title: A facile purification procedure of phospholipase D from cabbage and its characterization.
abstract: Phospholipase D (PLD), an enzyme predestined for the preparation of new phospholipids, was isolated from cabbage and purified in a highly efficient way by using a combination of hydrophobic chromatography and a specific calcium effect. In the presence of calcium ions (50mM), PLD is bound from the crude enzyme solution to Octyl-Sepharose and subsequently selectively eluted by removing the calcium ions. The obtained enzyme is electrophoretically pure (95%), its molecular mass and isoelectric point were determined to be 87,000 Da and 4.7, respectively. The purified enzyme was kinetically characterized by use of mixed phosphatidylcholine-SDS micelles as well as the short-chain lecithins 1,2-dihexanoyl- and 1,2-diheptanoyl-sn-glycero-3-phosphocholine as substrates. A hyperbolic upsilon/[S]-characteristic was obtained for the mixed micellar system, whereas the upsilon/[S] curves of the short-chain lecithins reflect the dependence of velocity on the physical state of the substrate. A small velocity increase was observed up to a critical substrate concentration near the critical micelle concentration, from where the velocity increases hyperbolically.
authors.affiliation: Arbeitsgruppe Angewandte Enzymologie des Fachbereichs Biochemie/Biotechnologie Universität Halle.
authors.forename: R
authors.initials: R
authors.lastname: Lambrecht
authors.forename: R
authors.initials: R
authors.lastname: Ulbrich-Hofmann
mesh_terms: D001937:Brassica / Q000201:enzymology*; D002850:Chromatography, Gel / Q000379:methods; D002852:Chromatography, Ion Exchange / Q000379:methods; D004591:Electrophoresis, Polyacrylamide Gel; D007700:Kinetics; D008970:Molecular Weight; D010739:Phospholipase D / Q000302:isolation & purification* / Q000378:metabolism
chemical_list: D010739:Phospholipase D