UBC Theses and Dissertations
Lipid-protein interactions: A., Semliki forest virus : B., CTP:phosphocholine cytidylyltransferase from rat liver Farren, S. Blake
A. Semliki Forest virus: Lipid Headgroup-Protein Interactions Semliki Forest virus is an alphavirus enveloped by a lipid bilayer that contains approximately 200 copies each of three glycoproteins: E₁, molecular weight 49,000, E₂, molecular weight of 52,000, and E₃, molecular weight of 10,000. Proton magnetic resonance measurements were done on intact Semliki Forest virus and virus that had been digested with thermolysin. The magnetic resonance line derived from a portion of the N-methyl groups of the choline containing phospholipids narrowed considerably after proteolytic digestion. This reduction in the N-methyl resonance is not inconsistent with increased motion of the headgroups of the phospholipids as a result of thermolysin removal of the viral glycoprotein spikes. To further investigate this phenomenon, experiments were planned using deuterium NMR. Tri-trideuteromethylcholine was chemically synthesized and added to the medium of BHK-21 cells with the hope of labelling a large majority of the choline containing lipids. However, no incorporation of this labelled species could be observed. Two other deuter-ated cholines, mono-trideuteromethylcholine and di-trideuteromethylchloline, were synthesized and easily incorporated into the BHK-21 cell choline containing lipids. Due to the low yields of Semliki Forest virus, deuterium NMR could not be performed, however, the lack of incorporation of tri-trideuteromethyl choline was of interest. From labelling studies it was concluded that this deuterated choline was not transported across the cell plasma membrane. B. CTP:phosphocholine Cytidylyltransferase: Lipid-Protein Interactions Early studies on the rat liver CTP:phosphocholine cytidylyltransferase (E.C. 126.96.36.199) (CT) reported that the enzyme isolated from the cytosolic fraction of"0.9% NaCl solution homqgena-tes increased in activty 4- to 5-fold upon aging several days at 0°C or incubation at 37°C for 3 hours. It was subsequently shown that the activating agent was lysophospha-tidylethanolamine (LPE). We have demonstrated that the purified rat liver CT is dependent upon lipid for acitvity and is activated by oleoyl-LPE and inhibited by oleoyl-lysophosphatidylcholine (LPC). The plot of CT activity at various concentrations of LPE yields a hyperbolic curve with a Ka of 0.3 mM. The activation of CT by LPE results in a decrease of the Km for CTP from 2 mM at 0.1 mM LPE, to 0.5 mM at 0.4 mM LPE. LPE had no effect on the Km for phosphocholine. Hence, the activation of CT by LPE is due to an influence on the Km for CTP. When CT is assayed in the presence of LPE, LPC inhibits the activity with a concentration for half-maximal inhibition of 0.16 mM. Inhibition by LPC was not competitive with phosphocholine.
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