title: Characterization of secretory phospholipase A₂ with phospholipase A₁ activity in tobacco, Nicotiana tabacum (L.).
abstract: A cDNA encoding protein with homology to plant secretory phospholipase A₂ (sPLA₂), denoted as Nt1 PLA₂, was isolated from tobacco (Nicotiana tabacum). The cDNA encodes a mature protein of 118 amino acid residues with a putative signal peptide of 29 residues. The mature form of Nt1 PLA₂ has 12 cysteines, Ca²⁺ binding loop and catalytic site domain that are commonly conserved in plant sPLA₂s. The recombinant Nt1 PLA₂ was expressed as a fusion protein with thioredoxin in E. coli BL21 cells and was purified by an ion exchange chromatography after digestion of the fusion proteins by Factor Xa protease to obtain the mature form. Interestingly, Nt1 PLA₂ could hydrolyze the ester bond at the sn-1 position of glycerophospholipids as well as at the sn-2 position, when the activities were determined using mixed-micellar phospholipids with sodium cholate. Both activities for the sn-1 and -2 positions of glycerophospholipids required Ca²⁺ essentially, and maximal activities were found in an alkaline region when phosphatidylcholine, phosphatidylglycerol or phosphatidylethanolamine was used as a substrate. The level of Nt1 PLA₂ mRNA was detected at a higher level in tobacco flowers than stem, leaves and roots, and was induced by salicylic acid.
authors: Graduate School of Biosphere Science, Hiroshima University, Higashi-Hiroshima, Hiroshima 739-8528, Japan.
authors: Yukichi
authors: Y
authors: Fujikawa
authors: Ritsuko
authors: R
authors: Fujikawa
authors: Noriaki
authors: N
authors: Iijima
authors: Muneharu
authors: M
authors: Esaka
mesh_terms: D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D054466:Phospholipases A1 / Q000737:chemistry* / Q000378:metabolism; D054497:Phospholipases A2, Secretory / Q000737:chemistry* / Q000378:metabolism; D010940:Plant Proteins / Q000737:chemistry* / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D016415:Sequence Alignment; D014026:Nicotiana / Q000201:enzymology* / Q000378:metabolism
chemical_list: D010940:Plant Proteins; D012333:RNA, Messenger; D011994:Recombinant Proteins; D054466:Phospholipases A1; D054497:Phospholipases A2, Secretory