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Cell Biology Test 2 Notes

by: Karen Notetaker

Cell Biology Test 2 Notes BIOL 231

Karen Notetaker
University of Louisiana at Lafayette

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This is a comprehensive study guide for Exam two for Cell Biology. It's in an outline format but still very fleshed out.
Cell and Molecular Biology
Patricia Mire-Watson
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This 25 page Study Guide was uploaded by Karen Notetaker on Saturday February 27, 2016. The Study Guide belongs to BIOL 231 at University of Louisiana at Lafayette taught by Patricia Mire-Watson in Spring 2016. Since its upload, it has received 65 views. For similar materials see Cell and Molecular Biology in Biological Sciences at University of Louisiana at Lafayette.

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Date Created: 02/27/16
Chapter 4 2/8 Friday, February 8, 2013 10:00 AM Figure 4-1 • 20 Amino Acids=1000's of proteins ○ Order of AA's ○ Number in chain • Dipeptide=20² = 400 possibleproteins (two AA attached) • Tripeptide=20 = 8000 possibleproteins (3 AA attached) • Most polypeptides= 100's of AA linked by peptide bonds • Protein structure ○ Primary structures= sequences of AA  Determines tertiary structure (3D shape) ○ Tertiary structure determines function  Cen formation determines function □ Polypeptide in flexibility,not rigid □ Covalent bonds are flexible • Polypeptide folding ○ Flexible backbone of alternative Amino group, C-H and a Carboxyl group ○ Side chains (R-group) protrude from backbone ○ R-groups change the polypeptide Figure 4-2 • In vitro, spontaneous, thermodynamically stable conformation appears • Crowded cytoplasm means proteins need help folding ○ Chaperones help proteins to fold  Prevent newly synthesized proteins from associating with wrong partners.  Makes folding more efficient and reliable • General structure of proteins and polypeptide ○ Polypeptide= AA bonded w/ peptide bonds Figure 4.3 Side Chains • Nonpolar/polar • Negative= acidic • Positive= basic • Uncharged  R groups can rotate about the peptide bond, but they can NOT "slide" Figure 4.4 • 3 degree structure ○ Interaction b/t backbone and R-groups  All but one type of structure involves non-covalent bonds  Ionic (AKA electrostatic) □ b/t R-groups (+ and -, polar) □ b/t parts of backbone □ b/t R-groups and backbone H-Bonds □ b/t R-groups and backbone  H-Bonds □ Areas of backbone of different uncharged polar AA's (amino-Carboxyl)  Van der Waals □ Temporary weak force  Happen because of ◊ Atom to atom proximity ◊ Can occur b/t any molecules Figure 4.5 • In aqueous/ polar environment, non-polar R groups face inside, polar R-groups +/- bond with water • Denaturing (unfolding)Conditions= 2 degree, 3 degree, 4 degree are weak interactions ○ Easy to cause peptide to change 3-D shape ○ Protein loosing 3-D shape, causes loss of function, and removal of denaturing condition restores function • Some conditions ○ Increase temperatures, increase K ○ Change pH of environment  Change [H+] to interfere w/ ionic bonds ○ Add or remove salts -> adds/ removes ions to solution interferes w/ ionic bonds • Reducing agents (uree, DTT, mercaptoethonal) ○ Breaks disulfidebridge (covalent bond) ○ Interrupts DS bridges b/t proteins (cysteine) Figure 4-26 • Reducing agent break S-S bonds by adding H's to the S's • Prions= proteins that are mutated ○ Mutation allows them to misfold to stable conformation ○ They are infections; Upon contact of other proteins of the same type, they cause it to misfold □ Misfold proteins tend to aggregate, they lose their function • Protein aggregates damage cells, tissues resists heat, pH, most reducing agent ○ From aggregate proteins you can get  BSE- Bovine Spongiform Encephalitis (Mad Cow)  CJD- Creutzfeldt Jacob Disease  Kury  Alzheimer's  Huntington's • 2 degree structure ○ Common holding patterns w/ in 3 degree  Alpha Helix  Beta Pleated sheets  Hydrogen bond b/t O and Carboxyl group (amino backbone) □ H-bonds b/t N-H and C=O of backbone (not AA specific) Chapter 4 2/15 Friday, February 15, 2013 10:00 AM Q: What is a primary structure? A: A chain of Amino Acid and it is the sequence of that chain. By sequence, the Amino Acids, which 20 are the first ones, 2nd, 3rd, etc. Q: What is the secondary structure? A: Alpha Helix and Beta Sheets are types; A folding of the amino acid sequence, put in particular patterns, the α and β Q: What is the tertiary Structure? A: The 3-D folding of the whole polypeptide Q: Does one structure depend on another? A: Yes, tertiary depends on primary, but secondary does not. (BC it is not AA specific), in other words, the bonds that hold 2 degree structure together are H-bonds that occur b/t the carboxyl and Amino groups, part of backbone Q: Can polar/Nonpolar sidechain effect it? A: Yes, to a certain extent. The primary structure does not determine precisely the 2 degree structure, there are some limitations. If you have particular types of AA in your primary structure, and those AA sides will have particular characteristics. They have to fit into the specific space the 2 degree structure will permit. The space for the R-groups to fit into the Alpha helix and that differs from the space permitted for the beta sheets. Also, the types of R-groups will determine, if that particular group can face water or not. There are some limitations but only a few, to prevent 2 degree from forming. So let's say it's substantially less dependent that tertiary structure is on folding, that's BC it's really dealing with interactions of H-bonds b/t the backbone, not b/t the side chains themselves. Figures 4-10 ac (A–C) In an α helix, the N–H of every peptide bond is hydrogen-bonded to the C=O of a neighboring peptide bond located four amino acids away in the same chain. (D–F) In the case of the β sheet, the individual polypeptide chains (strands) in the sheet are held together by hydrogen-bonding between peptide bonds in different strands, and the amino acid sidechains in each strand project alternately above and below the plane of the sheet. In the example shown, the adjacent peptide chains run in opposite directions, forming an antiparallel β sheet. (A) and (D) show all of the atoms in the polypeptide backbone, but the amino acid side chains are denoted by R. (B) and (E) show the carbon and nitrogen backbone atoms only, while (C) and (F) display the cartoon symbols that are used to represent the α helix and the β sheet in ribbon models of proteins (see Panel 4-2B, p. 129). • Secondary Structure = 2 degree ○ The folded sections w/in the tertiary structure  Types: alpha helix and beta sheets □ H-bonds b/t N-H and C=O, backbone (not AA specific) ○ Alpha helix= spiral shaped, in particular the alpha helix  H-Bonds occur b/t every 4 amino acids. So it’s the amino group of one amino acid that H-bonded to the carboxyl group of another AA that is four positions away from it. The more H-Bond, the stronger the structure. □ Takes about 20 AA to make it stable *** Polar=Phillic ***Non = Phobic Figure 4-12 The hydrophobic sidechains of the amino acids forming the α helix contact the hydrophobic hydrocarbon tails of the phospholipidmolecules, while the hydrophilic parts of the polypeptide backbone form hydrogen bonds with one another in the interior of the helix. About 20 amino acids are required to span a membrane in this way. • Happening in the lipid bilayer ○ The phospholipidhead are glycerol and phosphate, being polar, where the tails are non- polar. ○ The alpha helix is going to have side chains that are specifically non-polar. BC non-polar and polar do not like to be together, so in order for the R-groups to exist, they have to be nonpolar ○ We see alpha helix in trans membrane proteins  Ex: channels and receptors ○ The hydrophobic R groups are outside BC they are going to face the fatty acids which are also hydrophobic.  The backbone of a peptide chain is hydrophilic,BC the amino and the carboxyl groups are both polar covalent bonds, so both are polar. H-bonds has to be polar to bond  The polar backbone can exist going through the fatty acid tails BC, the R-groups are non-polar and it shields from the non-polar  If in non-lipid environment then roles are reversed Figure 4-13 In (A) a singleα helix is shown, with successiveamino acid side chains labeled in a sevenfold sequence “abcdefg.” Amino acids “a” and “d” in such a sequence lie close together on the cylinder surface, forming a stripe (shaded in red) that winds slowly around the α helix. Proteins that form coiled-coils typically have nonpolar amino acids at positions “a” and “d.” Consequently, as shown in (B), the two α helices can wrap around each other with the nonpolar sidechains of one α helix interacting with the nonpolar side chains of the other, while the more hydrophilicamino acid side chains are left exposed to the aqueous environment. (C) The atomic structure of a coiled-coil made of two α helices, as determined by X-ray crystallography. The red side chains are nonpolar. Coiled-coils can also form from three α helices (Movie 4.3). • Coiled coil ○ 2-3 alpha helices intertwined  Helices wrap around one another to minimizeexposure of hydrophobic residues to aqueous environment □ Ex: Heavy Myosin Chain (II)  They can coil around each other if there are AA side chains along the sides of them that want to interact □ Ex: If two hydrophobic chains come into contact with a watery environment then they will coil together to protect the hydrophobictails, by having the heads facing the water. The opposite happens in a lipid environment.  You see them in elongated proteins under mechanical strain. □ Ex: Keratin, muscle myosin (pulling mechanism) Figure 4 d-f • Beta sheets H-bonds b/t backbone of polypeptide lying side by side. H-bonds sideways to each other. A ○ H-bonds b/t backbone of polypeptide lying side by side. H-bonds sideways to each other. A horizontal bond pattern ○ Consists of several strands of the polypeptide chains, that are able to lie next to each other side by side to form H-bonds. ○ There are going to be some limitations on this. So any AA that have sidegroups that need to be close to each other would have to be AA that are both hydrophobic/hydrophilic. Figure 4-14 (A) Antiparallel β sheet (see also Figure 4–10D). (B) Parallel β sheet. Both of these structures are common in proteins. By convention, the arrows point toward the C-terminus of the polypeptide chain (Movie 4.4). • Strands antiparallel or parallel arrows point to C (carboxyl) terminus • The arrows by nature point to the C. The other end is A terminal (Amino) • The whole sheet is beta pleated, 3 strands or more ○ The more strands the stronger it is (stable). Figure 4-15 • B Sheets- Pleated ○ Rigid= more stable • Ex: proteins that require strength (silk)or platform surfaces (antifreeze proteins) ○ Anti-freeze proteins are found in a variety of organisms  Positioning hydrophilic AA, the same distance apart up the sheet and allows water molecules to come and H-bond with these areas and prevents the formation of ice crystals Figure 4-16 Elements of secondary structure such as α helices and β sheets pack together into stable, independently folding globular elements called domains. A typical protein molecule is built from one or more domains, linked by a region of polypeptide chain that is often relatively unstructured. The ribbon diagram on the right is of the bacterial gene regulatory protein CAP, with one large domain (outlined in blue) and one small domain (outlined in yellow). • Domains ○ Functional area, 2 degree structures plus unstructured regions. Proteins contain one or more. ○ Can have a domain made of Beta Sheets and Alpha helix, connected by loose regions, this is not whole protein only part. ○ Proteins can have more than one domain  Each domain has different function (depending on shape and change) ○ Domains are higher than secondary, but not a complete tertiary… Part of tertiary but made up of 2 degree *** Q: Could you have proteins w/ different 1 degree structures have the same/different domains? A: Yes, in order for a domain to exist it has to have 2 degree structure, BC they do not depend on the primary structure.  Figure 4.18 □ Different 1 degree structure= similar3 degree (SimilarAA/Domain) □ "Protein Families" ---> similarbut not identical function  Ex: Serine proteases, similar protolithic active sites; different substrates specificities ***Q: How can you have different primary, yet similar tertiary? ***Q: How can you have different primary, yet similar tertiary? ***A: BC of the categories (nonpolar/polar) if they are from the same group, then similarcharacteristics. And their R-groups are what make the folds. Chapter 4 2/18 Monday, February 18, 2013 10:00 AM • Quaternary Structure = 4 degree ○ Not all proteins have these structures ○ Formed from 2 or more polypeptides form functional proteins ○ Polypeptides = subunits (monomers of protein) ○ Each have 1, 2, and 3 degree structures Figure 4.19 • Primary sequence is identical • Non-covalent bonds • CAP proteins- homodimer (2 identical subunits) ○ Having domains  Binds DNA Figure 4.20 • Hemoglobin = heteroteramer (different 4) ○ 2 alpha globins ○ 2 beta globins ○ Found in blood • Each globin subunit has heme group associated in the middle of the polypeptide ○ Binds O2 in the hemoglobin protein, containing iron • Can bind 4 O2 molecules *** Cooperatively, that once one O2 binds, it makes the others easier to bind. Helped growth Figure 4.21 • Proteins ○ Globular or fibrous (shapes)  Globular=spherical  Fibrous=more linear • Come together to become more a complex, macromolecular, each protein=subunit • The shapes they become depend on the binding sites they have, the type of proteins they have and the location of the binding sites. Figure 4.22 • Actin Filament ○ Helix… Actin filament (micro filament/ F-Actin) an array of globular actin proteins (g-actin subunits) ○ Length nm-micro m, F-actin = 7nm wide ○ Same protein, G-actin working together to form the F active ○ They grow at one end and shrink at the other end ○ Dynamic structure, regulated by proteins inside. Figure 4.23 • Other globular proteins can form different structures • From different tubes can form microtubules • From different tubes can form microtubules • Spheres can form (virus coats) Figure 4.25 • Fibrous protein may form strong fibrils (left) (collagen) or elastic fibers (elastin) ○ Braided = collagen fibrils  Tissuestrength ○ Elastin- not braided, space b/t the protein, connected by disulfidebridges.  Elastic fibers, when mechanical force apply the proteins become straight but stay together. ○ Found in skin  Ex. Weenis, loose skin in elbow why skin stretches… Figure 4.27 • protein (substrate, enzyme) interacts w/ target molecules (ligand substrate) via complementary shapes/changes (usuallynon covalent) • Enzyme- substrate interaction (depends on shapes) • Receptor- ligand interactions ○ Receptors are proteins that bind to particular targets called ligands ○ Enzyme, the ligand is called substrate • Changing shapes, change the function, start, stop, etc. 4.29 • Antibodies ○ Immune response to foreign molecules (antigens) ○ Y-shaped made up of 2 HC and 2 LC w/ 2 identical binding sites at ends ○ Antibodies are fairly complex proteins ○ Specific to only antibodies, due to AA at binding sites ○ How your body knows which antibodies to make Table 4.1 • Ends in ase- usually enzyme • Prefix determines what it does ○ Hydrolase - catalyze hydrolysis (destroy) ○ Synthase - catalyze anabolic pathways, condensation (build) Figure 4.32 • 3 ways lower activation energy to start RXN ○ 2 or more Rxns come together- where they react together "comfy sofa" come together and react more easily ○ By changing the arrangement of charges, "electric chair" unlike charges next to each other ○ Dealing with substrate needs to bend in order to go into Rxn to change product. Puts in "traction device" to bend it to how it needs. Figure 4.34 • Regulation in cells (metabolic pathways) ○ Negative feedback inhibition-final product of pathway inhibits enzyme catalyzing first reaction reaction ○ Resources not used up ○ Efficient- no inhibitor synthesis ○ Self-regulating; product builds up, enzyme inhibited more ○ Fast: Not changing gene expression to up/down regulate enzyme Figure 4.36 • Competitive- inhibitor binds to active site • Noncom = inhibitor binds to allosteric site (not active) causes conformational (shape) change to active site • Feedback inhibition ○ Benefit of noncompetitive  Can attach to enzyme regard less of substrate concentration Figure 4.42 • Molecular Motors = allosteric proteins, shape changes by binding/hydrolysisof ATP and release of ADP • Hydrolysis irreversible w/o energy input; motor moves in one direction along cytoskeletal filament • Carry organelles or cause filament to slide Chapter 11 2/20 Wednesday, February 20, 2013 10:00 PM Figure 11-2 • Membrane functions 1. Receive information/initiate intracellular events (mostly proteins, lipids) 2. Import/export: transport hydrophilic(proteins), hydrophobic (lipids)substances 3. Provide flexibility for motility/growth (lipids) Figure 11-1 4. Selectively permeable barrier: Plasma membrane, cytosolic from extracellular. Internal membrane from organelles. • Plasma membrane enclosing cell Figure 11-3 • Types/concentration of molecules differ in cytosol compared to noncytosolic space ○ Ex: lysosomicmust contain an acidic pH compared to cytosol Figure 11-4 • Fluid mosaic model ○ Mainly non covalent interactions hold lipids/proteins w/in bilayer. Lipids/many proteins move w/in singlelayer (leaflet). Membrane flow, fuse, reseal if disrupted ○ Lipid bilayer is thermodynamically pleased a(5nm) ○ Proteins associated w/ plasma membrane must be amphipathic ○ Outer layer = extracellular leaflet ○ Inner layer = intracellular leaflet Figure 11.15 • Lipids ○ Move laterally, flex or rotate within a singleleaflet; they rarely flip-flop from one leaflet to another unless there is an input of energy Figure 11-5 • Lipids move laterally, flex, or rotate within singleleaflet; rarely flip flop from one leaflet to other (input of energy) • Cannot flip b/c heads must pass through tails • Lipid bilayer mostly phospholipids • Amphipathic: hydrophilicheads (phosphate (-)) and hydrophobic tails (fatty acids) • Double bond causes kink (unsaturated); less hydrogen to bond Figure 11-6 • Phosphotidycholine Most common; polar head (choline, phosphate/glycerol) and nonpolar tails (sat/unsat fats) ○ Most common; polar head (choline, phosphate/glycerol) and nonpolar tails (sat/unsat fats) Figure 11-7 • Other membrane lipids ○ Phosphotidylserine (phospholipid),cholesterol (sterol), and galactocerebroside (glycerol) ○ Amphipathic Figure 11-16 • Animal plasma membrane around 20% cholesterol (provides rigidity) • Decreases membrane fluidity (no cell wall), cholesterol small; easily inserts between unsaturated fats tails, 4 rings are rigid • Polar head groups • Rigid planar steroid ring structure • Nonpolar hydrocarbon tail Phospholipid vs cholesterol Polar head Cholesterol-stiffened region More fluid region Chapter 11 2/22 Friday, February 22, 2013 10:00 AM Q: Animal in warm or cold environment have higher cholesterol? A: Warm environment; BC the warmer climate would cause the lipids/fatty acids to become too fluid and fall apart. Higher temp causes more kinetic energy and movement in membrane and the falling apart of lipids. Having more cholesterols will cause the cell to be more rigid and less fluid. Q: How do lipidsmove around in the cell? A: They move laterally, they can shuffleright and left. However, they can't cut a flip spontaneously BC it causes too much energy. Figure 11-17 • Lipid bilayer is asymmetrical • Non-cytosol leaflet has different phospholipidsthan cytosol leaflets (types and concentrations) ○ Abundance of phospholipidswith read heads that face extra cellular space; they are dictating phospholipidColene ○ Brown headed ones are myelin • G's stand for sugar, so glycolipids ○ Cytosol lipids are higher in phospholipidcylene which are light green ones ○ Gray things are cholesterol, equal concentration between the two leaflets ○ Proteins are also, asymmetrical in both • Glycolipidsonly extracellular leaflet, except phosphotidyl inositol (sugar); internal signaling in cytosol leaflet. • Proteins asymmetrical distributed for function *Flipase-Cytosol Leaflet *Flopase-Non cytosol leaflet Figure 11.18 • Cell membrane synthesized in the ER (Smooth ER) ○ Unfavorable reaction, BC building (anabolic) ○ Couples w/ enzymes (flipase; an ATP-Ase) • Enzyme cytosol leaflet of the ER, it synthesizes phospholipids;adds to cytosol leaflet • Flipase flips particular phospholipidsto non-cytosol leaflet (ATP hydrolysis). Membrane grows evenly, of asymmetric in types of phospholipids. ○ Can flip in opposite direction (flopase) • Membrane grows evenly in size Figure 11.19 • Golgi is the source of adding the sugar groups, Golgi receives vesicle from the ER. ○ Motor proteins interact w/ cytoskeleton and carry vesicle around 1. In every organelle there will be a cytosolic leaflet (red) and a non-cytosolic leaflet (orange) a. When vesicles break off it fuses w/ plasmamembrane. b. Most glycolipids will end up on the extracellular membrane 2. The insideof the organelle is called the Lumen a. Enzymes adding sugar in lumen of Golgi,sugars added to lipidsin non-cytosol leaflet Types of membrane proteins based on their function 1. Transporters a. Diffusionof specific hydrophilicsubstance or moves substance up concentration gradient. b. Diffusion(passive) moves w/ gradient (more to less) can also be part of active 2. Anchors a. Links membrane to molecules or complexes on either side i. Intracellular linked to cytoskeleton filaments ii. Extracellular linked to collagen/elastin 3. Receptors a. Detect signals (ligand) from one side and relay info to molecules on other side i. Allosteric proteins (change shape = function change) ii. Binding to the other side changes the activity of the receptor at the other end. 4. Enzymes a. Catalyze or couple specific reactions on one side of membrane i. Catalyze reactions ii. Coupling favorable and unfavorable reactions iii. Specific to substrate based on active site *Table 11-1 (Will discuss more in future) Figure 11-21 • Membrane protein types w/ association to bilayer (connection b/t protein and membrane) 1. Trans membrane a. Extends through bilayer exposed on both sides (alpha helix and beta barrels) i. Useful for transporters 2. Mono-layered Association a. Hydrophobic imbedded in one leaflet; hydrophilicstands out 3. Lipid-Linked a. Covalent to lipid, lipid in one leaflet, protein extends from it 4. Protein attached a. Non covalent to another membrane protein i. To disrupt non covalent bonds you can 1) Increase kinetic energy (temp) 2) pH 3) Salt ii. PA= Peripheral Proteins (salt, pH, or temp to extract… Figure 11-26 • For lipid linked need to add detergent to extract, BC it has both hydrophilic/hydrophobicsides. Inserts b/t the lipids ○ Trans membrane, mono layered association and lipid layer  Equals integral proteins, detergent extractions • Detergent (amphipathic); linear CH chains w/ charged (SDS; SO4); or polar region (Triton X: OH) • Detergents interact with hydrophobic, hydrophilicor amphipathic substances ***DNA- Nucleotides (sugar, base, phosphate (-))**** Therefore DNA has negative (-) charge! Figure 11.34 • Intestinal epithelial cells; tight junctions restrict proteins to apical or basolatul. ○ Alsoprevent substances from passing b/t cells Figure 11.35 • Eukaryotic cells sugar-coated • Glycolipids,glycoproteins, proteoglycans on extracellular surface of plasma membrane (8+ sugar) • 3 main functions ○ Protection  Barrier to phys/ chem damage ○ Regnation  Cell type has sugar ID sugars of one cell bind recs on another cell. Selection on endothelial cells of blood vessels bind specific sugars on neutrophils during bac infections Chapter 11 2/25 Monday, February 25, 2013 10:00 AM Figure 11-27 • Detergents break up membranes ○ BC they are small anti-pathogens ○ Insert b/t lipidsand membrane BC proteins are weak (non-covalent bonds) • Physical perturbation: increase temp; detergent and lipid proteins form micelles ○ To separate the proteins from lipids  Centrifugation separates micelles by type protein, heavier  Then you can do SDS-PAGE, which separates denatured polypeptides by size. Figure 11-30 • Most membrane supported by protein mush work (cortex) attached by trans membrane anchors ○ Best studies with RBC's (Red Blood Cells)  Why? BC they need to keep their shapes and mature RBC do not have a nucleus Figure 11-31 (Electron Microscope to see) • RBC Cortex simple/ regular ○ Irregular array of proteins  Long/skinny- Spectrin (100nm flexible protein) Array attached to actin and other proteins held by TM (trans membrane) Anchors. ○ Other cells; cortex are more complex  Ex: Cell change shape or resist movement in proteins Figure 11-32 • Two types of studies (some proteins diffuse/somedo not) ○ Hybrid cell Epiflour  Mouse proteins G-ab, fusion of cells followmovement of dyes over time @ 37 degrees (mammalian body temp)  Fluorescent dyes used w/ EFM □ Rhodomein- emits red □ Fluorescence-Emits green  Human and mouse cells  Criticism over fact that human/mouse proteins together would not happen in nature. ○ Figure 11-36 FRAP (Fluorescence Recover After Photo Bleaching)  Label membrane proteins of singlecell with Fl-ab Bleach Dye in area w/ laser. Follow recover of Fl in bleached spot over time.  Dyed F, show up white  Proteins in membrane can diffuse laterally, in the membrane Figure 11.33 Non -DiffusionProteins • Restricting movements of membrane proteins localizes function • Stationary proteins tethered to cortex, extracellular matrix, surface proteins to another cell • Limited protein mov't by diffusion barriers in membrane Chapter 12 2/27 Wednesday, February 27, 2013 10:00 AM Table 12-1 Difference in particular ion per compartment for function (ex: Na+ in AP's, CO 2+ in sarcomere) • Higher outside vs in • Na, Mg, Ca, H, Cl • Showing free inons • Not part of another molecule • Important difference established concentration gradients for energy to move across membrane • ↑Ca inside= muscle contraction • Particular (ion) per compartment for function • Eg. Na in Aps, Ca2+ in sarcomere contraction • Organelles mitochondria concentration ↑ to cytosol H+ Figure 12-1 A. Protein free artificial lipid bilayer a. Ions can't cross lipid bilayer b/c charged-bilayer polar B. Cell Membrane a. Protein necessary to allow movement across membrane Transport proteins allow diffusionof charged polar molecules/ions. Move any molecule up concentration gradient. Figure 12.4 • Favorable ○ Passive transport involves molecule moving without cell energy; down concentration gradient ○ Move disordered ○ 2nd law of thermodynamics • Unfavorable ○ "Facilitated Diffusion"via channels and transporters  2 types □ Channel  One direction □ Transporter  2 way  Energy needed to go against concentration gradient ○ Active transport involves molecule moving with cell energy ○ More ordered ○ Binds to protein or not ○ Molecules bind to transporters Figure 12-3 • Channels ○ Pores select by size and charge ○ Passive ○ Most gated  No open pores • Transporters Greater than or equal 1 binding sites for molecules ○ Greater than or equal 1 binding sites for molecules ○ Allosteric (change shape) ○ Release molecule on other side of membrane ○ carries=passive ○ pump=active Figure 12-5 • Set of transporters determined by function of compartments ○ In lysoomes ○ More acidic in cytosol ○ 2 pyruvates per glucose ○ Oxidation = glycolysis Figure 12-16 • Transporter types ○ 1 type of molecule (uniport) ○ 2 different molecules in same direction (symport) ○ 2 different molecules in opposite direction (antiport) ○ Sometimes coupled transports ***Concentration gradients can be either a carrier or a pump!!! • Determined by the concentration gradient Figure 12-7 • Diffusionof ions depends on electrochemical (EC) gradient=charge and concentration difference across membrane • Electrostatic around particle • Combine concentration gradient and net charge • Synergistic: concentration gradient and net charge pull ion in same direction ○ Na influx into cytosol • Antagonistic: concentration gradient and net charge pull ion in opposite directions ○ K efflux from cytosol Chapter 12 3/1 Friday, March 1, 2013 10:00 AM • Channel ○ Passive transport only Transporter ○ Does NOT bind to substrate ○ Being transport one way only • Transporter ○ Passive or active Pathways ○ Binds substrate ○ Goes either ways Figure 12-8 • Carrier ○ Passive diff (no energy) ○ Binds substrate ○ ALWAYS down e-chem gradient • Pump ○ Active transport (energy used) ○ Binds substrate ○ Can be against e-chem gradient • Primary active transporters (ATP driven pump) ○ Use ATP hydrolysis to move substances up e-chem gradient ○ Must also be ATPase as well • Secondary active transporters (coupled transporter) ○ Using energy from other substrate going down e-chem gradient (couples favorable to unfavorable) • Active transporters require energy source ○ Primary (aka ATP-driven pump) use ATP hydrolysis ○ Secondary( aka coupled transporter) use energy from moving other substance down e-chem gradient Figure 12-9 • ATP driven pump (Na+/K+ pump) ○ 2 substances moving against e-chem gradient ○ Primary antiporter • Na+/K+ pump ○ Transport Na out and K in, both against e-chem gradient  Na (larger) □ Elec, conc gradient= synergistic(same direction)  K □ Elec, conc gradient= antagonistic (different directions) Figure 12-11 1. From cytosol 3 Na(s) bind to Na/K pump (on cytosolic site) 1. From cytosol 3 Na(s) bind to Na/K pump (on cytosolic site) 2. ATP hydrolysis,,brings E up a. Phosphate attaches to pump itself (high E link) 3. Pump changes conformation (cytosolic site closes ec site opens) a. B/c of phosphorylation (causes lack of affinity/fit to Na) 4. 2K's bind to new conf. of pump from EC 5. Pump loses P (DE phosphorylation) 6. Pump changes conf. to original, release K, good to bind Na again (EC site closes, C site opens, site of bind changes shape to orig.) ***Phosphate group is negative; weak non-covalent bonds in protein (pump) depend on dipole presence and disturbed by P (-) presence. Can continue as long as ATP present Figure 12-12, 12-13 • 3Na/2K movement benefits all: ○ 3 out vs. 2 in--> Delta= 1 out, prevents high concentration of solutes ○ In cell, prevents water moving into cell via osmosis ○ Animal cell:  Na/K pump maintains osmotic equilibriumby pumping out more ions that it pumps in ○ Solutes: (ions, organic mols) are high in cells ○ Water is relatively low  Water influx by osmosis would cause cell to swell, burst osmotic pressure balanced by 3Na out/2K in ○ Plant cells: vacuoles collect water and push against cell walls, stopping water influx(larger pressure)  Cell wall prevents bursting cells  rigid→push back cytosol = turgor  Rely on turgor to provide more rigidity ○ Protist: contractile vacuoles collect water and discharge water to outside of cell Figure 12-28 • Na/K pump also very important for maintaining charge dist. Across membrane ○ Membrane outside=positive ○ Membrane inside=negative ○ Both together = membrane potential ○ By pumping more + ions out than it take in, it makes outside more positive ○ Negative molecules insidethe cells  CL  Phosphate groups ○ K leak channels allow some k ions leak out the cell b/c of e-chem gradient. • Membrane Potential ○ Voltage difference across a membrane due to a slightexcess of positiveions on one side and negative on the other. A typical membrane potential for an animal cell plasma membrane is - 60mV (inside negative), measured relative to surrounding fluid. Chapter 12 3/4 Monday, March 4, 2013 10:00 AM Figure 12.9 • Na+/k+ pump+ transports Na outand K in; both against EC gradient Figure 12.28 • 2nd way potassium pump is helpful ○ Charge difference across plasma membrane: Na/K pump (1 net out), -mols inside, K- leak channels. Insidemore-than outside = membrane potential (difference in those charges) ○ Leak to potassium ion ○ Leak channels in membrane, leak out of cell ○ The difference b/t the positive and the negative is called the membrane potential Figure 12-17 • Big green thing is a transporter, you can tell bc things are binding to it • Third way potassium pump is helpful • Transporter- things can bid to • It's moving 2 things in the same direction (symporter) • Active transporter ○ BC it is being moved against the concentration/electrical chemical gradient ○ Secondary (doesn't use ATP Hydrolysis)  Gradient of Na, it being transported down it's gradient, so its favorable  They couple favorable/unfavorable reactions for energy • Na + EC grad. Used by secondary transport ○ Eg. Na+/glucose symport in apical membrane of epithelial Intestinal cells. Active glucose transport from lumen into cytosol coupled with passive Na+ transport. Figure 12.18 • Cartoon on intestine, so we can lookat epithelial cells ○ Coupling the transporter of Na/glucose into the epithelial cells  Showing us a conc. Gradient of glucose on the side  Low to high  Sodium is greater in the lumen then in the cell  As glucose comes in, the Na comes in as well ○ Active transporter • Na/K pump keeps cytosol {Na+} low • Basel membrane uniport (only one type molecule): ○ Passive glucose transport into bloodstream ○ Moving down it's conc. Gradient ○ It’s a carrier, BC it has binding sites • Tight Junction (diffusionbarriers) Figure 12-19 • In plants they have Proton pump H+ ○ Primary ○ Primary ○ Plant cells: H+ establish H+ gradient for secondary transport ○ Both animals and plants  Use H+ pumps to acidify organelles Figure 12.20 • Ion channels: Selectivity filers ○ Exist in the pore of the channel • Filter: ○ R-groups (from amino acids) create pore particular dia and charge for singleion type • Eg: K channel lined with C=O; O attracts cations; pore too narrow for Ca 2+ and too wide from Na Figure 12.22 • Channels faster rate of transport that transporters ○ Rush of ions into or out of cell creates electrical signal; rapidly cause response in cells  Ex. Venus fly trap Figure 12.23 • Patch clamp recording: ○ Detect electric currents through singleion channels. Sensitive and fast  So fast can measure through singleion channels Chapter 12 3/6 Wednesday, March 6, 2013 9:19 AM Chapter 15 36 Audio recording started: 9:59 AM Wednesday, March 6, 2013 Chapter 12 questions: Describe methods used to study ion channels Fig 12-23 An extremely sensitive type of recording, pico amp, record changes in a current in a mili second What kinds of ions are likely going through? How is the channel gated? Figure12-25 • 3 stimuli gate ion channels: voltage VG, ligand LG, mechanical stress MG 1. Voltage gated i. When at rest (inside -) outside + ii. Altered by the membrane potential (change in voltage) 2. Ligand gated i. Chemicals that bind to receptors ii. When channel binds the ligands, the proteins changes iii. Can come from the outside or the inside 3. Stress gated i. Opening is controlled by a mechanical force applied to the channel □ Inner Ear Hair Cells (in cochlea) □ Figure 12-26a □ In cochlea: Sound into fluid wave, moves basilar membrane, lifts hair cells in organ of Corti. Bundle of stereocillia on hair cell bends when bump against tectorial membrane. □ Stereocilla is an Actin filament (the hairs on the basilar membrane Fig 12-26b □ MG ion channels at stereocllia tips. Gates tethered to tips links. Pull open gates, cation influx, hair cell signals to auditory nerve □ The tip-linked model □ MG currents from Sea Anemone Hair Cells  Use hair for vibrations for communication Figure 12-31 • Neurons: receive (dendrites/soma), propagate (axon hillock, axon), and transmit (terminal) electric sigs. How? • "Giant" squid axons. Axons carry signals rapidly to muscles. ~ 1mm wide and 10 cm long. First studies of neurons • "Giant" squid axons. Axons carry signals rapidly to muscles. ~ 1mm wide and 10 cm long. First studies of neurons 1930's. • Fast reflexes, ,which cause mantle to close and jet propulsion away from danger Figure 12.36 a • Electrode placed inside the axon • Intracell recording: ○ Electrodes in/out axon monitor membrane potential. Ions flowing in/out change membrane potential. Figure 12-29 • Equilibrium • Rest: K+ leaks out until equil. Resting membrane potential-20 to -200mV ~equilibrium potential. Of K+ (other ion channels closed). Nerrnst equation: Calculate equil. Pot. Of ion V=RT/zF InCO/CI R= Gas constant T= Absolute temp Z= Charge F= Faraday's constant At 37C for K+ the equation simplifies to V=62log 10 (K+}o/[K+]i) If [K]I = 140 mM and [K+]o = 5mM Then V= 62 (-1.447) = -1.447 -89.28mV "polarized" Chapter 12 3/8 Friday, March 8, 2013 9:56 AM Figure 12-33 • Neuron depol to threshold at axon hillock (-40mV); AP froms. AP: Rapid depol (~+50mV) then rapid repolarization back to rest Figure 12-34 • Threshold: VG NA+ channels open. Voltage change exerts electric force on voltage sensor domain. (functional area of protein ,made up of secondary structures part of the tertiary structure) Figure 12.35 • Na enters, membrane potential depolarization. VG Na+ inactivate ~50 mV For ~1msec (stops Na influx). Channels return to closed configuration as membrane potential returns to rest. Figure 12.36 • Repol: "delayed" VG K+ channel opens as VG Na channel deactivates. VG K and leak K allow K efflux. Slight hyperpol until VG K close and Na/K pump restores resting concentration Figure 12.39 • Na/K channels throughout the axon (located in plasma membrane) • Propagation: AP travels down length of axon w/o diminishingfrom hillock to terminal. How? • Fig. A ○ Cytosol carries depolarized wave short distance. Threshold opens new VG Na channels unresponsive; AP only travels forward (away from hillock). Figure 12.40 • Synapse: axon terminal (presynaptic cell) relays sig to target cell (postsynaptic cell). Usually electric signal converted to chemical signal to cross space (cleft). Chemicals (NT) contained in presynaptic vesicles. • NT- neuro transmitters, released from vessicles Figure 12-41 • Terminal : AP opens VG Ca2+ channels. Ca influx causes NT vesicle fusion with presyn membrane. NT released into Cleft, diffuses, binds to receptor on postyn cell. Figure 12.42 • F NT rec is LG ion channel: NT (ligand) binding opens ion channel, creates electric signal in postsynaptic cell. ○ Ex: Accetocolyne  Occurs at  Muscle depolarises


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