Phospholipase D (PLD) is present in Leishmania donovani and its activity increases in response to acute osmotic stress

We report here that the signaling molecule phospholipase D (PLD) is present in the parasitic protozoan Leishmania donovani. In vitro enzymatic activit y is dependent on Ca2+ and Mg2+ ions. its basal activity is stimulated by p hosphatidyl-inositol-4,5-bisphosphate (PIP2), and its pH optima are pH 8.0 and pH 6.0. PLD activity increases 3-fold about 5 min after an abrupt decre ase in osmolality from 317 mOsm (isosmotic) to 155 mOsm and increases 1.5-f old in response to an abrupt increase in osmolality to 617 mOsM. Cells grow n for > 24 h under the anisosmotic conditions showed only marginal changes in activity compared to the controls grown under isosmotic conditions. indi cating an adaptation to long-term exposure to hypo- or hyper-osmolarity. Im munologically, two isoforms, PLD1 and PLD2, are present. An analysis of in vitro PLD activity in anti-PLD immunocomplexes revealed that either hypoton ic (cell swelling) or hypertonic stress (cell shrinking) causes an increase in PLD1 activation but a reduction in PLD2 activity. The interplay between these two isoforms results in a predominance for PLD1 in the observed incr ease when measuring total PLD activity. Finally, the increase in enzymatic activity in acute hyposmotic shock is accompanied by tyrosyl phosphorylatio n of the PLD1 isoform, suggesting a role for protein tyrosine kinase in the control of PLD activity in response to osmotic stress.