title: Identification of a secretory phospholipase A2 from Papaver somniferum L. that transforms membrane phospholipids.
abstract: The full-length sequence of a new secretory phospholipase A2 was identified in opium poppy seedlings (Papaver somniferum L.). The cDNA of poppy phospholipase A2, denoted as pspla2, encodes a protein of 159 amino acids with a 31 amino acid long signal peptide at the N-terminus. PsPLA2 contains a PLA2 signature domain (PA2c), including the Ca(2+)-binding loop (YGKYCGxxxxGC) and the catalytic site motif (DACCxxHDxC) with the conserved catalytic histidine and the calcium-coordinating aspartate residues. The aspartate of the His/Asp dyad playing an important role in animal sPLA2 catalysis is substituted by a serine residue. Furthermore, the PsPLA2 sequence contains 12 conserved cysteine residues to form 6 structural disulfide bonds. The calculated molecular weight of the mature PsPLA2 is 14.0 kDa. Based on the primary structure PsPLA2 belongs to the XIB group of PLA2s. Untagged recombinant PsPLA2 obtained by expression in Escherichia coli, renaturation from inclusion bodies and purification by cation-exchange chromatography was characterized in vitro. The pH optimum for activity of PsPLA2 was found to be pH 7, when using mixed micelles of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and Triton X-100. PsPLA2 specifically cleaves fatty acids from the sn-2 position of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine and shows a pronounced preference for PC over phosphatidyl ethanolamine, -glycerol and -inositol. The active recombinant enzyme was tested in vitro against natural phospholipids isolated from poppy plants and preferably released the unsaturated fatty acids, linoleic acid and linolenic acid, from the naturally occurring mixture of substrate lipids.
authors.affiliation: Department of Cell and Molecular Biology of Drugs, Faculty of Pharmacy, Comenius University in Bratislava, Kalinčiakova 8, 832 32 Bratislava, Slovakia.
authors.forename: Veronika
authors.initials: V
authors.lastname: Jablonická
authors.affiliation: Department of Cellular Biochemistry, Institute of Biochemistry and Biotechnology, Martin-Luther-University Halle-Wittenberg, Kurt-Mothes-Str.3, 06120 Halle (Saale), Germany.
authors.forename: Johanna
authors.initials: J
authors.lastname: Mansfeld
authors.affiliation: Department of Cellular Biochemistry, Institute of Biochemistry and Biotechnology, Martin-Luther-University Halle-Wittenberg, Kurt-Mothes-Str.3, 06120 Halle (Saale), Germany.
authors.forename: Ingo
authors.initials: I
authors.lastname: Heilmann
authors.affiliation: Department of Cell and Molecular Biology of Drugs, Faculty of Pharmacy, Comenius University in Bratislava, Kalinčiakova 8, 832 32 Bratislava, Slovakia. Electronic address: oblozinsky@fpharm.uniba.sk.
authors.forename: Marek
authors.initials: M
authors.lastname: Obložinský
authors.affiliation: Department of Cellular Biochemistry, Institute of Biochemistry and Biotechnology, Martin-Luther-University Halle-Wittenberg, Kurt-Mothes-Str.3, 06120 Halle (Saale), Germany.
authors.forename: Mareike
authors.initials: M
authors.lastname: Heilmann
mesh_terms: D000595:Amino Acid Sequence; D004926:Escherichia coli / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D019787:Linoleic Acid / Q000378:metabolism; D015394:Molecular Structure; D008970:Molecular Weight; D010207:Papaver / Q000201:enzymology* / Q000254:growth & development; D010713:Phosphatidylcholines / Q000737:chemistry*; D054497:Phospholipases A2, Secretory / Q000235:genetics / Q000378:metabolism*; D010743:Phospholipids / Q000737:chemistry / Q000302:isolation & purification; D020539:Sequence Analysis, Protein; D017962:alpha-Linolenic Acid / Q000378:metabolism
chemical_list: D010713:Phosphatidylcholines; D010743:Phospholipids; D017962:alpha-Linolenic Acid; D019787:Linoleic Acid; D054497:Phospholipases A2, Secretory; C028694:1-palmitoyl-2-oleoylphosphatidylcholine
keywords: Gene identification; Opium poppy phospholipid composition; Papaver somniferum L; Papaveraceae; Secretory phospholipase A(2); Substrate specificity