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Title: Lipid phosphate phosphatases : purification and investigation of their role in cellular lipid signalling
Author: Darroch, Peter Ian
ISNI:       0000 0001 3404 7665
Awarding Body: University of Strathclyde
Current Institution: University of Strathclyde
Date of Award: 2001
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Several isoforms of LPP have now been identified and cloned but remain to be purified. In the present study, a bacterial expression system was established and hexa- and deca-histidine epitope tagged-LPP1 and LPPla expressed in E. coli. In addition, a maltose binding protein (MBP) epitope tagged-LPP Ia was expressed in E. coli. Hexa- and deca-histidine LPP1 and LPPla, were partially purified using immobilised affinity chromatography. MBP-LPPla was expressed to higher levels than hexa- and deca-histidine LPP1 and LPPla in E. coli, most probably within insoluble inclusion bodies. In all cases, recovery of LPP activity was low. Membranes derived from HEK293 cells that stably over-express LPP I, LPP I a, LPP2 or LPP3 were used to demonstrate the differential hydrolysis of several molecular species of PA, LPA(18: 1), C8-CIP and SlP. Kinetic analysis using a multisubstrate assay system revealed that the LPP isoforms do not follow typical Michaelis-Menten kinetics towards most substrates under the assay conditions employed. The LPPs appear to show differential kinetics depending on the complement of substrates accessible to the enzymes. Stable over-expression of LPPI, LPPla, LPP2, but not LPP3, in HEK293 cells has previously been shown to attenuate the activation of ERK-1/2 by G-protein coupled receptors agonists such as SIP, LPA, PA and thrombin. The present study extended these observations by showing that basal growth rates were unaffected and that levels of mRNA transcript for the SIP, /EDGI receptor were reduced in the LPP stable cell lines but that this did not correlate with attenuation of the SIP-stimulated ERK-1/2 response. In addition, transient overexpression of LPPI, LPPIa and LPP2, but not LPP3 in HEK293 cells and GPASM cells also resulted in the attenuation of SIPinduced ERK-1/2 activation. Furthermore, transient transfection of a plasmid construct encoding the antisense sequence for LPP1 was also found to attenuate SIPinduced ERK-1/2 activation whereas the PMA-stimulated response was unaffected. Many questions remain to be fully answered in order to determine the physiological and pathophysiological. roles of the LPPs and the reason for the molecular diversity of the enzyme family.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
Keywords: Bacterial expression