Determination of the Inorganic Pyrophosphate Level and Its Subcellular Localization in Chara coraZZina

In order to determine the concentration of pyrophosphate (PPi) and its subcellular distribution in Chara corallina, a new method to concentrate PPi from cell extracts was developed. PPi was extracted and concentrated as CazPzO, under alkaline conditions. The amount of PPi in the precipitate was measured using an enzyme system containing pyrophosphate:fructose6-phosphate 1-phosphotransferase (EC 2.7.1.90) coupled to NADH oxidation in the presence of [ethylenebis(oxyethylenenitri10) Jtetraacetic acid. The subcellular localization of PPi and inorganic phosphate (Pi) was studied using the intracellular perfusion technique. The relative volumes of the cytoplasm (6.4%) and the vacuole (93.6%) were determined by perfusing Lucifer Yellow CH into the vacuole and by assuming that the Lucifer Yellow CH dead space represented the cytoplasmic volume. The volume of the chloroplast layer was determined microscopically, and it was found that it occupied 10% of the Chara cytoplasm. PPi was present predominantly in the cytosol at a level of 193 p ~ , while it existed in the vacuole at a level of only 2.20 pM and less than 1 pM in chloroplasts. By contrast, Pi was distributed almost equally in the cytosol (12.0 mM), chloroplasts (16.2 mM), and the vacuole (6.70 mM). The electrochemical potential gradient across the tonoplast for H+ (ASH+ = -11.6 to -18.0 KJ/mol) was nearly equal to the free energy release from the hydrolysis of PPi in cytoplasm (AGpp = -18.9 KJlmol), indicating that the H+-translocating inorganic pyrophosphatase can work as a H+ pump in C. corallina.