Download LECTURES 1,2 Membranes, lipids and phospholipases.ppt

Lipid membrane Learning outcomes
•  Describe the composition of the plasma membrane
•  Functions of the plasma membrane with reference
to specific molecules
•  Fatty acid composition and functions
•  Describe phospholipid structure
•  Phospholipid functions
Membrane
Thickness: 7.5-10 nm
Membrane Functions
1. Compartmentalization (Intracellular compartments)
2. Scaffold for biochemical activity
3. Selectively-permeable barrier
4. Transporting solutes
5. Responding to external signals
6. Intercellular interactions
7. Energy transduction
The membrane is
a lipid bilayer
Lipid bilayer comprises
Phospholipid head groups
(hydrophilic)
And
Associated fatty acids
(hydrophobic)
e
Saturated C16 or C18 FA
Phosphodiester
linkage
Derived from polar alcohol
•  smallest = H (from H-OH)
•  least common in membranes
•  phosphatidic acid
Unsaturated C16 – C22 FA
Saturated fatty acids
- No double bonds
- Space-saving
- More rigid membranes
Alpha linolenic acid
COOH
CH3
Arachidonic acid
DHA
EPA
Unsaturated fatty acids
- More than 1 double bond
- Each double bond confers ‘kink’
- Occupies more space
- Promotes fluidity ofmembranes
ω-3 fatty acids
ω-6 fatty acids
α-linolenic (18:3)
linoleic (18:2)
Δ-6-desaturase
Δ-6-desaturase
octadecatetraenoic (18:4)
γ-linolenic (18:3)
elongase
eicosatetraenoic (20:4)
dihomo-γ-linolenic (20:3)
Δ-5-desaturase
eicosapentanoic (20:5)
arachidonic (20:4)
PG3 and LT5
PG2 and LT4
The Importance of the fatty acids
•  Determine the fluidity of the membrane
length of chain (usually 18-22)
Level of unsaturation (# of C=C bonds)
•  Lipids rarely move from
one layer to another
•  Lipids exchange places with
their neighbors
•  Lipids rotate around their
axis
Membranes need to be fluid to:
Enable the membrane proteins to diffuse rapidly
Allow distribution of lipids and proteins properly
in the lipid bilayer
Ensure membranes can fuse with one another
when necessary
Cholesterol in the Membrane
•  Cholesterol is present where unsaturated lipids
predominate
•  It ‘fills the gaps’ and stabilizes the bilayer
•  It stiffens the bilayer ∴ decreases fluidity &
permeability
Extracellular face
75%
Outer layer stiffened by cholesterol
Inner layer (fluidity determined by PUFA)
5%
20%
Rigid
membrane
Fluidity determined by fatty acid composition of phospholipid
Saturated: Palmitic (C16:0), Stearic (C18:1)
Common
fatty acids
Fluid
membrane
Unsaturated: Arachidonic, Docosahexaenoic
AA (20:4)
DHA (22:6)
Membranes are Asymmetrical
Glycolipids are found only on
the extracellular surface
(Sugar added in the Golgi)
•  Inner & outer surfaces have different lipids
•  Proteins in the bilayer have a specific orientation due
to its function
Phospholipids
Phospholipids are Amphipathic
(Amphipathic molecules are mostly lipid-like in structure,
but have a hydrophilic region at one end
Choline
Ethanolamine
Serine
Inositol
BASE
Phosphatidylcholine
Phosphatidylethanolamine
Phosphatidylserine
Phosphatidylinositol
Phosphatidylinositol bisphosphate (PIP2)
Glycerol backbone
Inositol trisphosphate
Phospholipases
PLA1
PLA2
PLC
PLD
PLA1
CH2O..Stearic Acid
CH2O..Stearic Acid
CHO..AA
CHOH
CH2O..P..Base
CH2O..P..Base
Phospholipid
SA
Lysophospholipid
CH2O..Stearic Acid
CH2O..Stearic Acid
CHO..AA
CHOH
PLA2
CH2O..P..Base
Phospholipid
AA
CH2O..P..Base
Lysophospholipid
CH2O..Stearic Acid
CH2O..Stearic Acid
CHO..AA
CHO..AA
CH2O..P..Base
PIP2
CH2OH
PLC
IP3
Diacylglycerol
Action of phospholipase D
Phosphatidylinositol bisphosphate (PIP2)
Membrane
3 phosphate groups
ie PIP2
PLC hydrolyses PIP2
2 second messengers are produced
PIP2 hydrolysis
- stimulated by
ligand-receptor
Interactions
- G protein-mediated
- Leads to increased
intracellular calcium
concentration
PKC isozymes and some roles of PKC
1.  Conventional (require DAG, Ca2+ and Phospholipid)
PKCα, PKCβ (I and II), PKCγ
2. Novel (require DAG but not Ca2+)
PKCδ, PKCε (I and II), PKCη and PKCθ
3. Atypical (require neither DAG nor Ca2+)
PKCι, PKCζ (I and II) and PK-N1, N2
PKC is a serine-threonine kinase
Roles (tissue- and cell-specific) include
Receptor desensitization
Modulation of membrane structure events
Regulation of transcription
Modulation of immune responses
Regulation of cell growth
Learning and memory.
Some lipid molecules act as signalling molecules
Diacylglycerol
AA
Ceramide
Production of arachidonic acid
ACTIONS OF AA
Activates ion channels directly
Activates PKC
Precursor of many signalling lipids
Phospholipases A2
PLA2 – categorized in terms of localization and association with calcium.
(20 types identified; 9 in humans)
Secretory
Cytosolic
Ca2+-independent
(cytoplasmic)
PAF-acetyl hydrolyses
Roles: Inflammation, cell death, cell signaling, maintenance of membrane phospholipids.
Secretory PLA2 (sPLA2):
Originally from snake and bee venom
Found extracellular especially in damaged tissue
10 groups (at least)
Histidine residue at active site
Require Ca2+ for activity characterized by a serine residue
Roles:
Linked with rheumatoid arthritis*, atherosclerosis, CNS inflammation, inflammatory
bowel disease, skin inflammation, cancer, asthma
Causes lysis of gram-positive bacteria
*sPLA2-IIA primarily implicated in RA
Released by macrophages
Functions as a bacteriocidal
Cytosolic PLA2 (cPLA2):
4 groups (IVA, IVB, IVC and IVD named α, β, γ, δ).
Active site is characterized by a serine residue.
Ca2+-dependent
Method of action:
Ca2+-bound phosphorylated PLA2 translocates to intracellular membrane, cleaves
phospholipid and releases arachidonic acid
Pathological action (of head group/AA) can result in atherosclerosis, neuronal damage,
multiple sclerosis, alziehmers, epilepsy.
Groups IVA-C - high levels associated with colorectal, small bowel, and lung cancers
Ca2+-independent PLA2 (iPLA2):
Group members: VIA-1, VIA-2, VIB
Active site characterized by a serine residue
MW: 63 - 90 kDa (comparable in size to cPLA2).
Cytoplasmic
Roles:
Maintenance of homeostasis through remodeling of membrane phospholipids
Involvement in apoptosis, muscle contraction, and obesity.
No direct links to inflammation and role in cancer not clear
PLA2 and inflammation
Arachidonic acid and metabolites
NOTES
Arachidonic acid (C20:4) – 4 double bonds and therefore highly reactive
Activates PKC
Affects ions channels particularly K+ channels
Affects transmitter release
Double bonds mean it is a target for oxygenases
Cyclooxygenases
Lipoxygenases
Epoxygenase
1) Cyclooxygenase 1 and 2:
• Converts arachadonic acid into cycloperoxides
• Cycloperoxides can then form thromboxanes, prostaglandins, and prostacyclins.
• Ultimately roles of molecules include cell death, inflammation, vasoconstriction, and vasodilation in
platelets, the endothelium, and in smooth muscle.
• COX inhibitors eg VIOXX may be associated with increased risk of stroke.
• Abnormal COX activity linked with cancer
(2) Lipoxygenase (LOX):
Converts arachidonic acid into hydroperoxyeicosatetraenoic acid (HPETE) and subsequently leukotrienes.
(Leukotrienes have roles in vascular function and inflammation)
Abnormal LOX expression has been linked with cancers, including colon, pancreatic, lung, prostate,
bladder, skin, and liver cancer
(3) Cytochrome P450 monooxygenase (CYP450):
Converts arachidonic acid into 2 products, hydroxyeicosatetraenoic acid (HETE) and
epoxyeicosatrienoic acid (EET).
These molecules are peroxisomal proliferation activated receptor agonists (PPAR)
Roles in angiogenic and mitogenic signaling.
PPAR
PPAR receptor is located at the nuclear membrane and dimerizes with 9-cis retiniose acid receptor
following ligand binding; this causes binding to DNA at PPAR response elements.
PPAR response elements are located near genes involved in lipid metabolism
3 types PPAR receptors: alpha, beta, and gamma. PPAR gamma is a modulator of the inflammatory response
in peripheral macrophages, and monocytes (8).
AA gives rise to prostaglandins
1. NA, thrombin and bradykinin
activate PLA2 leading to AA
2. 
AA metabolites include
prostaglandins
thromboxanes
prostacyclin
3. PGH2 is parent prostaglandin
4. Subsequent metabolism is enzymedriven
Some enzymes
PGE2 is synthesized from PGH2 by PGE synthase (Several types; PGES1 is key enzyme).
PGD2 is synthesized from PGH2 by PGD2 synthases (2 forms known).
PGI2 is synthesized from PGH2 via prostacyclin synthase
PGF2a is synthesized from PGH2 by PGF synthase
PGs interact with specific receptors (pre- or postsynaptic) in brain
PGD2 interacts with DP1, 2
PGE2 interacts with EP1-4
PGF2 interacts with FPα, β
PGI2 interacts with IP
Thromboxanes interact with TPα, β
PG Receptors
Modulate physiological and pathological cellular functions
AA gives rise to leukotrienes
NOTES
AA is omega-6 fatty acid
DHA is omega-3 fatty acid
Omega-3 fatty acids
α-Linolenic acid (ALA) is parent fatty acid
EPA and DHA are precursors for lipid modulators
ALA .. plant sources (flaxseed, walnuts, pecans,
hazelnuts, and kiwifruit)
EPA and DHA … salmon, tuna, and herring
Omega-3 fatty acids
Linoleic acid (LA) is parent fatty acid
Most from vegetable oils (soybean oil, corn oil,
borage oil, and acai berry)
LA converted to γ-linolenic acid (GLA)
GLA converted to arachidonic acid
Lipid Analysis
Technologies:
Analytical techniques for fatty acids, phospholipids, sphingolipids, triglycerides and steroids.
Originally Thin layer chromatography (TLC) was used.
Now replaced by faster technologies with better resolution.
Gas chromatography-Mass Spectrometry (GC-MS)
Used for low molecular weight lipids of all different classes (<500 Da).
Enables routine profiling of ~ 100 lipid compounds (out of 800) in one single run
Can semi-quantify lipid-like substances contained in the FiehnLib mass spectral and retention
index database with high reliability.
Liquid chromatography-Mass Spectrometry (LC-MS) including HPLC-Chip/MS System, UPLC/
MS, UPLC/FT-MS, LC-TOF/MS
Used to generate high resolution lipid profiles for lipids in the mass range 100-2000 Da
Chromatography allows a better separation from plasma or tissue
Thin-layer chromatography (TLC)
Coat glass plates with silica gel (adsorption mode).
Migration in a chamber using solvents (mobile phases)
Separation of simple lipids and phospholipids by TLC
Separation uses solvent mixtures
eg chloroform-methanol-water (60:30:5) for X
& hexane-diethyl ether-acetic acid (80:20:1.5) for Y.
monogalactosyldiacylglycerols
diGDG
2-Dimensional TLC for complex lipids
diphosphatidylglycerol
High performance liquid chromatography (HPLC)
Analysis of fatty acids by HPLC
HPLC separates; detection by spectrophotometric analysis
Arachidonic Acid
α-linolenic
Palmitic
Linoleic
Stearic
Separation of rat lipids by HPLC with evaporative lightscattering detection (CE = cholesterol esters, TG =
triacylglycerols, C = cholesterol, PG, PE, PI, PC and SPH are
various phospholipids).
Liquid chromatography–mass spectrometry (LC-MS, or alternatively HPLC-MS)
combines the physical separation capabilities of liquid chromatography (or HPLC) with
the mass analysis capabilities of mass spectrometry.
Very high sensitivity and selectivity.
Main use: Detection/identification of chemicals in a complex mixture.
Mass spectrometry (MS)
Organic molecules are bombarded by electrons or other ionic
species causing them to ionize and fragment.
The ionic species produced by electron impact are separated
according to mass (strictly speaking mass/charge (m/z) ratio) in a
magnetic field, and a spectrum is obtained
the mass spectrum of methyl 6,9,12-octadecatrienoate (γ-linolenate).
2D representation of the lipid spectra for a sample obtained by Ultra
Performance Liquid Chromatography coupled to high resolution Mass
Spectrometry (UPLC-MS); Pietiläinen KH et al. 2007.
Niemann-Pick types A&B:
Lack of enzyme
to hydrolyze this bond
Phosphosphingolipids
Phosphosphingolipids:
Phosphate esterified to C1 OH.
Sphingomyelins:
Choline or ethanolamine
esterified to C1 phosphate
Glycosphingolipids
Glycosphingolipids:
Carbohydrate(s) on C1-OH
(instead of phosphate group)
Abundant in nerve cell membranes
Tay-Sachs disease: characterized by mental retardation, blindness, muscular weakness.
Abnormal ganglioside GM2 deposits in lysosomes. Death by age 3-4.
Niemann-Pick disease types A and B: Symptoms include enlarged liver and spleen, mental
retardation. Early death
Lack of sphingomyelinase. Required to hydrolyze phosphate ester linkage of phosphocholine
to ceramide
Tay-Sachs:
lack of enzyme
to hydrolyze
this bond