of kinesin-related protein complexes from eggs and embryos. Biophysical J 68:158s–162s

    CAS  Google Scholar 

  28. Wagner MC, Pfister KK, Bloom GS, Brady ST (1989) Copurification of kinesin polypeptides with microtubule- stimulated mg-ATPase activity and kinetic analysis of enzymatic properties. Cell Motil Cytoskel 12:195–215

    Article  CAS  Google Scholar 

  29. Ma YZ, Taylor EW (1997) Interacting head mechanism of microtubule-kinesin ATPase. J Biol Chem 272:724–730

    Article  CAS  PubMed  Google Scholar 

  30. Liu X, Yen L-F (1992) Purification and characterization of actin from maize pollen. Plant Physiol 99:1151–1155

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Homma K, Saito J, Ikebe R, Ikebe M (2001) Motor function and regulation of myosin X. J Biol Chem 276:34348–34354

    Article  CAS  PubMed  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Department of Biological, Geological and Environmental Sciences, University of Bologna, via Irnerio 42, 40126, Bologna, Italy

    S. Del Duca, I. Aloisi & D. Serafini-Fracassini

  2. School of Science and Technology, Nottingham Trent University, Clifton Lane, Nottingham, NG11 8NS, UK

    P. L. R. Bonner

  3. Department of Life Sciences, University of Siena, via Mattioli 4, 53100, Siena, Italy

    G. Cai

Authors
  1. S. Del Duca
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. P. L. R. Bonner
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. I. Aloisi
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. D. Serafini-Fracassini
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. G. Cai
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to S. Del Duca .

Editor information

Editors and Affiliations

  1. Department of Biology, Healthcare and Environment, Faculty of Pharmacy and Food Sciences, Section of Plant Physiology, University of Barcelona, Spain

    Rubén Alcázar

  2. Department of Biology, Healthcare and Environment, Faculty of Pharmacy and Food Sciences, Section of Plant Physiology, University of Barcelona, Spain

    Antonio F. Tiburcio

Rights and permissions

Reprints and permissions

Copyright information

© 2018 Springer Science+Business Media LLC

About this protocol

Cite this protocol

Del Duca, S., Bonner, P.L.R., Aloisi, I., Serafini-Fracassini, D., Cai, G. (2018). Determination of Transglutaminase Activity in Plants. In: Alcázar, R., Tiburcio, A. (eds) Polyamines. Methods in Molecular Biology, vol 1694. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7398-9_18

Download citation

  • DOI: https://doi.org/10.1007/978-1-4939-7398-9_18

  • Published:

  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-7397-2

  • Online ISBN: 978-1-4939-7398-9

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation