METABOLISM OF INOSITOL PHOSPHATES IN THE PROTOZOAN PARAMECIUM - CHARACTERIZATION OF A NOVEL INOSITOL-HEXAKISPHOSPHATE-DEPHOSPHORYLATING ENZYME

Basal and stimulated levels of inositol phosphates were determined in the protozoan Paramecium labelled with myo-[H-3]inositol. Under resting conditions, intracellular InsP6 (phytic acid), InsP5 and InsP4 concentrations were 140, 10 and 2-mu-M, respectively. InsP5 was comprised of 56% Ins(1,2,3,4,5)P5 and/or Ins(1,2,3,5,6)P5, 40% Ins(1,2,4,5,6)P5 and/or Ins(2,3,4,5,6)P5 and small amounts of Ins(1,3,4,5,6)P5 and Ins(1,2,3,4,6)Ps. InsP4 was mainly Ins(1, 4, 5, 6)P4 and/or Ins(3, 4, 5, 6)P4. Other inositol phosphates were not detected at a detection limit of 50 - 85 nM. Using various depolarizing and hyperpolarizing stimuli, no significant changes in level of inositol phosphates were observed in vivo, indicating that in the ciliate a contribution of inositol phosphates to signal-transduction mechanisms is unlikely. In homogenates prepared from myo-[H-3]inositol-labelled cells, a marked relative increase in InsP3 and InsP4 over the concentrations in vivo was observed. These inositol phosphates were identified as degradation products of endogenous InSP6. A novel separation methodology for inositol phosphates was established to allow unequivocal assignment of phosphate locations of all dephosphorylated InSP6-derived products. The dephosphorylation was catalyzed by a phytase-like enzyme with a molecular mass of 240 kDa, most likely of a hexameric structure. The enzyme had a pH optimum of 7.0 and did not require divalent cations for activity. Substrate concentrations above 300-mu-M were inhibitory. Dephosphorylation of InsP6 by the Paramecium enzyme differs from that of phytases from plants in that it proceeds via a sequential release of phosphate groups from positions 6, 5, 4 and 3 of the myo-inositol ring or/and positions 4, 5, 6 and 1.