SUBCELLULAR COMPARTMENTATION OF PYROPHOSPHATE AND DARK LIGHT KINETICS IN COMPARISON TO FRUCTOSE 2,6-BISPHOSPHATE

Subcellular compartmentation of pyrophosphate (PP(i)) was determined by rapid membrane filtration of evacuolated oat mesophyll protoplasts. By improving both the extraction procedure and its assay via bioluminescence, PP(i) recovery from samples was quantitative and linear down to below 200 fmol. Based on the content of the different fractions obtained after membrane filtration and compared to the respective pools of marker metabolites [cytosol, fructose 2,6-bisphosphate (F26BP); chloroplast stroma, ribulose bisphosphate] rather than enzymes, we found ca 2/3 of the total cellular content to be chloroplast-associated. Referred to compartmental volumes, cytosolic and stromal concentrations of PP(i) were nearly equal (70-100-mu-M). PP(i) was higher in evacuolated compared to vacuolated protoplasts which indicates a possible role of the tonoplast-located H4 pumping PP(i)ase in regulating the cellular pool size of PP(i). During dark-light-transition the pool sizes of PP(i) changed only marginally in both vacuolated and evacuolated protoplasts, while there were pronounced changes in those of F26BP, starch and sucrose. Thus our findings support the notion that the cellular pool size of PP(i) is kept rather constant. They are, however, in contrast to the assumption that appreciable PP(i) levels only exist in the cytosol.