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Journal of Experimental Botany, Vol. 54, No. 383, pp. 691-698, February 1, 2003
© 2003 Oxford University Press

In vitro distribution and characterization of membrane-associated PLD and PI-PLC in Brassica napus

Received 30 April 2002; Accepted 10 October 2002

Zuzana Novotná, Jan Martinec1, Bronislava Profotová, Stepánka Zdárová, Jean-Claude Kader2 and Olga Valentová3,

Department of Biochemistry and Microbiology, Institute of Chemical Technology Prague, 166 28 Prague 6, Czech Republic

1 Present address: Institute of Experimental Botany, Academy of Sciences of Czech Republic, Prague 6, Czech Republic.
2 Present address: Laboratoire de Physiologie Cellulaire et Moléculaire, Université Pierre et Marie Curie,UMR 7632, Paris Cedex 05, France.
3 To whom correspondence should be addressed: Fax: +420 2 24355167. E-mail: olga.valentova{at}vscht.cz

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 (PLD{alpha}). 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 PLD{alpha}, while the highest PI-PLC activity appears in the same stages of development of seeds and plants as for PLD{alpha}. 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 dextran- polyethylene glycol two-phase system, are also described. Direct interaction of PLD{alpha} with G-proteins under in vitro conditions was not confirmed.

Key words: Brassica napus, phospholipases, plasma membrane.


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