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123 Cards in this Set
- Front
- Back
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membrane functions
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1. Selectively permeable barriers
2. Compartmentalization/specialization 3. selective transport of solutes 4. Anchorage points 5. Energy transduction 6. Communication |
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Overton (1890)
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ist insight into lipid nature of membrane
"Overton Rule" |
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Langmuir (1905)
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Lipid monolayer model. Knew lipids were amphipathic, he reasoned that in a monolayer the polar hydrophilic head groups face the water and the non-polar hydrophobic fatty-acyl chains were oriented facing air.
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Davson & Danielli
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1935... lipid-protein sandwich model, which accounted for the association of protein with membrane
1950s... added aqueous pores as an attempt to explain why some polar solutes are able to cross membranes |
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Overton Rule (1899)
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The rate at which any molecule enters a cell through its membrane is directly proportional to its lipid solubility such that the more lipid soluble a substance, the more readily it can cross through a membrae
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Singer and Nicolson (1972)
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proposed the fluid-mosaic model
describe membranes as dynamic, fluid-like structures... each a mosaic of lipids and proteins |
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fluid mosaic model
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in this model the proteins are partly embedded in the lipid bilayer or completely traverse the lipid bilayer and display domains at each membrane face
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Unwin and Henerson (1975)
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1. Determined 3D structure of bacteriorhodopsin, which lead to first determination of 3D-structure of a transmembrane protein
2. Now realize that most transmembrane proteins have multiple membrane spanning domains (and most of the membrane-spanning regions are alpha-helical) |
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Bacteriorhodopin
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is actually a 1degree -active proton uniporter powered by light (a proton pump)
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Two types of Glycerolipids
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a) Glycerophospholipids (phosphoglycerides)
b) Glyceroglycolipids |
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Two Types of Sphingolipids
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a) sphingophospholipids
b) sphingoglycolipids |
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Glycerophospholipids
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Have glycerol as their backbone, all have a phosphate group esterified to the glycerol moiety, and all (except phosphatidic acids) have an additional polar group covalently-linked to the phosphate group forming phophodiesters:
Phosphatidylcholines (PC) Phosphatidylserines (PS) Phosphatidylethanolamines (PE) Phosphatidylinositols (PI) |
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Sphingophospholipids
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sphingomyelin most common example
all have a phosphate group esterified to the glycerol moiety and all have an additional polar group covalently-linked to the phosphate group |
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Types of sphingoglycolipids
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Cerebrosides (neutral ceramid monosaccharides)
globosides (neutral ceramide oligosaccharides) gangliosides (if one or more siA/NANA) |
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glycerolipids
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have a glycerol based backbone, all are modified diglycerides
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sterols
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are important determinants of membrane fluidity and have membrane lipid ordering effects
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cholesterol
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can be >50% of the lipid present in some animal cell membranes
very common in animal cell membranes, rare in plants, absent in bacteria (except mycoplasma); complex membrane effects |
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phytosterols
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B-sitosterol, stigmasterol of plants
ergosterols of fungi |
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Cholesterol and phytosterols
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are isoprenoids synthesized from squalene
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isoprenoids
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are compounds composed of isoprene units
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ubiquinone
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an electron carrier of the mitochondrial electron transport chain
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farnesyl and geranygeranyl
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lipid anchors added to some proteins to keep them associated with membranes and composed of 15C and 20C respectively
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Roles of membrane carboyhydrates
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cell-cell binding events (e.g. cadherins)
cell-ECM binding events (e.g. integrins) receptors for infectious agents M6P residues act like molecular address labels |
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3 basic classes of membrane proteins
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peripheral (extrinsic)
integral (intrinsic) and lipid linked |
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Ph domain
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(plekstrin homolgy) domain, recognize and bind to polar head group of phosphoinositieds
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phosphoinositides
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Phosphorylated derivatives of PI
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PLC
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Phospholipase C, which has a PH domain
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EDTA
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ethylene-diamine-tetra-acetic acid
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Integral membrane proteins are further classified into
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monotopic, bitopic and polytopic
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Transmembrane proteins
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Include bitopics and polytopics and the completely traverse the lipid bilayer
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Peripheral Membrane proteins
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are water-soluble, and noncovalently associated with either face of a membrane via weak interactions (eg. Hydrogen bonds, ionic bonds, and/or van der waals interactions)
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Who originally classified integral membrane proteins as either bitopic, polytopic or monotopic?
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Blobel (1980)
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Transmembrane proteins
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include bitopics and polytopics. They completely span the membrane while exposing hydrophilic mostly globular domains at each membrane face.
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What structures function as transporters, receptor, enzymes, cell adhesion molecules (CAMs, etc)?
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Many bitopics and polytopics
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porins
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belong to the class of transport proteins known as pores and channels
(eg. porins of mitochondrial and chloroplast outer membrane, and porins of outer membran of gram-negative bacteria) |
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Futher definition of porins
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are plytopic transmembrane proteins with a Beta-pleated sheet that forms a Beta-barrel
forms an aqueous channel walled by aniparallel Beta-strands. |
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Transmembrane proteins are much harder to extract from the lipid bilayer than are peripheral membrane proteins because...
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the release of transmembrane from a membrane requires a relatively harsh treatment such as the use of an amphipahtic detergent to disrupt the membrane
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non-ionic detergents
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Triton X-100 (TX-100). Tween 20 and tween 80, nonpolar end displaces/replaces membrane lipids, solubilizes proteins without denaturation
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Triton X-100 (TX-100)
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tween 20, tween 80, nonpolar end displaces/replaces membrane lipids, solubilizes protein without denaturation... protein remains in a nearly native conformation
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ionic detergents
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eg. sodium dodecyl sulphate (SDS)
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What does the ionic detergent SDS do?
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nonpolar end displaces/replaces membrane lipids, electrostatic repulsion between charged ends of SDS molecules causes proteins to unfold... ionic detergents denature proteins
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How does SDS work?
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gives virtually all proteins a similar charge/mass ration (~1.4g SDS/g protein). Can separate them in an electric field on the basis of their mass (on their chain length).
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Sodium Dodecyl sulphate polyacrylamide gel electrophoresis
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SDS- PAGE
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electrophoresis
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is a commonly used technique that takes advantage of the movement of charged molecules (such as proteins) through an electric field
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What happens in polyacrylamid gel electrophoresis?
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The gel matrix consists of cross-linked acrylamide molecules that form a molecular sieve.
1. Opposite ends of the gel are placed in reservoirs of a suitable buffer 2. The buffer is usually alkaline 3. Mixtures of proteins are then applied to sample wells at the cathodic (-) end of the gel and a voltage is applied |
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How does SDS apply to PAGE
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Protein samples are normally treated before and during electrophoresis with SDS
before loading onto the gel (sample is heated 100 degrees C or 50-65 for ~2-5min. In presence of a reducing agent, such as BME or DTT) and glycerol is often included in the sample |
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Charge density of proteins
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The charge per unit mass. The greater the charge density the greater the force on the protein (the greater its migration rate), but in SDS PAGE proteins have the same charge density.
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Characteristics of membrane selectively permeable barriers
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1. Very permeable to gases and hydrophobic molecules, but less permeable to small uncharged polar molecules (e.g. ethanol>urea>water>glycerol)
2. Essentially impermeable to large uncharged polar molecules (e.g. glucose, sucrose), inrganic ions (eg. Na+, K+, Cl-, etc.) and charged polar molecules (eg. ATP, DNA, RNA, amino acids and proteins) |
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Characteristics of membrane compartmentalization/specialization
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A membrane can establish a different environment in the enclosed space vs. exterior environment; a membrane can establish concentration, electrical or elecrochemical gradients; and allows different compartments to have different environments, constituents, structure, function
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Characteristics of membrane selective transport of solutes
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facilitated diffusion through channels (e.g. ion channels, porin proteins, aquaporins, gap junctions, plasmodesmata) and facilitated diffusion or primary/secondary active trasport via transporters (e.g. glucose transporter, sodium/potassium-ATPase, proton pumps, and many other porters)
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Characteristics of membrane anchorage points
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1. Anchorage to other cells (structural junctions between neighbouring cells, such as adherens junctions, tight junctions, and desomosomes)
2. Anchorage of ECM components to the plasma membrane ( e.g. hemidesmosomes, and focal adherens) 3. Anchorage of cytoskeleton components to the plasma membrane (scaffold for attachment or positioning of proteins/enzymes) |
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Characteristics of membrane energy transduction
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e.g. electron transport chain of inner mitochondrial membrane used during aerobic respiration, e.g. electron transport chain of chloroplast thylakoid membrane used during photosynthesis
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Characteristics of membrane communication
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e.g. gap junctions of animal cells or plasmodesmata of plant cells, endocrine, paracrine, and neuronal cell signalling
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Gorter and Grendel (1925)
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extracted lipids from known number of RBCs then used Langmuir's method to create lipid monolayers on water. The RBC lipids they created had twice the expected SA for the RBC used; thus, they proposed the LIPID BILAYER MODEL
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Bacteriorhodopin
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is actually a primary active proton uniporter powered by light (a proton pump)
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Jacobson (1995)
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Used single particle tracking (SPT) to demonstrate that the lateral mobility of many membrane proteins is more restricted than previously proposed by Singer and Nicolson
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glyceroglycolipids
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no phosphate group, have a carbohydrate in ether linkage to the glycerol moiety
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After electrophoresis the proteins can be visualized using such techniques as
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staining with a protein-specific dye solution or autoradiography if the proteins are radioactive
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Why is typical PAGE performed in SDS?
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to help minimize the difference in protein mobilities that are due to shape and/or charge density so that the proteins separate in the gel on the basis of their molecular mass
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In SDS-PAGE describe the rate of movement of a protein through the gel
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the protein's mobility is inversely proportional to its molecular mass
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One dye that is used to visualize proteins in SDS-PAGE
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Coomassie brilliant blue
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When proteins of known molecular weight are separated by SDS-PAGE their relative mobilities in a gel can be used to construct a Standard Curve that can be used to determine?
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the molecular weight of other proteins that run in the same gel
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monotopic proteins
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rare in comparison to transmembrane proteins, are usually partially embedded in just one membrane leaflet and thus espose a domain at just one membrane face (e.g. FAAH)
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FAAH
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fatty acid amid hyrolase, a monotopic with an alpha-helical hydrophobic plateau
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Other monotopics than FAAH
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are known to have one or more transmembrane domains, but they do not reach the opposite face of the membrane (e.g. MAO-B)
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MAO-B
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monoamine oxidase-B, a monotopic dimer with transmembrane helices
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Two examples of homodimers
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FAAH and MAO-B
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lipid-linked proteins
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covalently linked to a lipid embedded in a membrane leaflet, many have purification properties similar to that of transmembrane proteins (e.g. GPI-linked proteins and proteins with multiple lipid anchors), and others are relatively easy to release from a membrane if they have only a single lipid anchor
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GPI-anchors
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glycosylphophatidylinositol is a modified form of PI; the covalent addition of a GPI anchor occurs in the RER and is a post-translational process. The mechanism involves a cleavage event that generates a new C-terminus
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Where do GPI-anchored proteins occur
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only at the E-face of a membrane and mostly confined to clusters in lipid rafts
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How are mobility difference that might be due to differences in shape minimized by the denaturing effect of SDS?
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The detergent converts different proteins into a common rod-like shape
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Prenylation
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is a post-translational process that anchors proteins to the P-face of a membrane. Adds an isoprenoid anchor to a protein.
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Acylation
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adds fatty-acyl anchors. The two that are commonly used are palmitic acid (16C) and myristic acid (14C).
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Palmitoylation
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is a post-translational process. Covalently links the anchor to a cysteine at or near C-terminus creates a thioester linkage (S-acylation) and is the only reversible lipid modification
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Myristoylation
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is a co-translational process, links anchor to N-terminus, often to Gly (Glycine)B an N-amide bond. Both processes anchor proteins to the P-face.
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why can myristoylation be a co-translationaly
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The addition of the anchor is occuring at the N-end termins
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Membrane must be fluid for many basic cell processes to occur
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motion of cilia and flagella, endocytosis, vesical formation, cytokinesis, cell movement (ameboid motion/cell crawling), insertion of membrane components, simple diffusion of substance across membrane, membrane transport mechanisms, formation of intercellular junctions, formation of various membrane protein/enzyme complexes
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If a membrane is too fluid
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membrane components may not be oriented correctly, the membrane would lack needed structural organization and mechanical support, etc.
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If a membrane is to rigid
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mobility would be lacking/absent
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What two factors influence membrane fluidity?
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The temperature and the membrane's lipid composition
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In the fluid functional state the phospholipids are mobile and may exhibit 4 types of motion
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lateral diffusion, rotation, flexing of fatty-acyl chain, and spontaneous flip-flop (traverse diffusion); rare but can be catalyzed by flippases
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Membranes are less fluid when
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cooler tem, longer fatty-acyl chain length, saturated and there are trans bonds
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Membranes are more fluid
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im warmer temperatures, with shorter the fatty-acyl chain lengths, unsaturated, and cis
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hemidesmosomes
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specialized adhesive structures at the base of epithelial cells that function to attach the cells to the underlying basement membrane.
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focal adhesion
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adhesive structure characterized of cultured cells adhering to the surface of a culture dish.
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adherens junctions
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are a specialized type of adhesion junction particularly common in epithelia. The plasma membrane here is separated by 20 to 35nm and are sites where cadheren molecules are concentrated. The cells are held together by linkage of the extracellular domains of cadherin molecules that bridge the gap between neighbouring cells.
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cadherin
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A family of related glycoproteins that mediate CA2+ dependent cell to cell adhesion. They are transmembrane glycoproteins of the plasma membrane that bind other cadherins at the surface of neighbouring cells.
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paracrine
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release of a hormone or a sectretion by an endocrine cell into adjacent cells or the surrounding tissue rather than into the bloodstream
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lecithin
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phosphatidylcholine (PC) along with cholesterol were important components to the Overton Rule
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Davson and Danielli
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proposed lipid sandwich model (1935)
modified lipid sandwich model with aqueous pores (1950s) |
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aquaporins
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are proteins embedded in the cell membrane that regulate the flow of water
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bacteriorhodopsin
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first determinant of the 3D-structure of a transmembrane protein done by Unwin &Henderson and found it is a "7-pass" integral membrane protein . Primary active proton uniporter powered by light (a proton pump)
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Glyceroglycolipids
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no phosphate group; instead, have a carbohydrate ether linked to the glycerol moiety
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sphingophospholips
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all have a phosphate group esterified to the ceramide moiety and all have an additional polar group covalently-linked to the phosphate group
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ceramide
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a sphingolipids that has a sphingosine based "backbone" and one fatty-acyl chain esterified to sphingosine
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sphingoglycolipids
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no phosphate group and two broad categories: globosides and cerebrosides
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cerebroside
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are neutral ceramide monosaccharides
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globosides
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are neutral ceramide oligosaccarides
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ganglioside
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if one or more sialic acid residue is present on a cerebroside or globoside
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Dolichols
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used in the production of N-linked glycoproteins
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Examples of steroid hormones
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Cortisol, estradiol, and testosterone, used in cell signaling
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What does the polymerization and/or cyclization of isoprene units synthesize?
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1. Cholesterol, erogosterol, and stigmasterol
2. ubiquinone 3. fanesyl and geranylgeranyl 4. steroid hormones 5. dolichols |
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Almost all integral membrane proteins are?
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Glycolipids
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The sugar coat that glycolipids form at the outer surface of animal cell is called ____?
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glycocalyx
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Integrins
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are transmembrane glycoproteins of the plasma membrane that bind components of the extracellular matrix (ECM)
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What important roles in recognition events at the exterior surface of the cell (e-face of the plasma membrane) do the carbohydrate moieties of glycolipids and glycoproteins play?
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Cell-cell binding events (performed by cadherins)
Cell-ECM binding events (performed by integrins) and receptors for infectious agents |
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What important roles do the carbohydrate moieties of glycolipids and glycoproteins play in the role of molecular recognition events at internal membranes?
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Newly made lysosomal enzymes are tagged with M6P residues. These modified carbohydrates can then act like moleculare address labels (they are recognized and bound by M6P receptors of the trans-Golgi network (TGN)
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What two types of linkages exist between carbohydrates and proteins
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N-linked to asparagine (Asn) and O-linked to serine (Ser) or threonine (Thr)
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N-linked to asparagine (Asn)
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The reaction that produces these types of covalently modified proteins occurs on the lumenal side of the RER
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Where do most of the reactions that produce O-linked to serine (Ser) or threonine (Thr) occur?
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these types of covalently-modified proteins occur on the cisternal side of a Golgi (some also in the ER and even in the cystol)
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Where are membrane carbohydrates almost always located?
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at the noncytosolic (e-) face of a membrane
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dolichol-PP glycoside
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is a sugar carrier formed at the p-face of RER during the synthesis of N-linked oligosaccharides
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Zovirax
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Acyclovir
is an antiviral medicine. It is used to treat or prevent infections caused by certain kinds of viruses. Examples of these infections include herpes and shingles |
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phosphoinositides
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are important binding sites for lipid-binding proteins that have PH domains, such as phosholipase C
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Phospholipase C
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(PLC) an ezyme which is involved in the production of second messengers within cells during signal transduction events
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What are the membrane spanning domains of integral membrane proteins like?
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They are usually alpha-helical with mostly hydrophobic R-groups oriented towards the hydrophobic fatty-acyl chains of membrane lipids
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How are polar channels formed?
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Some polytopics form one or more polar channels by associating several alpha-helices such that the polar R-groups of their amino acids are oriented towards the interior of the channel while their nonpolar R-groups are oriented towards the hydrophobic core of the membrane
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Porins
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belong to the class of transport proteins known as pores and channels, different porins have different channel sizes and different solute selectivities although they tend to allow the free passage of many different types of solutes (e.g. porins of mitochondrial and chloroplast outer membrane, and porins of outer membrane of Gram-negative bacteria)
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What is glycerol's role in SDS-PAGE?
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It is usually included in the sample cell, because it makes the sample more dense than the electrode buffer and that helps to keep the sample in the well when it is being applied.
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What does bromophenol blue do in SDS-PAGE?
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because the proteins are invisible during the process, the progress of the electrophoretic separation can be followed by watching the movement of a this tracking dye
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autoradiography
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if the proteins were readioactively labeled an X-ray film can be applied to the gel for a period of time and then develope into an autoradiograph in much the same way as a photographic negative
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prenylation
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The process that add isoprenoid-anchors (such as farnesyl & geranylgeranyl) to a protein. It is a post-translational process that anchors proteins to the P-face of a membrane and covalently links the anchor to the C-terminus of a protein
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Proteins with multiple lipid anchors
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caveolin of lipid rafts - has 3 lipid anchors
Galpha of heterotrimeric G-proteins is palmitoylated near its C-terminus and myristoylated at its N-terminus |
