In vitro distribution and characterization of membrane-associated pld and pi-plc in brassica napus

Abstract

Two types of phospholipid degrading enzyme, phospholipase D (PLD; EC 3.1.4.4) and phosphatidyl-inositol-specific phospholipase C (PIP2-PLC; PI-PLC 3.1.4.11) were studied during the development of seeds and plants of Brassica napus. PLD exhibits two types of activity; polyphosphoinositide-requiring (PIP2-dependent PLD) and polyphosphoinositide-independent requiring millimolar concentrations of calcium (PLDα). Significantly different patterns of activity profiles were found for soluble and membrane-associated forms of all three enzymes within both processes. Membrane-associated PIP2-dependent PLD activity shows the opposite trend when compared to PLDα, while the highest PI-PLC activity appears in the same stages of development of seeds and plants as for PLDα. In subcellular fractions of hypocotyls of young plants, phospholipases were localized predominantly on plasma membranes. The biochemical characteristics (Ca2+, pH) of all three enzymes associated with plasma membrane vesicles, isolated by partitioning in an aqueous dextranpolyethylene glycol two-phase system, are also described. Direct interaction of PLDα with G-proteins under in vitro conditions was not confirmed.