Lecture title: BIOMEMBRANE 2018/2019

Dr Ajayi Olulope Olufemi   (Published 2019)

Dr Olulope Olufemi
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Lecture Note

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EDO UNIVERSITY IYAMHO
Department of Biochemistry
BCH 316: Biomembrane
Instructor: Dr. Olulope Ajayi, email: olulope.olufemi@edouniversity.edu.ng
Lectures: Wednesday (2-3 pm), Friday (10 am-12 pm). LT6. Phone (+234)8063737930
Office hours: Monday-Friday (8.00 am-4.00 pm). Office: New College of Medical Sciences and
Faculty of Engineering Office Block, Floor 2 Rm. AD 82
General overview of the Course
This course covers the central dogma of membrane biology (the fluid mosaic model), Membrane
functions, types and composition: Lipid structure, properties and formation of the bilayer; protein
and carbohydrates. Membrane structure and integrity. Membrane asymmetry and movements,
diffusion, rotation and fluidity. Isolation and identification electron microscopy and marker
enzyme assays. Introduction to receptor function: antigenicity of membrane components. Cell
membrane and toxins, transport processes, action of polymyxin and ionophores. Introduction to
neurotransmission. Membrane transport system- active versus passive transport systems.
Transport of sugars and amino acids. Defence mechanism in parasites. Biomembranes of
parasites.
Intended Learning Outcomes
At the end of this course, students should be able;
1. To define biomembrane
2. To list and discuss constituents of biomembrane
3. To discuss the functions of biomembrane
4. To discuss diffusion of biomembranes lipids
5. To discuss the roles of integral proteins
6. To discuss artificial membrane and its applications
7. To discuss membrane fusion
8. To explain the interaction of toxins with biomembranes
9. To describe defense mechanisms in parasites
Assignments: We expect to have 5 individual homework assignments throughout the course in
addition to a Mid-Term Test and a Final Exam. Home works are due at the beginning of the class
on the due date. Home works are organized and structured as preparation for the midterm and
final exam, and are meant to be a studying material for both exams. There will also be 2 term
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papers are expected to be written by individuals taking this course. This is aimed at broadening
student’s knowledge of the course.
Grading: We will assign 10% of this class grade to homeworks, 10% for the term papers, 10%
for the mid-term test and 70% for the final exam. The Final exam is comprehensive.
Textbooks: The recommended textbooks for this class are as stated:
Title: Lehninger Principles of Biochemistry
Authors: David L. Nelson, Michael M. Cox
Publisher: Addison-Wesley 2nd edition
ISBN-13: 9781464126116
Title: Lippincott’s Illustrated Reviews Biochemistry
Author: Denise R. Ferrier
Publisher: Lipincott Williams & Wilkins
ISBN: 978-1-4511-7562-2
Title: Harper’s Illustrated Biochemistry. 28th edition
Authors: Robert K. Murray, Daryl K. Granner, Victor W. Rodwell
Publisher: McGraw Hill Lange
MAIN LECTURE
Topic: Membrane Biochemistry: Introduction
What are membranes?
Membranes are complex molecules composed of proteins, lipids and carbohydrate molecules.
They are sheet-like enclosed structures consisting of an asymmetric lipid bilayer with distinct
inner and outer surfaces. These sheet-like structures are formed spontaneously in water due to
the amphipathic nature of lipids. The membranes contain numerous proteins that carry out
specific functions. They are structures that define and control the composition of the space that
they enclose.
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Membrane Lipids
The major lipids in mammalian membranes are; Phospholipids, Glycosphingolipids and Sterol
(Cholesterol)
The Phospholipids
There are two major classes of phospholipids; Phosphoglycerides and Sphingophospholipids.
Phosphoglycerides
They are the more common class of phospholipid and consist of a glycerol backbone to which
two fatty acids are attached in ester and a phosphorylated alcohol. The fatty acid constituents are
usually even-numbered carbon molecules, most commonly containing 16 or 18 carbons. They
are unbranched and can be saturated or unsaturated with one or more cis double bonds. The
simplest phosphoglyceride is phosphatidic acid
Sphingophospholipids
This is the second major class of phospholipids. Sphingophospholipids contain the sphingosine
backbone rather than glycerol. Sphingomyelin is prominent in myelin sheaths. A fatty acid is
attached by an amide linkage to the amino group of sphingosine forming a ceramide. The
primary hydroxyl group (-OH) of sphingosine is esterified to phosphorylcholine forming
sphingomyelin.
Sterols
Cholesterol is the most common sterol in mammalian membranes. It is found in the plasma
membranes of mammalian cells and to a lesser extent in certain organelles such as mitochondria,
golgi complexes and also in nuclear membranes. Cholesterol intercalates among the
phospholipids of the membrane with its hydroxyl group at the aqeous interface and the remainder
of the molecule within the leaflet. Saturated fatty acids have straight tails, whereas unsaturated
fatty acids, which generally exist in the cis form in membranes, make kinked tails. As more
kinks are inserted in the tails, the membrane becomes less tightly packed and therefore more
fluid.
Lipid bilayer: This is a thin bimolecular sheet of mainly phospholipid molecules that form the
structural basis for all cell membranes. The amphipatic nature of phospholipids suggests that the
two regions of the molecule (hydrophilic head and hydrophobic tail) have incompatible
solubilities.
Membrane proteins
Proteins are the major functional molecules of membranes and consist of; enzymes, pumps,
channels, structural components, antigens, receptors. They are vital components of the
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membrane lipid bilayer. The hydrophilic nature of peptide bond is minimized by the helical
structure of proteins. Their hydrophilic regions protrude at the inside and outside faces of the
membrane but connected by a hydrophobic region that traverses the hydrophobic core of the
membrane bilayer. Membrane proteins are of two types
Integral: They are located within the membrane
Peripheral: They are located on the surface of membrane
The Integral Proteins
They constitute a high percentage of membrane protein, interact extensively with the
phospholipids and require the use of detergents for their solubilization. They span the membrane
bilayer .They are asymmetrically distributed across the membrane bilayer. They are usually
globular and are amphipathic
The Peripheral Proteins
They are bound to the hydrophilic regions of specific integral proteins. They do not directly
interact with the hydrophobic cores of the phospholipids in the bilayer.
Functions of the Biomembranes
1. They keep toxic substances out of the cell
2. They contain receptors and channels that allow specific molecules, such as ions,
nutrients, wastes, and metabolic products, that mediate cellular and extracellular activities
to pass between organelles and between the cell and the outside environment
3. They separate vital but incompatible metabolic processes conducted within organelles.
Topic: Membrane Fluidity
Membrane fluidity is the ability of membrane to take on a more liquid-like or fluid arrangement.
Membrane fluidity is a function of temperature and lipid composition
Temperature
The hydrophobic chains of the fatty chains can be aligned or ordered to provide a rather stiff
structure in a bilayer lipid.
Increase in temperature causes the hydrophobic side chains to undergo a transition from ordered
state to a fluid arrangement. The temperature at which the structure undergoes the transition from
the ordered state to disordered state is called the transition temperature (Tm).
Lipid Composition
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The multiple (unsaturated) bonds that exist in the cis configuration increase the fluidity of a
bilayer by decreasing the compactness of the side chain. Cholesterol reduces the fluidity of
membranes. Transition temperature is however indistinguishable at a high cholesterolphospholipid
ratio.
The fluidity of a membrane significantly affects its functions. As membrane fluidity increases, so
does its permeability to water and other small hydrophilic molecules. The lateral mobility of
integral proteins increases as the fluidity of the membrane increases.
Diffusion of membrane lipids
Flexibility is a notable feature of biomembranes. Membrane flexibility is their ability to change
shape without losing their integrity and becoming leaky. It is a peculiar quality of biomembranes.
Membrane flexibility is made possible by the non-covalent interactions among the lipid bilayers
and the motions allowed to individual lipids. Sterols reduce membrane flexibility. The rigid
planar structure of the steroid nucleus inserted between fatty acyl side chains reduces the
freedom of neighboring fatty acyl chains to move by rotation about their fully extended
conformation.
Types of diffusion of membrane lipids
1. Flip-flop diffusion/ transbilayer: This is uncatalyzed transverse diffusion
2. Catalysed transverse diffusion
3. Uncatalysed lateral diffusion
Flip-flop diffusion
Flip-flop occurs slowly within a temperature range of 20-40oC. It requires that a polar/charged
head group leave its aqueous environment and move into the hydrophobic interior of the bilayer.
This type of movement becomes necessary during the synthesis of the bacterial plasma
membrane, phospholipids are produced on the inside surface of the membrane and must undergo
flip-flop diffusion to enter the outer leaflet of the bilayer. The flippases facilitates flip flop
diffusion this provides transmembrane path that is energetically more favourable and much faster
than the uncatalyzed movement
Topic: Artificial membrane and membrane fusion
Artificial membranes are synthetically made membranes usually for separation purpose. They are
produced from organic compounds i.e. cellulose nitrate or acetate, inorganic compounds i.e.
alumina etc. Separation depends factors such as the chemical properties, physical properties,
nature of separated particles and choice of driving force
Application of artificial membrane
1. Dehydrogenation of natural gas
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2. Removal of microorganisms from products (dairy product)
3. Water purification-reverse osmosis,
4. Microfilteration: Membrane pore size (0.1-10?m) Process of separating material of
colloidal size. Ultrafilteration. Membrane pore size (0.1-0.01 ?m)
5. Dialysis
Advantages of artificial membranes
1. The possibility of varying the lipid content of the membranes. This allows systematic
assessment of the effects of varying lipid composition on certain function
2. Purified membrane proteins/enzymes can be integrated into these vesicles in order to
assess the required factors for the proteins to reconstitute
3. The environment of these systems can be rigidly controlled and systematically varied
(e.g. ion concentrations, ligands)
Liposomes
They are small artificial vesicles of spherical shape that can be created from cholesterol and
natural non-toxic phospholipids.
Their usefulness in drug delivery is as a result of its size, hydrophobic and hydrophilic
characters. Liposomes can also be made to entrap certain compounds inside themselves.
Examples are drugs and isolated genes. Liposomes can be used to distribute drugs to certain
tissues. If certain antibodies to certain cell surface molecules could be incorporated into
liposomes so that they would be targeted to specific tissues or tumors, the therapeutic impact
would be considerable. DNA entrapped inside liposomes appears to be less sensitive to attack by
nucleases.
Membrane Fusion
This is the coming together of two membranes without loss of continuity. Mechanisms involving
membrane fusion are seen in exocytosis (release of neurotransmitters), endocytosis, cell division,
fusion of egg and sperm cells and entry of membrane-enveloped virus into its host cell
Requirements for membrane fusion
1. They must recognize each other
2. Their bilayer structures become locally disrupted resulting in fusion of the outer leaflet of
each membrane (hemifusion)
3. Their surfaces become closely apposed which requires the removal of water molecules
normally associated with the polar head groups of lipids
4. The fusion process is triggered at the appropriate time or in response to specific signal
5. Their bilayers fuse to form a single continuous bilayer
Instances of membrane fusion
1. The entry of an enveloped virus (influenza virus) into a host cell
2. The release of neurotransmitters
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The entry of an influenza virus into a host cell
The influenza virus is enveloped by a membrane containing hemagglutination (HA) protein. The
virus enters the host cell by inducing endocytosis at a pH of about 5. The low pH causes a
conformational change in the HA which exposes a sequence within the HA protein, this enables
the protein penetrate the endosomal membrane. Thus, the endosomal membrane and the viral
membrane are connected through the HA protein. The HA protein bends at its middle forming a
hairpin shape, bringing its two ends together, this pulls the two membranes into close apposition,
causes fusion of the viral membrane and the endosomal membrane
The release of neurotransmitters
Neurotransmitters are released at synapses when intracellular vesicles loaded with
neurotransmitter fuse with the plasma membrane. This process involves a family of proteins
called SNARES.
Types of SNARES
1. v-SNAREs: SNAREs in the cytoplasmic face of the intracellular vesicles
2. t-SNAREs: those in the target membranes with which the vesicles fuse
3. Other proteins involved: SNAP25 and NSF
During fusion, v- and t- SNAREs bind together and undergo a structural change that produces a
bundle of long thin rods made up of helices from both SNARES and two helices from SNAP25.
This structural change pulls the two membranes into contact and initiates the fusion of their lipid
bilayers.
Topic: Communication through Biomembranes (Part 1)
Intended learning outcomes
1. To discuss the transport types in membranes
2. To discuss transporters
Transport types
1. Passive (Diffusion); Simple or facilitated via ion channels and transporters
2. Active transport
Passive transport (Diffusion)
Simple Diffusion
The passive flow of a solute from a higher to a lower concentration due to random thermal
movement. It depends on factors such as the thermal agitation of the specific molecule,
concentration gradient across the membrane i.e. transmembrane gradient of the substrate and the
solubility of that solute in the hydrophobic core of the membrane bilayer.
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Facilitated diffusion
The passive transport of a solute from a higher to a lower concentration mediated by specific
protein transporter. Hydrophilic molecules that cannot freely pass through the lipid bilayer
membrane do so passively by facilitated diffusion or by active transport. Facilitated diffusion is
explained by the ping-pong mechanism. In the ping state, it is exposed to high concentrations of
solute and molecules of the solute bind to specific sites on the carrier protein. Binding induces a
conformational change that exposes the carrier to a lower concentration of solute (pong state).
This process is reversible and the net flux across the membrane depends upon the concentration
gradient.
Rate of entrance of solutes into cell by facilitated diffusion is determined by the
1. Concentration gradient across the membrane
2. Amount of carrier available
3. Affinity of the solute-carrier interaction
4. Rapidity of the conformational change for both the loaded and the unloaded carrier
Factors affecting diffusion of a substance
1. Concentration gradient across the membrane. Solutes move from high to low
concentration
2. The electrical potential across the membrane toward the solution that has the opposite
charge. The inside of the cell usually has a negative charge
3. The permeability coefficient of the substance for the membrane
4. The hydrostatic pressure gradient across the membrane. Increased pressure will increase
the rate and force of the collision between the molecules and the membrane
5. Temperature. Increased temperature will increase particle motion and thus increase the
frequency of collision between external particles and the membrane
6. Facilitated diffusion involves certain transporters or ion channels. A multitude
transporters and channels exist in biological membranes that route the entry of ions into
and out of cells
Active transport
This is the transport of a solute across a membrane against a concentration gradient. It requires
energy (frequently derived from the hydrolysis of ATP).
Resemblance of Passive and active transport with substrate-enzyme interaction
1. There is a specific binding site for the solute
2. The carrier is saturable, so it has a maximum rate of transport
3. There is a binding constant (Km) for the solute and so the whole system has a Km
4. Structurally similar competitive inhibitors block transport.
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Transporters
Are membrane proteins that speed the movement of a solute across a membrane by facilitating
diffusion. They are classified into carriers and channels
Carriers
Catalyze transport at rates below the limits of free diffusion, bind substrates with high stereo
specificity, are saturable just like enzymes and function as monomeric proteins
Channels
They show less stereospecificity than carriers. They are usually not saturable. They generally
allow transmembrane movement at higher rates than carriers. They are oligomeric complexes of
several identical subunits. The permeability of a channel depends on the size, extent of hydration
and extent of charge density on the ion
Transport systems
Uniport system: Moves one type of molecule bi-directionally
Co-transport system: the transfer of one solute depends upon the stoichiometric simultaneous or
sequential transfer of another solute
Symport system: moves two solutes in the same direction. Examples are the proton-sugar
transporter in bacteria and the Na+ -sugar transporters (for glucose and certain other sugars) and
Na+ -amino acid transporters in mammalian cells.
Antiport system: move two molecules in opposite directions (eg, Na+ in and Ca2+ out).
Ion channels
They are very selective, in most cases permitting the passage of only one type of ion (Na+, Ca2+,
etc).
Properties of Ion Channels
1. They are composed of transmembrane protein subunits.
2. Most are highly selective for one ion; a few are nonselective
3. They allow impermeable ions to cross membranes at rates approaching diffusion limits.
4. They can permit ion fluxes of 106–107/s.
5. Their activities are regulated.
6. The main types are voltage-gated, ligand-gated, and mechanically gated.
7. They are usually highly conserved across species.
8. Mutations in genes encoding them can cause specific diseases.
9. Their activities are affected by certain drugs.
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Ionophores
Are molecules that act as membrane shuttles for various ions. They contain hydrophilic centers
that bind specific ions and are surrounded by peripheral hydrophobic regions. This allows the
molecules to dissolve effectively in the membrane and diffuse transversely therein. Aquaporins
are proteins that form water channels in certain membranes.
Topic: Communication through Biomembranes (Part 2)
Intended learning outcomes
1. To discuss active transport system
2. To discuss endocytosis and exocytosis
Active Transport System
This transport system requires that molecules are transported against concentration gradients.
They require energy which can come from hydrolysis of ATP, electron movement and light.
About 4 major classes of ATP-driven active transporters have been recognized (P, F, V and ABC
transporters). Examples of the P class is the Na+K+ ATPase and Ca2+ATPase of muscle. The
second class is referred to as F-type (example is mt-ATP synthase. The V-type active
transporters pump protons into lysosomes and other structures. ABC transporters include Cystic
fibrosis transmembrane regulator protein (CFTR protein), a chloride channel implicated in the
causation of cystic fibrosis. Multidrug resistance-1 protein (MDR-1 protein) which pump a
variety of drugs, including manyanti-cancer agents out of the cells
Plasma membrane Na+-K+- ATPase
This is an important enzyme that regulates the intracellular concentration of Na+ and K+.
Usually, cells maintain a low intracellular Na+ concentration and a high intracellular K+
concentration coupled with a net negative electrical potential inside. ATPase is the pump that
maintains these ionic gradients and is activated by Na+ and K+. It pumps Na+ out and K+ into
cells. The ATPase is an integral membrane protein containing a transmembrane domain allowing
the passage of ions and cytosolic domains that couple ATP hydrolysis to transport. It has
catalytic centers for both ATP and Na+ on the cytoplasmic side of the plasma membrane with
K+ binding sites located on the extracellular side of the membrane.
The phosphorylation by ATP of three Na+-binding sites on the cytoplasmic surface of the cell
induces a conformational change in the protein leading to transfer of 3 Na+ ions from the inner
to the outer side of the plasma membrane. 2 molecules of K+ bind to sites on the protein on the
external surface of the plasma membrane resulting in dephosphorylation of the protein and
transfer of the K+ ions across the membrane to the interior. Therefore, 3 Na+ ions are
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transported out for every 2 K+ ions entering. This creates a charge difference between the intra
and extracellular compartments, making the intracellular compartment more negative. ATPase is
inhibited by cardiac drugs such as oubain and digitalis. The Na+-K+-ATPase can be coupled to
various other transporters such as those involved in transport of glucose
Endocytosis
This is a process by which cells take up large molecules. It is a mechanism for regulating the
content of certain membrane components e.g. hormone receptor. It can be used to study how cell
function. DNA transfection depends on endocytosis. It is responsible for the entry of DNA into
the cell.
Endocytosis requires energy (usually from ATP), Ca2+, and contractile elements in the cell
(possibly the microfilament system). Endocytotic vesicles are generated when segments of the
plasma membrane invaginate, enclosing a small volume of extracellular fluid and its contents.
The vesicle pinches off when the fusion of plasma membranes seals the neck of the vesicle at the
original site of invagination. This vesicle fuses with other membrane structures thereby ensuring
the transport of its contents to other cellular compartments. Most endocytotic vesicles fuse with
primary lysosomes to form secondary lysosomes which contain hydrolytic enzymes hence, are
specialized organelles for intracellular disposal
Types of Endocytosis
1. Phargocytosis
2. Pinocytosis
Phargocytosis
Phargocytosis involves the ingestion of large particles such as viruses, bacteria, cells or debris
and granulocytes. It occurs only in specialized cells such as macrophages. Macrophages may
ingest 25% of their volume in an hour
Pinocytosis
This is a property of all cells that leads to the cellular uptake of fluid and fluid contents.
Types
1. Fluid-phase pinocytosis. It is a non selective process in which the uptake of a solute by
formation of small vesicles is simply proportionate to its concentration in the surrounding
extracellular fluid. Fibroblasts are examples, they internalize their plasma membrane at
about one third the rate of macrophages
2. Absorptive Pinocytosis. This is a receptor-mediated selective process primarily
responsible for the uptake of macromolecules for which there are fixed numbers of
binding sites on the plasma membrane. The vesicles formed are derived from
invaginations that are coated on the cytoplasmic side with a filamentous material called
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coated pits. Absorptive pinocytosis of extracellular glycoproteins requires that the
glycoproteins carry specific carbohydrate recognition signals. These recognition signals
are bound by membrane receptor molecules which play a role analogous to that of the
LDL receptor. A galactosyl receptor on the surface of hepatocytes is helpful in the
absorptive pinocytosis of asialoglycoproteins from circulation. Moreover, acid hydrolases
taken up by absorptive pinocytosis in fibroblasts are recognized by their mannose 6-
phosphate moieties. Viruses which cause hepatitis, poliomyelitis and AIDS initiate their
damage by absorptive pinocytosis. Moreover, iron toxicity also begins with excessive
uptake due to endocytosis
Exocytosis
This releases certain macromolecules from the cell to extracellular space. It is involved in
membrane remodeling when the components synthesized in the golgi apparatus are carried in
vesicles to the plasma membrane. Signal for exocytosis is usually hormone which when it binds
to a cell-surface receptor, induces a local and transient change in Ca2+ concentration. Ca2+
triggers exocytosis.
Neurotransmission
Membranes of the neurons have an asymmetry of inside-outside voltage (electrical potential).
They are electrically excitable as a result of the presence of voltage-gated channels. Hence,
appropriate stimulation by a chemical signal mediated by a specific synaptic membrane receptor,
opens the channels in the membrane to allow the rapid entry of Na+ or Ca2+ so that the voltage
difference rapidly collapses and the segment of the membrane is depolarized. The gradient is
quickly restored by the action of the ion pumps present in the membrane. When large areas of the
membrane are depolarized in this manner, the electrochemical disturbance propagates in wavelike
form down the membrane, generating a nerve impulse.
Myelin sheets, formed by Schwann cells, wrapped around nerve fibers provide an electrical
insulator that surrounds most of the nerve and greatly speeds up the propagation of the wave
(signal) by allowing ions to flow in and out of the membrane only where the membrane is free of
the insulation (at the nodes of Ranvier). The myelin membrane is highly rich in lipids, this
confers a tremendous insulating property on it. Demyelination and impaired nerve conduction
are features of certain diseases such as Multiple sclerosis and Guillain-Barr syndrome
Glucose transport
The entrance of glucose into the cell is the first step in energy utilization. A number of glucose
transporters are involved depending on each tissue. Glucose enters the erythrocyte by facilitated
diffusion called GLUT1. In the skeletal muscle and adipocytes, glucose enters by a specific
transport system enhanced by insulin. Glucose transport in the small intestine involves the
binding of Na+ and glucose to different sites on a Na+-glucose symporter located at the apical
surface. Na+ moves into the cell down its electrochemical gradient and drag glucose with it.
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Therefore, the greater the Na+ gradient the more glucose enters and if Na+ in extracellular fluid
is low, glucose transport stops. In a bid to ensure a steep Na+ gradient, this Na+-glucose
symporter is dependent on gradients generated by the Na+-K+- ATPase which maintains a low
intracellular Na+ concentration. The transcellular movement of glucose in this case involves one
additional component; a glucose uniporter: which allows the glucose accumulated within the cell
to move across the basolateral membrane
Topic: Isolation of sub-cellular organelles
Intended learning outcomes
1. To review cellular organelles
2. To discuss the process involved in the isolation of cellular organelles
Sub-cellular organelles
Organelles are organized structures within the cell e.g. the nucleus, mitochondria, ribosomes,
golgi bodies, endoplasmic reticulum etc. They are separated by centrifugation based on the
difference in their size, density, shape
Steps in the isolation of cell organelles
1. Homogenization
2. Sub-cellular fractionation by centrifugation
3. Marker assays
Homogenization
This is the disruption of cells under conditions that prevent deterioration aimed at isolation of
morphologically intact and functionally active organelles and microsomal fractions. It is the first
step in the isolation of sub-cellular organelles. It involves breaking the cell membrane
Common homogenization techniques
1. Dounce homogenization: crushing of cells between two revolving solid surfaces
2. Filteration: cells are forced through smaller pores in a filter
3. Grinding: where cells are ground by swirling with glass beads
4. Sonication: where cells are bombarded with ultrasonic vibrations
5. Solubilization: in which cell membranes are dissolved by detergents such as triton X-100
Enzyme digestion is also used to remove cell-wall constituents. The method of choice depends
on the type of tissue to be homogenized and the specific purpose of the experiment. During
homogenization, isotonic sucrose is added to the homogenization buffer to prevent osmotic
rupture of organellar membranes. After homogenization, the homogenate is spurn at low speed to
remove any intact cells along with large cellular debris. This is followed by subcellular
fractionation
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Differential centrifugation
This is the movement of subcellular particle in a centrifugal field. This movement
(sedimentation) results from the interaction between a particle’s weight, the resistance it
encounters in moving through a suspension medium and the relative centrifugal force exerted on
the particle. Under a given centrifugal force, particles that are relatively large or dense will
sediment more rapidly than particles that are smaller and lighter. The order of sedimentation is
typically from most to least dense; nuclei, mitochondria, lysosomes, plasma membrane,
endoplasmic reticulum and contractile vacuoles. It precedes density-equilibrum centrifugation
Density-equilibrum centrifugation
subcellular organelles are layered on a density gradients and subjected to a very high centrifugal
force. The density gradient is formed by layering increasing concentration of sucrose solutions in
a centrifuge tube. Other solutions such as cesium chloride, Percoll can be used. These two
solutions when spun spontaneously set up a density gradient, thereby alleviating the need
manually layer sucrose solutions of varied concentration.
During centrifugation, organelles initially layered on the density gradient will sediment until they
arrive at the region of the gradient where the density of the suspension is equal to their own
Marker enzymes
Indicate the presence of an enzyme. Acid phosphatase is located in the lysosomes, succinate
dehydrogennase is located in the mitochondria. ATP synthase is present in the inner
mitochondrial membrane. Galactosyl transferase is present in the golgi apparatus. Glucose-6-
phosphatase is present in microsomes. Galactosyl transferase is present in the Golgi apparatus.
This helps to monitor where each enzyme activity is found during a cell fractionation protocol.
Marker enzymes also provide information on the biochemical purity of the fractionated
organelles. The presence of unwanted marker enzyme activity in the preparation indicates the
level of contamination by other organelles. The degree of enrichment for the desired organelle is
determined by the specific activity of the target marker enzyme. Electron microscopy is
generally used as a final step to assess the preparation’s purity and the morphology of the
isolated organelle
Question
Discuss the isolation of subcellular organelles
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Topic: Cell membranes and toxins
Intended learning outcomes
1. To discuss the mechanisms by which toxins interfere with the host’s cell membrane
2. To discuss the defense mechanisms in parasites
3. To discuss mode of action of polymyxins
Cell membranes play significant roles in the interaction of pathogenic microbes with the host
cells. About 50% of the membrane volume is made up of transmembrane, peripheral and lipidlinked
proteins arranged in a bilayer. Membrane lipid is important in providing cellular
signaling, membrane trafficking as well as membrane microdomain organization. Infectious
organisms rely on the multifunctional role of lipids to modulate cell processes to ensure their
survival
Toxins
Toxins are powerful pathogenicity factor produced by macro and microorganisms which mediate
drastic interactions of the pathogens on the organism’s host. Classically, bacteria toxins are
divided into endotoxins and exotoxins. Endotoxins: membrane compounds of gram negative
bacteria which elicits inflammation in the host. Exotoxins: secreted proteins which act locally
and at distance of the bacteria colonization site. The invasion of cells by pathogens/toxins is
marked by binding to carbohydrate moieties exposed by a lipid or a protein in the plasma
membrane of target cells. Cholera toxins binds with its ?-subunit to the ganglioside GM1 in the
intestinal cells, Pseudomonas aeruginosa attaches to respiratory cells by binding to asialoGM1
and asialo-GM2 through type IV pili. Influenza A virus initiates its uptake by binding to sialic
acids in the host cell membranes. Glycosphingolipids are important for self-induced endocytosis
of toxins and viruses. Phosphatidylinositol-phosphate (PIP) is also important for the uptake of
pathogens. They are essential components of the cell membranes involved in signaling events;
vesicle trafficking. One strategy by which invasive bacteria manipulate the PIP is the
translocation of effector proteins, which act as phosphatidylinositol phosphatases. A second
option to interfere with the PIP metabolism is the engagement of specific host cell receptors
Shigella dysenteriae
It secretes two types of enterotoxins; Shigella toxins I and II. In humans they cause serious
complications in the GIT such as haemolytic colitis. The binding affinity of the ?-subunit of
Shiga toxin (StxB) and cholera toxin (CtxB) to individual Gb3 and GM1 molecules respectively
is very low. But the cooperative binding of multiple lipid molecules markedly increases the
apparent affinity of the toxin to its receptor. After binding, Stx is internalized by clathrindependent
as well as clathrin-independent endocytosis. Cholera toxin has been found to be
associated with caveolae and is efficiently endocytosed into cells devoid of caveolin-1
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Defense mechanisms in parasites
The interplay between parasite survival strategies and the host defense mechanisms is the basis
for the relationship that exists between the host and the infecting parasite. Parasites have devised
strategies for their survival while in the host; the parasite aims at propagating within the host and
be transmitted to subsequent host, while the goal of the host is to limit the infection. Parasites
have evolved mechanisms for evading the immune system of the host. Below are some of the
mechanisms
Protozoan immune evasion strategies
Antigenic variation
In Plasmodium, different stages of the life cycle express different antigens. In trypanosomes,
there is a whole range of variant specific surface groups (VSG) which protects the underlying
membranes.
As the level of antigen increases, a small fraction of the population switches to producing a coat
of a new VSG with an antigenic character circulating antibodies are no longer able to recognize
Strategy of avoidance
Some pathogens reduce antigenicity so that they are not recognized as foreign. The ?2-
macroglobulin is present on the surface of adult schistosomes and has a potent anti-protein
activity. If attached to a molecule with the appropriate conformation, it may also act as
aproteinase inhibitor preventing breakdown of parasite tissues. Plasmodium lives inside Red
Blood Cells (RBCs) which have no nucleus, when infected, it is not recognized by immune cells.
Other stages of Plasmodium live inside liver cells. Plasmodium ookinetes develop in serosal
membrane & are beyond reach of phagocytic cells (hemocytes). Leishmania parasites and
Trypanosoma cruzi live inside macrophages
Enzymatic role
Enzymes are produced that are capable of inactivating reactive oxygen intermediates resulting
from the host inflammation defense systems. The antioxidant enzymes counteract oxidative
burst. Some of the enzymes are superoxide dismutase, catalase, glutathione peroxidase,
glutathione S- transferase (GST). Glutathione S- transferase is located in the external tegument
of schistosomes
Shedding or replacement of surface
Example: Entamoeba histolytica.
Immunosupression
17
Manipulation of the immune response e.g. Plasmodium.
Anti-immune mechanisms
Leishmania produce anti-oxidases to counter products of macrophage oxidative burst
Helminth immune evasion mechanisms in the vertebrate host
Large size
It is difficult for immune system to eliminate large parasites. Primary response is inflammation
to initiate expulsion, often worms are not eliminated.
Coating with host proteins
Tegument of cestode & trematode worms, is able to adsorb host components e.g. RBC Ags, thus
giving the worm the immunological appearance of host tissue. Schistosomes take up host blood
proteins, e.g. blood group antigens & MHC class I & II molecules, therefore, the worms are seen
as “self”.
Molecular mimicry
Synthesis of surface proteins similar to host proteins by the parasite which are unrecognized as
foreign. In tapeworm, synthesis of antigen that resemble blood group of MHX antigen of the
host. Depression of the host immune response
Anatomical seclusion
Trichinella spiralis can live inside mammalian muscle cells for many years.
Shedding or replacement of surface
Examples; hookworms, trematodes
Immunosupression
Manipulation of the immune response. High burdens of nematode infection often carried with no
outward sign of infection.
Polymyxins
They are antibiotics produced by Paenibacillus polymyxa. There are two main types; polymyxin
B and polymyxin E (colistin). They are usually used in treating gram negative bacterial
infections. They however have little or no effect on gram positive bacteria. This is because the
cell wall of gram positive bacteria is too thick to permit access to the membrane. Their neuro and
nephrotoxicities make them last line therapy.
18
Summary and Conclusion
This course has informed students on the central dogma of membrane biology, functions, types
and composition of the biomembrane. It also covered lipid structure, properties and formation of
bilayers. It also covered the isolation and identification electron microscopy and marker enzyme
assays. Introduction to receptor function: antigenicity of membrane components. Cell membrane
and toxins, transport processes, action of polymyxin and ionophores. Introduction to
neurotransmission. Membrane transport system- active versus passive transport systems.
Transport of sugars and amino acids. Defence mechanism in parasites. Biomembranes of
parasites.
Interaction and Questions
1. What is membrane fluidity?
2. Explain the physiological roles of integral protein
3. Write an essay on Immunoglobin-like proteins
4. Discuss the isolation of subcellular organelles
5. What is endocytosis and exocytosis?
6. Describe the active transport system
7. List the advantages of artificial membranes
8. What is membrane fusion?
9. Mention two instances of membrane fusion
10. List other applications of artificial membrane
11. Distinguish between active and passive transport
12. What are the factors that affect diffusion of a substance?
13. What are transporters?
14. What are the differences between channels and carriers?
Bibliography/ Further Reading
1. Leninger Principles of Biochemistry
2. Harpers illustrated Biochemistry
3. Padh, H. 1992. Organelle isolation and marker enzyme assay. Pages 129–146, in Tested
studies for laboratory teaching, Volume 13 (C. A. Goldman, Editor). Proceedings of the
13th Workshop/Conference of the Association for Biology Laboratory Education
(ABLE), 191 pages
4. Dr. Michele Loewen. Proteins and enzymes, ultracentrifugation. 24 pages.
www.cbr.nrc.ca/loewen/Home.html
5. http://www.sfu.ca/biology/courses/bisc318/2015 pdfs/lecture_33_Apr-
02_Evasion of immunity.pdf


Item Type: Lecture note(non-copyrighted)
Format: PDF document,   194.65 KB
Copyright: Creative Commons LicenseCreative Commons license
Keywords: BIOCHEMISTRY
Department: Natural Science
Field of Study: Biochemistry
Uploaded By: Uwaifo Ferdinand
Date Added: 04 Feb 2019 8:06am
Last Modified: 04 Feb 2019
Lecture URL: https://www.edouniversity.edu.ng/oer/lecturenotes/biomembrane_20182019


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