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P-31 NMR saturation transfer experiments in Chlamydomonas reinhardtii : evidence for the NMR visibility of chloroplastidic Pi

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Abstract

ATP synthesis and consumption in respiring cells of the green alga Chlamydomonas reinhardtii were measured with 31P in vivo NMR saturation transfer experiments to determine the intracellular compartmentation of inorganic phosphate. Most of the observed flux towards ATP synthesis was catalyzed by the coupled enzymes glyceraldehyde-3-phosphate dehydrogenase/phosphoglycerate kinase (GAPDH/PGK). The attribution of the measured flux to these enzymes is supported by the observation, that (i) the magnetization transfer was strongly reduced by iodoacetate, an irreversible inhibitor of GAPDH and that (ii) the unidirectional flux was much greater than the net flux through the mitochondrial F0F1-ATPase as determined by oxygen consumption measurements. In Chlamydomonas, glycolysis is divided into a chloroplastidic and a cytosolic part with the enzymes GAPDH/PGK being located in the chloroplast stroma (Klein 1986). The 31P-NMR signal of inorganic phosphate must, therefore, originate from the chloroplast. The life time of the magnetic label transferred to Pi by these enzymes is too short for it to be transported to the cytosol via the phosphate translocator of the chloroplast envelope. When the intracellular compartmentation of Pi was taken into consideration the calculated unidirectional ATP synthesis rate was equal to the consumption rate, indicating operation of GAPDH/PGK near equilibrium. The assignment of most of the intracellular Pi to the chloroplast is in contradiction to earlier reports, which attributed the Pi signal to the cytosol. This is of special interest for the use of the chemical shift of the Pi signal as an intracellular pH-marker in plant cells.

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Abbreviations

3-PGA:

3-phosphoglycerate

CW:

continuous wave

dG6P:

2-deoxyglucose-6-phosphate

GAPDH:

glyceraldehyde-3-phosphate dehydrogenase

MO:

equilibrium z-magnetization

M0 :

instantaneous z-magnetization after selective saturation for time t

MDP:

methylene-diphosphonic acid

PDE:

phosphodiester

PGK:

phosphoglycerate kinase

Pi :

inorganic orthophosphate

polyP:

polyphosphate

T1 :

longitudinal relaxation time

τ1 :

longitudinal relaxation time with chemical exchange

TCA cycle:

tricarboxylic acid cycle

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Authors and Affiliations

  1. Fachbereich 1 - Physik, Universität Bremen, W-2800, Bremen 33, Germany

    S. Hentrich & A. Mayer

  2. Fachbereich 2 - Biologie, Universität Bremen, W-2800, Bremen 33, Germany

    M. Hebeler & L. H. Grimme

  3. Fachbereich 2 - Chemie, Universität Bremen, W-2800, Bremen 33, Germany

    D. Leibfritz

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  1. S. Hentrich
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  2. M. Hebeler
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  3. L. H. Grimme
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  4. D. Leibfritz
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  5. A. Mayer
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Correspondence to: A. Mayer

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Hentrich, S., Hebeler, M., Grimme, L.H. et al. P-31 NMR saturation transfer experiments in Chlamydomonas reinhardtii : evidence for the NMR visibility of chloroplastidic Pi . Eur Biophys J 22, 31–39 (1993). https://doi.org/10.1007/BF00205810

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