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Measurement of chloroplast ATP synthesis activity in Arabidopsis.

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 pKa (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 ΔμH+ 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.

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  • Publisher Full Text
  • Authors

    Grennan AK, Ort DR

    Institution

    Department of Plant Biology, University of Illinois, Urbana, IL, USA.

    Source

    Methods in molecular biology (Clifton, N.J.) 775: 2011 pg 343-55

    MeSH

    Adenosine Diphosphate
    Adenosine Monophosphate
    Adenosine Triphosphate
    Arabidopsis
    Chloroplasts
    Color
    Firefly Luciferin
    Hydrogen-Ion Concentration
    Luciferases, Firefly
    Luminescent Measurements
    Phosphorus Radioisotopes
    Phosphorylation

    Pub Type(s)

    Journal Article

    Language

    eng

    PubMed ID

    21863453