Plant phospholipase Dα is an acidic phospholipase active at near-physiological Ca2+ concentrations

The conventional plant phospholipase D (PLD) requires Ca2+ for activity; however, the most distinct and puzzling feature of this PLD is its in vitro need for 20 to 100 mM Ca2+, This noncytoplasmic Ca2+ requirement has raised doubt about the role of Ca2+ in regulating its function in vivo, Using the cloned conventional castor bean PLD, PLD alpha, expressed in Escherichia coli, this study demonstrates that this PLD is active at micromolar, near-physiological concentrations of Ca2+, and this activity at low Ca2+ requires an acidic pH (4.5-5.5), By comparison, the newly cloned PLD beta and -gamma were active only at neutral pH under the same Ca2+ concentrations. This study also shows that PLDa activity at low Ca2+ needs substrates presented as a mixture of membrane lipids. Phosphatidylinositol 4,5-bisphosphate and phosphatidylinositol 4-phosphate are equally effective in stimulating the acidic PLD alpha activity, whereas phophatidylinositol is inactive. These results suggest that the conventional plant PLD in vivo is an acidic phospholipase that is active at near-physiological Ca2+ concentrations. The possible physiological significance of these findings will be discussed. (C) 1999 Academic Press.