Purification and characterisation of rape seed phospholipase D

• Published in: Plant physiology and biochemistry 1999-07, Vol.37 (7-8), p.531-537
• Main Authors: Novotná, Zuzana, Káš, Jan, Daussant, Jean, Sajdok, Jiří, Valentová, Olga
• Format: Article
• Language: English
• Subjects: Agronomy. Soil science and plant productions; Biological and medical sciences; BRASSICA NAPUS; characterisation; CHEMICOPHYSICAL PROPERTIES; Economic plant physiology; ENZYMATIC HYDROLYSIS; Enzymes; ESTERASAS; ESTERASE; ESTERASES; Fundamental and applied biological sciences. Psychology; HIDROLISIS ENZIMATICA; HYDROLYSE ENZYMATIQUE; Metabolism; Nutrition. Photosynthesis. Respiration. Metabolism; OIL CROPS; phospholipase D; Plant physiology and development; PLANTAS OLEAGINOSAS; PLANTE OLEAGINEUSE; PROPIEDADES FISICOQUIMICAS; PROPRIETE PHYSICOCHIMIQUE; PURIFICACION; PURIFICATION
• ISSN: 0981-9428; 1873-2690
• DOI: 10.1016/S0981-9428(00)80104-7

Phospholipase D (PLD, phosphatidylcholine:phosphatidohydrolase, EC 3.1.4.4) has been isolated from matured dry winter rape seed (Brassica napus L.), variety Lirajet). Final purification of the soluble enzyme was achieved by two-step ammonium sulphate precipitation, hydrophobic interaction chromatography and native PAGE followed by electroelution. The specific activity of the final electrophoretically homogeneous preparation was increased about 700 times during the purification process with an overall yield of 4.6%. The activity of purified soluble PLD depends strictly on the presence of Ca2+ (120 mM). The pH optimum of rape seed PLD was in the range 5.5–6. The Km value for phosphatidylcholine depends on the ratio between SDS and substrate concentration. No polymorphism of PLD was detected by SDS-PAGE and size exclusion chromatography of the purified enzyme. The purified enzyme was a monomer with a molecular mass of 105 000 Da determined by SDS-PAGE and of 90 000–100 000 Da assessed by size exclusion chromatography. The amino acid composition of PLD was also determined. Similar intensities of immunochemical cross reactions were demonstrated between PLD extracted from rape seed, soybean, castor bean and sunflower using immunoabsorption technique with the immune serum previously prepared against partially purified rape seed PLD. Data obtained in this study and those gathered from the literature indicate close similarities in molecular, enzymatic and antigenic characteristics between PLDs of oil seeds of different species.