Term Test 2 Review.docx

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University of Toronto St. George
Stavroula Andreopoulos

BCH210 Midterm #2 Review Dr. Reithmeier Lipids - Phospholipd bilayer: the basis of a biological membrane, made up of lipids, proteins and sugars Fatty Acids - Composed of a hydrocarbon tail (hydrophobic) and an alcohol head (hydrophilic) head is able to lose/gain a proton because of its negative charge - Carried in the bloodstream by triacylglycerols - Saturated made up of all single bonds (e.g. Palmitate, 16:0) Allows for very tight packing, have stronger interactions that hold them together (van der Waals) - Unsaturated contains at least one double bond (e.g. Oleate, 18:1) Double bonds in nature tend to be in cis orientation; trans fatty acids are very rare in nature Does not pack together closely, double bonds produce kinks in the chain; changes physical properties Can be polyunsaturated: multiple double bonds (e.g. linoleate 18:2) - The last carbon in the tail is referred to as the omega carbon; double bonds get their name according to this carbon E.g. omega 3 fatty acid: double bond is on the third carbon from the end We cant make omega-3 and omega-6 polyunsaturated fatty acids in our body, they need to be taken in through our diet essential fatty acids - Melting point: saturated fatty acids have a higher melting point because of their strong van der Waals (can pack tightly) Increases with addition of carbons, decreases with addition of double bonds Palmitate C16:0 MP 63C Stearate C18:0 MP 70C Oleate C18:1 MP 13C Palmitoleate C16:1 MP -0.5C Linoleate C18:2 MP -9C Phospholipids - Structure: 2 fatty acids + glycerol + phosphate + alcohol Glycerol: 3 C + 3 OH; phosphate joins to OH on C3, alcohol joins to O on phosphate (phosphodiester bond) OH groups can be phosphorylated - Can be zwitterionic (neutral, net charge = 0) or negatively charged depending on alcohol head group (always have different structures) Phosphate group (PO ) is 4lways negatively charged - Neutral: + phosphatidylcholine (PC): OCH CH N2(CH2) 3 3 phosphatidylethanolamine (PE): OCH CH NH 2 2 3+ - Anionic: + - phosphatidylserine (PS): OCH CH(N2 )(COO 3 phosphatidylglycerol (PG): OCH CH(O2)CH (OH) 2 *All referred to as glycerophospholipids Sphingomyelin: starts out as spingosine (serine component) ceramide when a chain is attached at a N on C2 sphingomyelin when phosphate head group is attached to C1 - Phospholipids are amphipathic: hydrophobic tail and hydrophilic head Cholesterol - Very important component of biological membranes, sometimes makes up half the lipid in a membrane Modulates fluidity of lipid bilayer - We make 90% of the cholesterol I our diet; statins inhibit cholesterol production in the body - Planar and rigid: disrupts packing & slows motion of acyl chains Ring structure contributes to rigidity Very hydrophobic tail - Precursor for all steroid hormones (e.g. testosterone) and bile salts (e.g. sodium cholate) Eicosanoids - Arachidonic acid (C20:4, omega-6 precursor from diet) - Prostaglandin E2 (blood vessel constriction) - Thromboxane A2 (blood clotting) - Leukotriene D4 (lung smooth muscle contraction) Biological Membranes - Consist of mobile globular proteins embedded in a lipid fluid bilayer - Brain: myelin membrane consists of 75% lipid (insulator) and 25% protein - Membranes are different in composition; they define the cell and the organelles within the cell - Asymmetric: PC and SM are enriched in the outer leaflet, PE and PS are in inner leaflet Inner: layer of negative charge attracts protons and ions (K, Na); creates a microenvironment When calcium-activated proteins from the cytosol are moved to the membrane, they are turned on ([Ca] is much higher in the membrane than in cytosol by 100 fold) Phospholipid Bilayer - Two phospholipid molecules-thick - Choline head group (hydrophilic) and non-polar acyl chain (hydrophobic) form spontaneously - Thickness of the bilayer depends on the length of acyl chains, degree of unsaturation, etc. Bacterial membranes are thinner The Lipid Bilayer - Membrane for compartments: cells, ER, Golgi, lysosome, mitochondria - Provides a hydrophobic barrier Water, sugars, amino acids, sodium, potassium Proteins form channels, transporters, pumps, etc. that allow things to move across the membrane - Maintains cellular homeostasis Na+/K+ pump takes Na out and K in, driven by ATP High intracellular K, low intracellular Na 1/3 of what you eat is used to fuel this pump - Forms a vesicle or closed compartment (aqueous, cytosol) that separates two regions Phospholipids - Amphiphilic: have a polar (head group) and non-polar (tail) region - Spontaneously form bilayers - Bury their fatty acid tails and expose polar head groups to water Cholesterol: does not form a lipid bilayer on its own; the OH group pokes out facing water and acyl chains turn inwards - Can help diffuse molecules in two ways: Lateral diffusion: very fast Transverse diffusion: very slow (flipping occurs through enzymes); polar head group must be moved through the hydrophobic region energetically unfavourable - Red blood cells: 50% of blood, surrounded by a plasma membrane Contain hemoglobin in very high concentrations (carries oxygenblood, CO2lungs) and a few enzymes Membrane consists of multiple phospholipids in different quantities Phospholipid Structure - Unsaturated lipids modulate fluidity, provide kinks which disrupt packing - Hydrocarbon chain length affects fluidity - Cholesterol content modulates fluidity - These factors help regulate T m the gel-to-liquid crystalline state of the membrane Above a certain temperature, the membrane changes from a solid to a liquid crystal state Heat induces a disordered liquid-crystalline state, but the bilayer is still intact Above 37C: solid gel phase; below 37C: liquid phase The longer the acyl chain, the higher the T m E.coli: changes lipid composition when temp drops below 37C (adds shorter chains or more unsaturated chains in the membrane) - Experiment: human cell w/ red fluorescent markers + mouse cell w/ green fluorescent markers Immediately after fusion: fluorescent markers remain localized on either side 40 minutes after fusion: fluorescent markers were randomly distributed over entire surface Done at 37C; if it was done at 0C, it would stay as the second state (right after fusion) - Freeze-fracture electron microscopy: most proteins stay in the inner leaflet of the bilayer membrane when frozen SDS Gel Electrophoresis - Developed in the early 70s to separate proteins based on molecular weight - SDS: detergent, extracts protein from membrane, solubilizes membrane, dissociates subunits and denatures proteins Replaces lipid, allows the protein to be removed from the membrane Breaks apart dimers into monomers Other detergents (e.g. soap) do NOT denature - Polyacrylamide: polymeric gel matrix used to separate proteins - SDS-PAGE: sodium dodecyl sulfate-polyacrylamide gel electrophoresis separates proteins based on their size Sodium dodecal disrupts lipid and lipid-protein interactions SDS-treated samples are loaded in wells inside SDS-polyacrylamide gel between two glass plates Direction of migrationdecreasing molecular weight SDS-PAGE of the Erythrocyte Membrane - Top to bottom (largest to smallest): Glycophorin A: major protein, 60% sugar (so much that it doesnt bind very well, invisible on SDS gel) Band 3: anion exchanger Band 4.5: glucose transporter Actin: inner leaflet of RBC, gives shape Spectrin Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) - The whole glycolytic complex of RBC are bound to the RBC membrane they do not float freely Glucose comes in through a glucose transporter and is immediately shuttled to this pathway to produce ATP Membrane Proteins: Properties - Peripheral proteins (extrinsic) can be released from the membrane by extraction in a high ionic strength medium - Integral proteins (intrinsic) require more forceful means, such as detergents Most intrinsic proteins span the bilayer because they function as receptors, transporters, channels, etc. that move things through the membrane - Single spanning protein: glycophorin A (single polypeptide chain, single transmembrane helix) 60% weight by carbohydrate; units located at the N-terminal, on the outer face of the membrane N-terminal out, C-terminal in Part of the chain on the inside of the cell contains both basic and acidic amino acids; amino acids in the membrane bilayer are hydrophobic RULE: Glycoproteins always contain sugar units on external face of the membrane Intrinsic Membrane Proteins - Transmembrane segment: one cro
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