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.
Abousalham
A
A
Centre de Biochimie et de Biologie Moleculaire, Centre Nationale de la Recherche Scientifique, Marseille, France.
Riviere
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M
Teissere
M
M
Verger
R
R
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D001937:Brassica / Q000201:enzymology*; D002413:Cations, Divalent; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D006863:Hydrogen-Ion Concentration; D007526:Isoelectric Point; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010739:Phospholipase D / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism
D000596:Amino Acids; D002413:Cations, Divalent; D010739:Phospholipase D