lecture 18 - 17-30 Demonstration of lateral diffusion of...

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Demonstration of lateral diffusion of lipids Attach a fluorescent group (= fluorophore) to a specific bilayer component on a cell Bleach (destroy) fluorophore in a small patch with an intense laser pulse. Monitor bleached area (by fluorescence microscopy) See recovery of fluorescence in bleached area as bleached molecules diffuse out and fluorescently-labeled molecules diffuse in. By FRAP: “fluorescence recovery after photobleaching” Fluorescence recovery rate depends on diffusion rate of labeled molecule Phospholipids can diffuse at ~ 1-2 μ m/sec 17-30
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MCB 354 Lecture 18 Membrane Asymmetry Membrane Proteins Reading in Lehninger : Chapter 11 , pp. 369-385 18-1
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Phospholipid distribution between bilayers is asymmetrical ow done ? Treat intact cell with phospholipase C (can’t reach lipids in inner monolayer), but moves headgroups of lipids in outer monolayer. Determine total P-lipid composition of lated membrane. Proportion of each headgroup released by PLC gives fraction in outer lay 18-2 ERYTHROCYTE PM
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Membrane asymmetry-1 18-3 When synthesized, P-lipids are inserted in one leaflet of bilayer Rate of unassisted transbilayer “flip-flop” is very low (huge free energy barrier)…. b/c polar or charged headgroup must leave aqueous environment and move through hydrophobic interior of bilayer
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18-4 Translocation of specific phospholipids from one leaflet to the other is postulated to be assisted by “ flippases Membrane asymmetry-2 Because v. slow flipping of some lipids, and selective specific flipping of others, the distribution of P-lipids in bilayer is asymmetric
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Reagent = TNBS a membrane impermeant probe that specifically reacts with and labels PE Experiment to demonstrate the asymmetry of membrane phospholipid synthesis (e.g. PE) membrane of E. coli cell ADD TO CELLS: ONLY LABELS PE ON EXTERNAL FACE OF MEMBRANE 18-5
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Demonstration of asymmetry of membrane PE synthesis Pulse label PE w. [32P]O43- (tags newly synthesized PE as it’s made inside the cell) Immediate incubation w. TNBS Delayed incubation w. TNBS membrane of E. coli cell No [32P]PE double labeled with TNBS : PE must have been synthesized on cytoplasmic face of membrane [32P]PE double labeled with TNBS now also present on external face of membrane 18-6
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in both their lipid and protein composition. Fluid mosaic model for
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This note was uploaded on 01/29/2012 for the course MCB 354 taught by Professor Spees during the Spring '09 term at University of Illinois, Urbana Champaign.

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lecture 18 - 17-30 Demonstration of lateral diffusion of...

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