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 (PIP(2)-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 (PIP(2)-dependent PLD) and polyphosphoinositide-independent requiring millimolar concentrations of calcium (PLDalpha). Significantly different patterns of activity profiles were found for soluble and membrane-associated forms of all three enzymes within both processes. Membrane-associated PIP(2)-dependent PLD activity shows the opposite trend when compared to PLDalpha, while the highest PI-PLC activity appears in the same stages of development of seeds and plants as for PLDalpha. In subcellular fractions of hypocotyls of young plants, phospholipases were localized predominantly on plasma membranes. The biochemical characteristics (Ca(2+), pH) of all three enzymes associated with plasma membrane vesicles, isolated by partitioning in an aqueous dextran- polyethylene glycol two-phase system, are also described. Direct interaction of PLDalpha with G-proteins under in vitro conditions was not confirmed.

Mutual regulation of plant phospholipase D and the actin cytoskeleton

“…In contrast to Kusner et al. (2003), who used recombinant Arabidopsis PLDβ1 or a crude enzymatic preparation (commercial PLD from Brassica oleracea ), we utilized a purified plasma membrane (PM) fraction for in vitro measurements of distinct PLD activities (Novotná et al. , 2003).…”

“…The enriched plasma membrane fraction was prepared from a tobacco cell suspension culture by partitioning microsomes in an aqueous dextran/polyethylene glycol two‐phase system (Novotná et al. , 2003).…”

Aluminium ions inhibit the formation of diacylglycerol generated by phosphatidylcholine‐hydrolysing phospholipase C in tobacco cells

“…Cytosolic, microsomal and plasma membrane‐enriched fractions were prepared as described by Novotná et al. (2003).…”

Biology and Biochemistry of Plant Phospholipases

“…It has also been demonstrated that PIs can be targeted though vesicle trafficking to different locations throughout the cell (Kim et al, 2001), opening up the possibility that localization provides a means for discrimination between PLC/DGK and PLD signaling. Indeed, in developing Brassica napus seeds, PLC and PLD activities have been shown to differ in subcellular location (Novotna et al, 2003b). Studies on PA receptors that have been identified in plants to date have shed little light on how PA interacts with the receptors or to what extent structure or location are important for discrimination between different intracellular sources of PA (Testerink and Munnik, 2005).…”