Molecular cloning, expression, and characterization of secretory phospholipase A2 in tobacco.
Phospholipase A2 (PLA2) activity was investigated in various tissues of tobacco (Nicotiana tabacum). PLA2 activity in the flower was 15 times higher than that in the leaf, stem, and root. PLA2 activity in the flower appears to have originated from both Ca2+-dependent and -independent PLA2. A cDNA clone for protein with homology to animal secretory PLA2 (sPLA2), denoted as Nt PLA2, was isolated from the tobacco flower. The cDNA of Nt PLA2 encoded a mature protein of 127 amino acid residues with a putative signal peptide of 30 residues. The amino acid sequence for mature Nt PLA2 contains 12 cysteines, a Ca2+ binding loop, and a catalytic domain that are commonly conserved in animal sPLA2. The Nt PLA2 mRNA was mainly expressed in the root and stem of tobacco. The recombinant Nt PLA2 was expressed as a fusion protein with thioredoxin in Escherichia coli. From the bacterial cell lysate, the fusion protein was recovered in soluble form and cleaved by Factor Xa proteinase. Then the recombinant mature Nt PLA2 was purified by ion exchange chromatography. It was discovered that the purified Nt PLA2 essentially requires Ca2+, for the enzyme activity when the activity was determined using mixed-micellar phospholipid substrates with sodium cholate. The optimal activity of Nt PLA2 was at pH 8-10 when PC was used as a substrate.
Fujikawa
Ritsuko
R
Graduate School of Biosphere Sciences, Hiroshima University, Higashi-Hiroshima, Japan.
Fujikawa
Yukichi
Y
Iijima
Noriaki
N
Esaka
Muneharu
M
D000595:Amino Acid Sequence; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism; D003001:Cloning, Molecular; D018076:DNA, Complementary; D004926:Escherichia coli / Q000235:genetics; D035264:Flowers / Q000201:enzymology; D015971:Gene Expression Regulation, Enzymologic; D018506:Gene Expression Regulation, Plant; D054501:Group II Phospholipases A2; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D010741:Phospholipases A / Q000235:genetics* / Q000378:metabolism*; D054467:Phospholipases A2; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013879:Thioredoxins / Q000235:genetics / Q000378:metabolism; D014026:Nicotiana / Q000201:enzymology* / Q000235:genetics
D018076:DNA, Complementary; D011993:Recombinant Fusion Proteins; D013879:Thioredoxins; D010741:Phospholipases A; D054501:Group II Phospholipases A2; D054467:Phospholipases A2; D002118:Calcium