P-31 NMR SATURATION-TRANSFER EXPERIMENTS IN CHLAMYDOMONAS-REINHARDTII- EVIDENCE FOR THE NMR VISIBILITY OF CHLOROPLASTIDIC-P(I)

ATP synthesis and consumption in respiring cells of the green alga Chl amydomonas reinhardtii were measured with P-31 in vivo NMR saturation transfer experiments to determine the intracellular compartmentation o f inorganic phosphate. Most of the observed flux towards ATP synthesis was catalyzed by the coupled enzymes glyceraldehyde-3-phosphate dehyd rogenase/phosphoglycerate kinase (GAPDH/PGK). The attribution of the m easured flux to these enzymes is supported by the observation, that (i ) the magnetization transfer was strongly reduced by iodoacetate, an i rreversible inhibitor of GAPDH and that (ii) the unidirectional flux w as much greater than the net flux through the mitochondrial F0F1-ATPas e 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 P-31-NMR signal of inorganic phosphate must, therefore, ori ginate from the chloroplast. The life time of the magnetic label trans ferred to P(i) by these enzymes is too short for it to be transported to the cytosol via the phosphate translocator of the chloroplast envel ope. When the intracellular compartmentation of P(i) was taken into co nsideration the calculated unidirectional ATP synthesis rate was equal to the consumption rate, indicating operation of GAPDH/PGK near equil ibrium. The assignment of most of the intracellular P(i) to the chloro plast is in contradiction to earlier reports, which attributed the P(i ) signal to the cytosol. This is of special interest for the use of th e chemical shift of the P(i) signal as an intracellular pH-marker in p lant cells.