Purification, characterization and cloning of phospholipase D from peanut seeds.
We purified phospholipase D (PLD) enzyme from peanut seeds, and the PLD enzyme eluted as two distinct peak fractions on Mono-Q chromatography, the first of which was characterized. N-terminal sequencing indicated that the N-terminus was blocked. The molecular mass of the purified enzyme was estimated to be 92 kDa by SDS-PAGE. The pH optimum of the enzyme was 5.0, and the Km value against its substrate phosphatidylcholine (PC), in the presence of 10 mM CaCl2 and 4 mM deoxycholate, was estimated to be 0.072 mM. The enzyme catalyzed two reactions, i.e., hydrolysis of PC generating phosphatidic acid (PA) and choline, and transphosphatidylation of the PA-moiety in the PC molecule to the acceptor glycerol, generating phosphatidylglycerol. Furthermore, we cloned two types of full-length cDNA, Ahpld1 and Ahpld2, each encoding distinct PLD molecules having 794 and 807 residues, respectively. The partial amino acid sequence of the purified PLD was consistent with the deduced sequence of AhPLD2.
Nakazawa
Yozo
Y
Department of Applied Biology and Chemistry, Faculty of Applied Bioscience, Tokyo University of Agriculture, 1-1-1 Sakuragaoka, Setagaya-ku, Tokyo, 156-8502, Japan.
Sato
Hiroaki
H
Uchino
Masataka
M
Takano
Katsumi
K
D000595:Amino Acid Sequence; D010367:Arachis / Q000201:enzymology* / Q000235:genetics; D001483:Base Sequence; D002852:Chromatography, Ion Exchange; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D017343:Genes, Plant; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010713:Phosphatidylcholines / Q000737:chemistry*; D010739:Phospholipase D / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D010802:Phylogeny; D010940:Plant Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D012117:Resins, Synthetic / Q000737:chemistry; D012639:Seeds / Q000201:enzymology*; D013379:Substrate Specificity
D010713:Phosphatidylcholines; D010940:Plant Proteins; D012117:Resins, Synthetic; C042855:Mono Q; D010739:Phospholipase D