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 ar ound 1,200-fold to homogeneity by acetone precipitation, Macro-Prep Hi gh Q anion exchange, and octyl-Sepharose CL-4B affinity chromatography . The purified enzyme released 1,600 mu mol of choline per min per mg of protein. The enzyme is monomeric with a molecular mass of 92 kDa, a s estimated by SDS-PAGE. One of the most interesting characteristics o f the purified soybean phospholipase D was the dependence of the pH op timum on the Ca2+ ion concentration in the assay. With 10 mM, 20 mM an d 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 s uggests 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 th ose previously published for cabbage leaf and castor bean endosperm en zymes. Western blots of the soybean phospholipase D showed an immunore activity with antibodies raised against a synthetic peptide correspond ing to the 15 N-terminal aminoacid residues of phospholipase D from ca bbage leaves.