Phospholipase D from soybean (Glycine max L.) suspension-cultured cells: purification, structural and enzymatic properties.
Phospholipase D (phosphatidylcholine phosphatidohydrolase EC 3.1.4.4) from soybean (Glycine max L.) suspension-cultured cell was purified around 1,200-fold to homogeneity by acetone precipitation, Macro-Prep High Q anion exchange, and octyl-Sepharose CL-4B affinity chromatography. The purified enzyme released 1,600 mumol of choline per min per mg of protein. The enzyme is monomeric with a molecular mass of 92 kDa, as estimated by SDS-PAGE. One of the most interesting characteristics of the purified soybean phospholipase D was the dependence of the pH optimum on the Ca2+ ion concentration in the assay. With 10 mM, 20 mM and 40 mM Ca2+ ions, the optima were at pH 7.5, 6 and 5.5, respectively. The specific adsorption of phospholipase D onto octyl-Sepharose gel suggests that the molecule becomes more hydrophobic in the presence of Ca2+ ions. The amino acid sequence of the first 18 N-terminal residues of soybean phospholipase D revealed a high degree of homology with those previously published for cabbage leaf and castor bean endosperm enzymes. Western blots of the soybean phospholipase D showed an immunoreactivity with antibodies raised against a synthetic peptide corresponding to the 15 N-terminal amino acid residues of phospholipase D from cabbage leaves.
Laboratoire de Lipolyse Enzymatique du CNRS, UPR 9025, CNRS, Marseille, France.
A
A
Abousalham
M
M
Teissere
A M
AM
Gardies
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R
Verger
G
G
Noat
D000595:Amino Acid Sequence; D002118:Calcium / Q000378:metabolism; D002384:Catalysis; D002478:Cells, Cultured; D002846:Chromatography, Affinity; D002852:Chromatography, Ion Exchange; D003429:Cross Reactions; D004591:Electrophoresis, Polyacrylamide Gel; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D010739:Phospholipase D / Q000037:antagonists & inhibitors / Q000276:immunology / Q000302:isolation & purification* / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013025:Glycine max / Q000166:cytology / Q000201:enzymology*; D013347:Subcellular Fractions / Q000201:enzymology
D010739:Phospholipase D; D002118:Calcium