Use this URL to cite or link to this record in EThOS: https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.776198
Title: Partial purification and characterisation of a membrane-associated phospholipase D from bovine spleen
Author: Paul, Andrew
Awarding Body: University of Glasgow
Current Institution: University of Glasgow
Date of Award: 1995
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Abstract:
Phospholipase D (PLD; EC 3.1.4.4) catalyses the hydrolysis of the phosphate ester bond of phospholipid molecules, releasing the hydroxyl containing head group with the concomitant formation of phosphatidic acid (PtdOH). Evidence now exists that PLD catalyses the hydrolysis of membrane phospholipids, particularly PtdCho, in an agonist-dependent manner. This may serve as a novel source of the established second messenger diacylglycerol with its immediate lipid product, phosphatidic acid, functioning as a putative second messenger. The relationship between agonist- stimulated PLD activity in vivo and observed in vitro activity remains unclear. This study examines the purification and characterisation of membrane-associated PtdCho-PLD activity from bovine spleen. PLD activity was primarily associated with the membrane compartment of the bovine spleen homogenate. Incubation of these freeze-thawed membranes at high pH resulted in efficient solubilisation of the membrane-associated PLD activity. Purification of the solubilised PLD was subsequently investigated. Use of cation- exchange chromatography resolved two PtdCho-hydrolysing PLD activities, the major one being further purified by successive chromatographic separation on heparin-agarose, hydroxyapatite, cation-exchange and gel-filtration chromatography. SDS-PAGE revealed the final protein preparation to be heterogeneous and it was not possible to identify a particular protein that co-migrated with the PLD activity on gel- filtration which displayed an apparent native molecular weight of 69 kDa. The solubilised PLD remains to be purified to homogeneity. Using mixed micellar methodology the catalytic activity of the partially purified enzyme towards PtdCho was characterised. At all substrate concentrations the post heparin enzyme displayed pseudo-first order kinetics and values for Vmax and Km could not be determined by this assay methodology, however, the reaction velocity displayed a pH optimum of 7.0 and was independent of Ca2+ and Mg2+. Finally, investigation of catalytic activity towards PtdCho, PtdEtn, Ptdlns and PtdSer confirmed the partially purified preparation to be a distinct PLD activity that was selective between the major phospholipids and displayed substrate specificity towards PtdCho.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.776198  DOI:
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