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Characterization of Secretory Phospholipase A 2 with Phospholipase A 1 Activity in Tobacco, Nicotiana tabacum (L.)
Author(s) -
Fujikawa Yukichi,
Fujikawa Ritsuko,
Iijima Noriaki,
Esaka Muneharu
Publication year - 2012
Publication title -
lipids
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.601
H-Index - 120
eISSN - 1558-9307
pISSN - 0024-4201
DOI - 10.1007/s11745-011-3632-3
Subject(s) - nicotiana tabacum , biochemistry , phosphatidylethanolamine , biology , phospholipase c , complementary dna , glycerophospholipids , phospholipase a , phospholipase , phosphatidylcholine , phospholipid , enzyme , phospholipase a2 , membrane , gene
Abstract A cDNA encoding protein with homology to plant secretory phospholipase A 2 (sPLA 2 ), denoted as Nt1 PLA 2 , 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 PLA 2 has 12 cysteines, Ca 2+ binding loop and catalytic site domain that are commonly conserved in plant sPLA 2 s. The recombinant Nt1 PLA 2 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 PLA 2 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 Ca 2+ essentially, and maximal activities were found in an alkaline region when phosphatidylcholine, phosphatidylglycerol or phosphatidylethanolamine was used as a substrate. The level of Nt1 PLA 2 mRNA was detected at a higher level in tobacco flowers than stem, leaves and roots, and was induced by salicylic acid.