Skip to main content
SpringerLink
Log in

Extraction of the Functional Manganese and Calcium From Photosystem II

  • Protocol
Photosynthesis Research Protocols

Part of the book series: Methods In Molecular Biology™ ((MIMB,volume 274))

  • 1768 Accesses

Abstract

Manganese (Mn) and calcium (Ca) are both metal cofactors required for photosynthetic oxygen evolution (1). The functional roles for these ions in the O2-evolving reactions are not completely known. They are studied by comparative spectroscopic measurements between intact and metal-depleted samples. In this chapter, we outline three experimental procedures used for the various removal of Mn and Ca from photosystem (PS) II-containing (i.e,. O2-evolving) preparations: the complete Mn extraction using a strong alkaline Ches buffer (pH 9.4)/MgCl2 wash, partial Mn extraction using a mild hydroxylamine (pH 6.8) wash, and specific Ca extraction through a low pH/citrate (pH 3) wash. The O2 evolution activities (measured by a Clarke-type oxygen electrode), protein composition (determined by sodium dodecyl sulfate- polyacrylamide gel electrophoresis), and the relative Mn and Ca content (measured by atomic absorption spectroscopy) are reported for each extraction procedure.

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

Access this chapter

Log in via an institution
Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 74.99
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Bricker, T. M. and Ghanotakis, D. F. (1996) Introduction to oxygen evolution and the oxygen-evolving complex. In: Oxygenic Photosynthesis: The Light Reactions (Ort, D. R. and Yocum, C. F., eds.), Kluwer Academic Publishers, Dordrecht, pp. 113–136.

    Google Scholar 

  2. Zouni, A., Witt, H. T., Kern, J., Fromme, P., Krauß, N., Saenger, W., and Orth, P. (2001) Crystal structure of photosystem II from Synechococcus elongatus at 3.8 Å resolution. Nature 409, 739–743.

    Article  PubMed  CAS  Google Scholar 

  3. Kamiya, N. and Shen, J.-R. (2003) Crystal structure of oxygen-evolving photosystem II from Thermosynechococcus vulcansus at 3.7 Å resolution. Proc. Natl. Acad. Sci. USA 100, 98–103.

    Article  PubMed  CAS  Google Scholar 

  4. Berthold, D. A., Babcock, G. T., and Yocum, C. F. (1981) A highly resolved, oxygen-evolving photosystem II preparation from spinach thylakoid membranes. FEBS Lett. 13, 231–233.

    Article  Google Scholar 

  5. Porra, R. J., Thompson, W. A., and Kriedemann, P. E. (1989) Determination of accurate extinction coefficients and simultaneous equations for assaying chlorophylls a and b extracted with four different solvents: verification of the concentration of chlorophyll standards by atomic absorption. Biochim. Biophys. Acta 975, 384–394.

    Article  CAS  Google Scholar 

  6. Hunziker, D., Abramowicz, D. A., Damoder, R., and Dismukes, G. C. (1987) Evidence for an association between 33 kDa extrinsic membrane protein, manganese and photosynthetic oxygen evolution in correlation with the S2 multiline EPR signal. Biochim. Biophys. Acta 890, 6–14.

    Article  CAS  Google Scholar 

  7. Kuwabara, T. and Murata, N. (1983) Quantitative analysis of the inactivation of photosynthetic oxygen evolution and the release of polypeptides and manganese in the photosystem II particles of spinach chloroplasts. Plant and Cell Physiol. 24, 741–747.

    CAS  Google Scholar 

  8. Tamura, N. and Cheniae, G. (1987) Photoactivation of the water-oxidizing complex in photosystem II membranes depleted of Mn and extrinsic proteins. I. Biochemical and kinetic characterization. Biochim. et Biophys. Acta 890, 179–194.

    Article  CAS  Google Scholar 

  9. Latimer, M. J., DeRose, V. J., Mukerji, I., Yachandra, V. K., Sauer, K., and Klein, M. P. (1995) Evidence for the proximity of calcium to the manganese cluster of photosystem II: determination by x-ray absorption spectroscopy. Biochemistry 34, 10,898–10,909.

    Article  PubMed  CAS  Google Scholar 

  10. Walker, D. (1990) “The Use of the Oxygen Electrode and Fluorescence Probes in Simple Measurements of Photosynthesis,” Oxygraphics Limited, Sheffield, UK.

    Google Scholar 

  11. Ananyev, G. M. and Dismukes, C. G. (1996) Assembly of the tetra-Mn site of photosynthetic water oxidation by photoactivation: Mn stoichiometry and detection of a new intermediate. Biochemistry 35, 4102–4109.

    Article  PubMed  CAS  Google Scholar 

  12. Vrettos, J. S., Stone, D. A., and Brudvig, G. W. (2001) Quantifying the ion selectivity of the Ca2+ site in photosystem II: evidence for direct involvement of Ca2+ in O2 formation. Biochemistry 40, 7937–7945.

    Article  PubMed  CAS  Google Scholar 

  13. Laemmli, U. K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680–685.

    Article  PubMed  CAS  Google Scholar 

  14. Hansson, Ö. and Wydrzynski, T. (1990) Current perceptions of Photosystem II. Photosynth. Res. 23, 131–162.

    Article  CAS  Google Scholar 

  15. Shen, J.-R. and Inoue, Y. (1991) Plant Cell Physiol. 32, 453–457.

    CAS  Google Scholar 

  16. Kashino, Y., Satoh, K., and Katoh, S. (1986) A simple procedure to determine the Ca in oxygen-evolving preparations from Synechococccus sp. FEBS Lett. 205, 150–154.

    Article  CAS  Google Scholar 

  17. Shen, J.-R., Satoh, K., and Katoh, S. (1988) Calclium content of oxygen-evolving photosystem II preparations from higher plants: effect of NaCl treatment. Biochim. Biophys. Acta 933, 358–364.

    Article  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Photobioenergetics, Research School of Biological Sciences, The Australian National University, Canberra, Australia

    Joel Freeman, Garth Hendry & Tom Wydrzynski

Authors
  1. Joel Freeman
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Garth Hendry
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Tom Wydrzynski
    View author publications

    You can also search for this author in PubMed Google Scholar

Editor information

Editors and Affiliations

  1. Départment de Chimie-Biologie (GREIB), Université du Québec à Trois-Rivières, Trois-Rivières, Québec, Canada

    Robert Carpentier

Rights and permissions

Reprints and permissions

Copyright information

© 2004 Humana Press Inc., Totowa, NJ

About this protocol

Cite this protocol

Freeman, J., Hendry, G., Wydrzynski, T. (2004). Extraction of the Functional Manganese and Calcium From Photosystem II. In: Carpentier, R. (eds) Photosynthesis Research Protocols. Methods In Molecular Biology™, vol 274. Humana Press. https://doi.org/10.1385/1-59259-799-8:205

Download citation

  • DOI: https://doi.org/10.1385/1-59259-799-8:205

  • Publisher Name: Humana Press

  • Print ISBN: 978-1-58829-232-2

  • Online ISBN: 978-1-59259-799-4

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation