Measurement of chloroplast ATP synthesis activity in Arabidopsis

Aleel K. Grennan, Donald Richard Ort

Research output: Chapter in Book/Report/Conference proceedingChapter

Abstract

There are numerous options for monitoring ATP synthesis in chloroplasts using isolated thylakoid membranes, intact chloroplasts, and even whole leaves. Currently, the most commonly used method employs isolated thylakoids coupling the synthesis of ATP to light emission from luciferin in a reaction catalyzed by luciferase. The luciferin-luciferase assay can be highly sensitive and is a direct measure of ATP. Another direct measurement of ATP is the incorporation of 32P into ATP, which, while more technically difficult, has the advantage over the luciferin-luciferase assay of being able to distinguish newly synthesized from total ATP. The phosphorylation of ADP results in a net decrease in pK a (acid disassociation constant) between the reactants and the product ATP, resulting in an increase in the pH of the assay media, which can be used as a convenient, continuous measurement of ATP synthesis. The formation of Δμ H+ across the thylakoid membrane and its concomitant dissipation as ATP is synthesized can be measured by an electrochromic absorption band shift (ECS) of thylakoid pigments measured at 518 nm (Witt, Biochim. Biophys. Acta 505:355-427, 1979; Petty and Jackson, Biochim. Biophys. Acta: Bioenergetics 547:463-473, 1979). The first-order decay time of the ESC can be used to estimate the rate of ATP synthesis providing a noninvasive, indirect method for measuring ATP synthase activity that can be used with intact leaves.

Original languageEnglish (US)
Title of host publicationChloroplast Research in Arabidopsis
Subtitle of host publicationMethods and Protocols, Volume II
Pages343-355
Number of pages13
Volume775
DOIs
StatePublished - Oct 24 2011

Publication series

NameMethods in Molecular Biology
Volume775
ISSN (Print)1064-3745

Fingerprint

Chloroplasts
Arabidopsis
Adenosine Triphosphate
Thylakoids
Luciferases
Adenosine Diphosphate
Energy Metabolism
Phosphorylation
Light
Acids

Keywords

  • ATP synthase
  • Electrochromic shift
  • pH change
  • Proton motive force
  • Thylakoid

ASJC Scopus subject areas

  • Molecular Biology
  • Genetics

Cite this

Grennan, A. K., & Ort, D. R. (2011). Measurement of chloroplast ATP synthesis activity in Arabidopsis. In Chloroplast Research in Arabidopsis: Methods and Protocols, Volume II (Vol. 775, pp. 343-355). (Methods in Molecular Biology; Vol. 775). https://doi.org/10.1007/978-1-61779-237-3_19

Measurement of chloroplast ATP synthesis activity in Arabidopsis. / Grennan, Aleel K.; Ort, Donald Richard.

Chloroplast Research in Arabidopsis: Methods and Protocols, Volume II. Vol. 775 2011. p. 343-355 (Methods in Molecular Biology; Vol. 775).

Research output: Chapter in Book/Report/Conference proceedingChapter

Grennan, AK & Ort, DR 2011, Measurement of chloroplast ATP synthesis activity in Arabidopsis. in Chloroplast Research in Arabidopsis: Methods and Protocols, Volume II. vol. 775, Methods in Molecular Biology, vol. 775, pp. 343-355. https://doi.org/10.1007/978-1-61779-237-3_19
Grennan AK, Ort DR. Measurement of chloroplast ATP synthesis activity in Arabidopsis. In Chloroplast Research in Arabidopsis: Methods and Protocols, Volume II. Vol. 775. 2011. p. 343-355. (Methods in Molecular Biology). https://doi.org/10.1007/978-1-61779-237-3_19
Grennan, Aleel K. ; Ort, Donald Richard. / Measurement of chloroplast ATP synthesis activity in Arabidopsis. Chloroplast Research in Arabidopsis: Methods and Protocols, Volume II. Vol. 775 2011. pp. 343-355 (Methods in Molecular Biology).
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