Effects of Lipid on Membrane Protein Function
Effects of Lipid on Membrane Protein Function
The effects of phospholipids on the functioning of the membrane proteins diacylglycerol kinase (DGK) of Escherichea coli and Ca2+-ATPase of sarcoplasmic reticulum have been investigated. A procedure has been developed for the reconstitution of DGK into phospholipid bilayers containing dihexanoylglycerol (DHG) as substrate. DGK activity was found to be dependent on fatty acyl chain length, with highest activity in dioleoylphosphatidylcholine (di(C18:1)PC). Low activities in di(C14:1)PC followed from an increase in Km values for DHG and ATP with no effect on vmax, whilst low activity in di(C24:1)PC followed from a decrease in vmax with no effect on Km. In mixtures of two phosphatidylcholines with different chain lengths, the activity corresponded to that expected for the average chain length of the mixture. Cholesterol increased activity in di(C14:1)PC by possibly changing the bilayer thickness. In mixtures of di(C18:1)PC and anionic phospholipids, the presence of cardiolipin, dioleoylphosphatidic acid (di(C18:1)PA), or dioleoylphosphatidylserine (di(C18:1)PS) all resulted in lower activities for DGK than in di(C18:1)PC, whilst the presence of dioleoylphosphatidylglycerol (di(C18:1)PG had little effect on activity. In some cases low kinase activity in the presence of anionic phospholipids followed from a decrease in vmax, in some cases it followed from an increase in Km for DHG, and for di(C18:1)PA, from both. The presence of anionic phospholipids had little effect on Ca2+-ATPase activity. However, inclusion of 10 mol % anionic phospholipid (di(C18:1)PA or di(C18:1)PS) led to higher levels of accumulation of Ca2+ by the Ca2+-ATPase reconstituted into sealed vesicles, 10 mol % being the optimum concentration. Cardiolipin or phosphatidylinositol 4-phosphate were more effective than di(C18:1)PA or di(C18:1)PS in increasing accumulation of Ca2+.
University of Southampton
Pilot, Jeffrey David
0bc043cb-80ea-4222-82a7-980dcda9dd8a
2001
Pilot, Jeffrey David
0bc043cb-80ea-4222-82a7-980dcda9dd8a
Pilot, Jeffrey David
(2001)
Effects of Lipid on Membrane Protein Function.
University of Southampton, Doctoral Thesis.
Record type:
Thesis
(Doctoral)
Abstract
The effects of phospholipids on the functioning of the membrane proteins diacylglycerol kinase (DGK) of Escherichea coli and Ca2+-ATPase of sarcoplasmic reticulum have been investigated. A procedure has been developed for the reconstitution of DGK into phospholipid bilayers containing dihexanoylglycerol (DHG) as substrate. DGK activity was found to be dependent on fatty acyl chain length, with highest activity in dioleoylphosphatidylcholine (di(C18:1)PC). Low activities in di(C14:1)PC followed from an increase in Km values for DHG and ATP with no effect on vmax, whilst low activity in di(C24:1)PC followed from a decrease in vmax with no effect on Km. In mixtures of two phosphatidylcholines with different chain lengths, the activity corresponded to that expected for the average chain length of the mixture. Cholesterol increased activity in di(C14:1)PC by possibly changing the bilayer thickness. In mixtures of di(C18:1)PC and anionic phospholipids, the presence of cardiolipin, dioleoylphosphatidic acid (di(C18:1)PA), or dioleoylphosphatidylserine (di(C18:1)PS) all resulted in lower activities for DGK than in di(C18:1)PC, whilst the presence of dioleoylphosphatidylglycerol (di(C18:1)PG had little effect on activity. In some cases low kinase activity in the presence of anionic phospholipids followed from a decrease in vmax, in some cases it followed from an increase in Km for DHG, and for di(C18:1)PA, from both. The presence of anionic phospholipids had little effect on Ca2+-ATPase activity. However, inclusion of 10 mol % anionic phospholipid (di(C18:1)PA or di(C18:1)PS) led to higher levels of accumulation of Ca2+ by the Ca2+-ATPase reconstituted into sealed vesicles, 10 mol % being the optimum concentration. Cardiolipin or phosphatidylinositol 4-phosphate were more effective than di(C18:1)PA or di(C18:1)PS in increasing accumulation of Ca2+.
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Published date: 2001
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Local EPrints ID: 464502
URI: http://eprints.soton.ac.uk/id/eprint/464502
PURE UUID: 74def3f3-0afc-4af8-b056-2b3b3ccc7ec5
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Date deposited: 04 Jul 2022 23:42
Last modified: 16 Mar 2024 19:33
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Author:
Jeffrey David Pilot
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