Improved purification and biochemical characterization of phospholipase D from cabbage

Phospholipase D (phosphatidylcholine phosphatidohydrolase, EC 3.1.4.4) was purified from cabbage leaves. The two step purification procedure involved hydrophobic chromatography on Octyl-Sepharose followed by a Mono-Q/FPLC-column with a total yield of 23% and a purification factor of 1000. A zymographic assay was used to detection of PL D activities at various stages of purification under non denaturing PAGE. The molecular mass was determined to be 90 kDa using the SDS/PAGE method, and 90,200 Da as calculated from the amino acid analysis. The isoelectric point of the enzyme is acidic (pI = 4.7). The amino-acid composition and 29 residues of the NH2-terminal amino-acid sequence were determined.