Phospholipase D from Soybean (Glycine max L.) Suspension-Cultured Cells: Purification, Structural and Enzymatic Properties

Phospholipase D (phosphatidylcholine phosphatidohydrolase EC3.1.4.4[EC]) from soybean (Glycine max L.) suspension-cultured cellwas purified around 1,200-fold to homogeneity by acetone precipitation,Macro-Prep High Q anion exchange, and octyl-Sepharose CL-4Baffinity chromatography. The purified enzyme released 1,600µmol of choline per min per mg of protein. The enzymeis monomeric with a molecular mass of 92 kDa, as estimated bySDS-PAGE. One of the most interesting characteristics of thepurified soybean phospholipase D was the dependence of the pHoptimum on the Ca²⁺ ion concentration in the assay. With 10mM, 20 mM and 40 mM Ca²⁺ ions, the optima were at pH 7.5, 6and 5.5, respectively. The specific adsorption of phospholipaseD onto octyl-Sepharose gel suggests that the molecule becomesmore hydrophobic in the presence of Ca²⁺ ions. The amino acidsequence of the first 18 N-terminal residues of soybean phospholipaseD revealed a high degree of homology with those previously publishedfor cabbage leaf and castor bean endosperm enzymes. Westernblots of the soybean phospholipase D showed an immunoreactivitywith antibodies raised against a synthetic peptide correspondingto the 15 N-terminal aminoacid residues of phospholipase D fromcabbage leaves. (Received March 13, 1995; Accepted May 29, 1995)