Medical Biochemistry

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Medical Biochemistry

• Membranes Bilayer Properties, Transport
• Lecture 71

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Membrane function

• Serve as barriers to separate contents of cell
  from external environment or contents of
  organelles form remainder of the cell
• Proteins in cell membrane have many functions
• transport of substances across the membrane
• enzymes that catalyze biochemical reactions
• receptors on exterior surface that bind external
  ligands (e.g., hormones, growth factors)
• mediators that aid ligand-receptor complex in
  triggering sequence of events (second messengers
  that alter metabolism are produced inside the
  cell)

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Plasma membrane has selective permeabilities

• Channels and pumps
• for ions and substrates
• Specific receptors
• for signals (e.g., hormones)
• Exchange materials with extracellular environment
• exocytosis and endocytosis

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Membranes form specialized compartments

• Organelles with specialized functions
• e.g., mitochondria, ER, Golgi complex
• Localize enzymes
• Excitation-response coupling
• Energy Transduction
• photosynthesis, oxidativephosphorylation

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Internal Water Is Compartmentalized

• Intracellular Fluid (2/3 of total water)
• rich in K and Mg2, phosphate major anion
• protein higher
• Extracellular Fluid (1/3 of total water)
• high Na and Ca, chloride major anion
• glucose higher

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Composition of membranes varies within and
between cells

• Major lipids in mammalian membranes
• Phospholipids
• Glycosphingolipids
• Cholesterol

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• Phospholipids - two major classes
• 1. phosphoglycerides are more common
• glycerol backbone
• two fatty acids in ester linkage
• usually even-numbered carbons (C16, C18)
• unbranched, either saturated or unsaturated
• C18 or 204,?5,8,11,14
• phosphorylated alcohol
• phosphatidic acid (1,2-diacylglycerol
  3-phosphate) is simplest -- key intermediate in
  formation of all other phospholipids

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• Phospholipids - two major classes
• 2. sphingomyelins
• sphingosine backbone (rather than glycerol)
• fatty acid attached by amide linkage
• primary hydroxyl group of sphingosine esterified
  to phosphocholine
• prominent in myelin sheaths

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• Glycosphingolipids
• sugar-containing lipids
• e.g., cerebrosides and gangliosides
• also derived from sphingosine
• differ from sphingomyelin in group attached to
  primary hydroxyl group of sphingosine
• sphingomyelin - phosphocholine
• cerebroside - single hexose (glucose or
  galactose)
• ganglioside - chain of 3 or more sugars (at least
  one is sialic acid)

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• Sterols
• most common sterol ? cholesterol
• almost exclusively in plasma membrane
• lesser amounts in mitochondria, Golgi, nuclear
  membranes
• generally more abundant toward outside of plasma
  membrane
• intercalates among phospholipids of membrane with
  its hydroxyl group at aqueous interface and
  remainder of molecule within leaflet

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Membrane lipids are amphipathic

• Contain both hydrophobic and hydrophilic regions
  (like detergents)
• polar head group
• nonpolar tails
• Saturated fatty acids - straight tails
• Unsaturated fatty acids (generally cis) - kinked
  tails

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What is the effect of unsaturated fatty acids?
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What is the effect of unsaturated fatty acids?

• as more kinks added, membrane becomes less
  tightly packed, more fluid

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Membrane lipids form bilayers

• Amphipathic phospholipids have two regions with
  incompatible solubilities
• in aqueous solvent, organize into
  thermodynamically favorable form (e.g., micelle)

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Membrane lipids form bilayers

• Bimolecular layer (bilayer) can also satisfy
  thermodynamic requirement of amphipathic molecule
• only ends or edges of bilayer sheet exposed to
  unfavorable environment
• can eliminate by folding sheet back upon itself
  to form enclosed vesicle with no edges.
• Closed bilayer is essential property of membrane
• impermeable to most water-soluble molecules

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Lipid-soluble materials

• Gases (oxygen, CO2, nitrogen)
• little interaction with solvents, readily diffuse
  through hydrophobic regions of membrane
• Lipid-derived molecules (e.g., steroid hormones)
• readily transverse bilayer
• Organic nonelectrolyte molecules
• diffusion dependent upon oil-water partition
  coefficients (the greater lipid solubility, the
  greater its diffusion rate across membrane)

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Non-lipid-soluble molecules

• Proteins are also amphipathic molecules
• inserted into lipid bilayer
• form channels for movement of ions and small
  molecules
• serve as transporters for larger molecules

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Non-lipid-soluble molecules

• Side chains determine hydrophobic nature
• 6 strongly hydrophobic side chains, few weakly
  hydrophobic, remainder hydrophilic
• amphipathic proteins have hydrophobic region
  transversing bilayer and hydrophilic regions
  protruding inside and outside of membrane
• protein content varies with membrane
• enzymes, transport proteins, receptors

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Membranes and components are dynamic structures

• Lipids and proteins in membranes turn over
• different lipids and proteins have individual
  turnover rates, may vary widely
• membrane may turn over more rapidly than any of
  its constituents

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Membranes Are Asymmetric Structures

• Irregular distribution of proteins within
  membrane
• External location of carbohydrates attached to
  membrane proteins
• Regional asymmetries
• villous border of mucosal cells
• gap junctions, tight junctions,synapses

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Membranes Are Asymmetric Structures

• Phospholipid asymmetry
• choline-containing phospholipids located mainly
  in outer leaflet
• phosphatidylcholine, sphingomyelin
• aminophospholipids preferentially located in
  inner layer
• phosphatidylserine, phosphatidylethanolamine
• cholesterol generally present in larger amounts
  on the outside

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Membranes Are Asymmetric Structures

• Must be limited transverse mobility (flip-flop)
• half-life of asymmetry in synthetic bilayers is
  several weeks
• enzymes for phospholipid synthesis are located on
  cytoplasmic side of microsomal membranes
• flippases
• phospholipid exchange proteins

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Integral and peripheral proteins

• Integral membrane proteins
• interact with phospholipids, require detergents
  for solubilization
• usually globular, amphipathic
• may span bilayer many times
• asymmetrically distributed across bilayer
• orientation determined during insertion in bilayer

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Integral and peripheral proteins

• Peripheral proteins
• do not interact directly with phospholipids
• do not require detergent for release
• weakly bound to hydrophilic regions of specific
  integral proteins

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Integral and peripheral proteins

• e.g., ankyrin, bound to integral protein band 3
  of erythrocyte membrane
• spectrin, a cytoskeletal structure within
  erythrocyte, bound to ankyrin
• plays important role in maintenance of biconcave
  shape of erythrocyte

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Artificial membranes model membrane function

• Mixtures of one or more phospholipids treated
  (e.g., sonication) to form spherical vesicles ?
  liposomes
• can control lipid content to examine effects of
  lipid composition on certain functions
• purified membrane proteins can be incorporated
  into these vesicles to access factors required
  for function
• environment can be controlled and varied (e.g.,
  ion concentrations)
• can be made to entrap compounds inside (e.g.,
  drugs, isolated genes) for drug delivery, gene
  therapy

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Fluid mosaic model

• Singer and Nicolson (1972)
• icebergs (membrane proteins) floating in a sea of
  predominantly phospholipid molecules
• translational diffusion - integral proteins and
  phospholipids can move within the plane of the
  membrane

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Fluid mosaic model

• phase changes (fluidity) of membrane are
  dependent upon lipid composition
• hydrophobic chains of fatty acids can be highly
  ordered ? rigid structure
• with ? temperature, side chains undergo
  transition from ordered state (gel-like or
  crystalline phase) to disordered (liquid-like or
  fluid) phase
• transition temperature (Tm)
• longer, more saturated fatty acid chains interact
  more strongly, cause higher Tm
• unsaturated chains tend to ? fluidity, ?
  compactness

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Fluid mosaic model

• Cholesterol modifies fluidity of membranes
• At temperatures below Tm it interferes with the
  interaction of hydrocarbon tails of fatty acids
  and increases fluidity
• At temperatures above Tm it limits disorder
  because it is more rigid than tails of fatty
  acids and cannot move in membrane to same extent,
  thus limits fluidity
• At high cholesterolphospholipid ratios,
  transition temperatures are abolished

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Fluid mosaic model

• Fluidity significantly affects membrane functions
• As membrane fluidity ?, so does permeability to
  water and other small hydrophilic molecules
• Lateral mobility of integral proteins increases
• If active site of integral protein resides
  exclusively in hydrophilic regions, changing
  fluidity probably has little effect on activity
• If protein involved in transport, with transport
  components span membrane, lipid phase effects may
  significantly alter transport rate.
• EXAMPLE Insulin receptor - As concentration of
  unsaturated fatty acids in membrane increased
  (grow in unsaturated. fatty acid rich medium),
  fluidity increases, receptor binds more insulin

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Fluid mosaic model

• Some protein-protein interactions within plane of
  membrane can restrict mobility of integral
  proteins

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Asymmetry of proteins and lipids maintained
during membrane assembly

• Fusion of a vesicle with the plasma membrane
  preserves the orientation of any integral
  proteins embedded in the vesicle bilayer

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Signal Sequences Target Many Proteins

• Many proteins carry signals that target them to
  their destination
• Major sorting decision - synthesis on free or
  membrane-bound polyribosomes
• cytosolic branch
• no signal peptide, delivered tocytosol
• can be directed to mitochondria, nuclei,
  peroxisomes by specific signals

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Signal Sequences Target Many Proteins

• rough ER branch (Secretory or exocytotic pathway)
• contain signal peptide
• many destined for various membranes (ER, Golgi,
  lysosomes, and plasma membrane) and for secretion
• certain proteins sorted in Golgi for delivery to
  lysosomes
• proteins destined for secretion carried in
  secretory vesicles
• regulated secretion (secretory vesicles)
• constitutive secretion (transport vesicles)

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Signal Hypothesis - Entry into ER

• Blobel and Sabatini - explanation for difference
  between free and membrane-bound ribosomes
• All ribosomes have the same structure,
  distinction dependent upon protein possessing
  signal sequence

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Synthesis of secretory proteins
1. N-terminal signal sequence is synthesized 2.
Signal bound by SRP, complex docks with SRP
receptor on ER membrane 3. Signal sequence binds
to translocon, internal channel opens, inserted
into translocon
4. Polypeptide elongates, signal sequence
cleaved 5. ER chaperones prevent faulty folding,
carbohydrates added to specific residues 6.
Ribosomes released, recycle 7. C-terminus of
protein drawn into ER lumen, translocon gate
shuts, protein assumes final conformation