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Membrane dynamics
• Degree of fluidity is similar in all membranes– Cells regulate composition based on growth
conditions
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Membrane dynamics• Experiment on lateral diffusion
– Attach fluorescent probes to head group to monitor
– Tag all lipids on outer membrane– Bleach the probe– Measure fluorescence return
– FRAP: fluorescence recovery after photobleaching
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Areas of lower dynamics?
• Structural proteins within the cell ‘anchor’ certain proteins in place
• Lower degree of lateral diffusion
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Areas of lower dynamics?
• ‘Rafts’– Thick, more ordered
microdomains– Exclude
glycerophospholipids (shorter sat/unsat chains)
• Sphingolipids (long FA chains…mostly saturated)
• Cholesterol• Integral membrane
proteins
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Lipid rafts
• Typically detergent-insoluble
• Serves to sequester a subset of proteins together…other reasons?
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Extreme case of membrane segregation
• Caveolae: physical distortion of the membrane
• Segregates transmembrane signaling molecules, etc.
• eg. insulin receptor
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Exam review
• Questions?
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Kinetic parameters
• Km
– Meaning– Usage– Experimental determination– Manipulation?
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• Vmax
– Meaning– Usage– Experimental determination– Manipulation?
Kinetic parameters
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• kcat (turnover number)
• kcat/Km (specificity constant)
• Inhibition constants– Competitive– Non-competitive– Irreversible?
Kinetic parameters
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General aspects of catalysis
• How do catalysts affect kinetics?
• Mechanisms:– Give two (more?) ways a positively-charged
residue in an active site might influence a reaction