title: Purification, sequencing and characterization of phospholipase D from Indian mustard seeds.
abstract: Phospholipase D (PLD; E.C. 3.1.4.4) is widespread in plants where it fulfills diverse functions in growth and in the response to stresses. The enzyme occurs in multiple forms that differ in their biochemical properties. In the present paper PLD from medicinally relevant Indian mustard seeds was purified by Ca(2+)-mediated hydrophobic interaction and anion exchange chromatography to electrophoretic homogeneity. Based on mass-spectrometric sequence analysis of tryptic protein fragments, oligonucleotide primers for cloning genomic DNA fragments that encoded the enzyme were designed and used to derive the complete amino acid sequence of this PLD. The sequence data, as well as the molecular properties (molecular mass of 92.0 kDa, pI 5.39, maximum activity at pH 5.5-6.0 and Ca(2+) ion concentrations ⩾60 mM), allowed the assignment of this enzyme to the class of α-type PLDs. The apparent kinetic parameters Vmax and Km, determined for the hydrolysis of phosphatidylcholine (PC) in an aqueous mixed-micellar system were 356±15 μmol min(-1) mg(-1) and 1.84±0.17 mM, respectively. Phosphate analogs such as NaAlF4 and Na3VO4 displayed strong inhibition of the enzyme. Phosphatidylinositol 4,5-bisphosphate had a strong activating effect at 2-10 mM CaCl2. PLD was inactivated at temperatures >45 °C. The enzyme exhibited the highest activity toward PC followed by phosphatidylethanolamine and phosphatidylglycerol. PCs with short-chain fatty acids were better substrates than PCs with long fatty acid chains. Lyso-PC was not accepted as substrate.
authors.affiliation: Institute of Biochemistry and Biotechnology, Martin-Luther University Halle-Wittenberg, Kurt-Mothes-Str. 3, D-06120 Halle, Germany.
authors.forename: Hafeeza
authors.initials: H
authors.lastname: Khatoon
authors.affiliation: Institute of Biochemistry and Biotechnology, Martin-Luther University Halle-Wittenberg, Kurt-Mothes-Str. 3, D-06120 Halle, Germany.
authors.forename: Johanna
authors.initials: J
authors.lastname: Mansfeld
authors.affiliation: Institute of Biochemistry and Biotechnology, Martin-Luther University Halle-Wittenberg, Kurt-Mothes-Str. 3, D-06120 Halle, Germany.
authors.forename: Angelika
authors.initials: A
authors.lastname: Schierhorn
authors.affiliation: Institute of Biochemistry and Biotechnology, Martin-Luther University Halle-Wittenberg, Kurt-Mothes-Str. 3, D-06120 Halle, Germany. Electronic address: renate.ulbrich-hofmann@biochemtech.uni-halle.de.
authors.forename: Renate
authors.initials: R
authors.lastname: Ulbrich-Hofmann
mesh_terms: D000595:Amino Acid Sequence; D007700:Kinetics; D008970:Molecular Weight; D009149:Mustard Plant / Q000737:chemistry*; D010713:Phosphatidylcholines / Q000378:metabolism; D010739:Phospholipase D / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D012639:Seeds / Q000737:chemistry; D013379:Substrate Specificity
chemical_list: D010713:Phosphatidylcholines; D010739:Phospholipase D
keywords: Amino acid sequence; Brassica juncea; Calcium; Inhibitors; Phosphatidylinositol 4,5-bisphosphate; Phospholipase D; Purification; Substrate specificity