Partial Purification and Characterisation of the Membrane-Associated DAG Kinase of Rat Brain

Paterson, Andrew (1993) Partial Purification and Characterisation of the Membrane-Associated DAG Kinase of Rat Brain. PhD thesis, University of Glasgow.

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The sn-1,2-Diacylglycerol produced on receptor-stimulated hydrolysis of PtdIns(4,5)P2 hydrolysis is metabolised primarily by phosphorylation in a reaction catalysed by DAG kinase. The DAG kinase active in this role has been localised to the membrane-associated compartment and has been shown to display catalytic selectivity towards different sn-1,2-diacylglycerols. This study examines the purification and characterisation of membrane-associated DAG kinase from rat brain. DAG kinase activity was predominantly associated with the soluble component of rat brain homogenate. Incubation of the homogenate with phospholipase C (B. cereus) affected a redistribution of the soluble DAG kinase activity toward the particulate fraction, thus providing a higher specific activity starting material for the purification of the membrane-associated enzyme. Incubation of such phospholipase C-treated membranes with hypertonic concentrations of KC1 resulted in the efficient solubilisation of the DAG kinase activity. The protein preparation solubilised in this manner was found to contain two resolvable enzymes: (1) a KC1-independent DAG kinase which remains soluble in the absence of KC1, and (2) a KC1-soluble DAG kinase which requires the presence of at least 300 mM KC1 for continued solubility. The purification of the KC1-soluble enzyme was further investigated. It was possible to purify this enzyme, in the presence of 300 mM KC1, by successive chromatographic separation on AffiGel blue, butyl-Sepharose 4B/phenyl-Sepharose CL-4B, Sephacryl S-300 SF, heparin-agarose, and hydroxyapatite. SDS-PAGE revealed the final protein preparation to be heterogeneous. Furthermore, it was not possible to identify any proteins which co-migrate with DAG kinase activity on hydroxyapatite chromatography of the partially purified protein preparation. Although, the KC1-soluble DAG kinase remains to be purified to homogeneity, it migrates on gel filtration with an apparent native molecular mass ozf 160 kDa. The substrate dependence of the partially purified enzyme was characterised by mixed micellar assay methodology. The post gel filtration enzyme preparation was found to have a Vmax of 29.9 nmol/min/mg and Km values of 1.7 mol% and 330 muM for sn-1-stearoy1-2-arachidonylglycerol and ATP, respectively. Furthermore, when the Km and Vmax 12r of the KCl-soluble DAG kinase was determined with sn-1,2-dioleoylglycerol and stz-1,2-dilaurylglycerol they were found not to be significantly altered from the values determined with sn-l-stearoy1-2-arachidonylglycerol. Therefore, the KC1-soluble enzyme was not found to be selective amongst sn-1,2-diacylglycerol substrates. The reaction velocity of the KC1-soluble DAG kinase is independent of Ca2+ and PtdSer, but the enzyme would appear to require 7.0 mM, or greater, free Mg2+ for maximal catalytic rates. Finally, although the Vmax of the DAG kinase did not alter, its Km for ATP was observed to decrease by greater than 4-fold in the presence of 20 mM potassium phosphate.

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Additional Information: Adviser: R M Smellie
Keywords: Biochemistry
Date of Award: 1993
Depositing User: Enlighten Team
Unique ID: glathesis:1993-74805
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 13 Nov 2019 15:58
Last Modified: 13 Nov 2019 15:58

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