Fructose-2,6-bisphosphate, metabolites and 'coarse' control of pyrophosphate: fructose-6-phosphate phosphotransferase during triose-phosphate cycling in heterotrophic cell-suspension cultures of Chenopodium rubrum

Abstract

Experiments were carried out to determine whether pyrophosphate: fructose-6-phosphate phosphotransferase (PFP) catalyses the rapid recycling of triose phosphates that is found in the cytosol of heterotrophic cell cultures of Chenopodium rubrum L. (W.-D. Hatzfeld, M. Stitt, 1990, Planta, 180, 198-204). Oxygen uptake, carbohydrate turnover, fructose 2,6-bisphosphate (Fru2,6bisP), glycolytic intermediates, adenine and uridine nucleotides, pyrophosphate and the activity of PFP and glycolytic enzymes were monitored for 48 h after subculturing carbohydrate-depleted cells onto glucose. Immediately after transfer there was an increase in the amount of Fru2,6bisP, and of the hexose phosphate. The triose phosphates, fructose-1,6-bisphosphate and inorganic pyrophosphate increased gradually over the next 24 h. This was accompanied by a tripling in the extractable activity of PFP, but not of phosphofructokinase. The activity of fructose-1,6-bisphosphatase was 20-50fold lower than that of PFP. It is calculated that the activity of PFP is high enough to catalyse the observed rate of cycling between the triose phosphates and the hexose phosphates, based on the measured Vmax capacity of the enzyme, the known kinetic properties, and the measured levels of its reactants and Fru2,6bisP. The changes in the levels of Fru2,6bisP were not correlated with the rate of respiration. Instead, the rate of O2 uptake was inversely related to the phosphoenolpyruvate level, showing that pyruvate kinase or phosphoenolpyruvate carboxylase are regulating the use of glucose for respiration. There was also no relation between Fru2,6bisP, and partitioning to sucrose or starch. It is proposed that the main function of the cycle in these cells is to maintain high levels of inorganic pyrophosphate and triose phosphates, which are necessary for the remobilisation of sucrose and for biosynthesis in the plastid, and that 'coarse' and 'fine' control of PFP play an important role in regulating this cycle.