Download CH2O -OCH CH2O- - f.a. #1 f.a.#2 f.a.#3 f.a. = fatty acid.

IX. LIPIDS & MEMBRANES
Most biomolecules are readily soluble in water. However there is one very important class of biomolecules
that are water-insoluble; these are the LIPIDS. Every day we come in contact with solid lipids (FATS) and liquid lipids
(OILS).
Lipids are the water-insoluble compounds that can be extracted from cells and tissues with organic solvents
such as chloroform. They play a variety of roles in cells. This semester we will focus on two major classes of lipids.
These are:
(i). The PHOSPHATIDES or phospholipids, a major, frequently the dominant, structural component of
biological membranes.
(ii). The TRIACYLGLYCEROLS or (electrically) NEUTRAL LIPIDS. These function as the principal
storage device for energy reserves, fatty acids being the principal fuel of the cell. This arises because (i) -CH bond is
such a good source of energy; and (ii) fats are not hydrated, so there's no wasted weight. Why not store pure fatty
acids rather than triglyceride esters? Because fatty acids are potent detergents. The tri-acylglycerols are the largest class
by weight (approximately 20-25% of body tissue).
We will not consider lipids that have specialized functions, though from the following list you should deduce that they
are very important:
a) They can form a protective coat (a wax) on the surface of certain organisms e.g. the tuberculosis bacterium
(Mycobacter).
b) Some cell surfaces bear modified lipids as a device to effect cell-cell recognition.
c) Some important vitamins, coenzymes and hormones are lipids; e.g. Vitamins A (for visual pigments) and D
(derived from cholesterol), ubiquinone and the steroid hormones.
d) Cholesterol is an important component of membranes and the oxidation products of
cholesterol, (cholate and deoxycholate (the bile salts)), function as detergents to emulsify other
lipids present in the food so that the lipid-digesting enzymes released into the intestine can function
effectively.
In the first half of this semester we will concentrate on the first class-lipids as structural elements of the
membrane. However as an introduction to the lectures on the metabolism of fats (after mid-term) we will also cover
the structural chemistry of the second class.
There are important structural similarities and differences between the neutral lipids and phospholipids. Both
classes are esters between glycerol, or derivatives of glycerol, and long chain fatty acids. The simplest example is
provided by the neutral lipids and so we will begin by considering the structures of these compounds.
The schematic structure of a triacylglycerol is:
CH2O - f.a. #1
f.a.#2 -OCH
f.a. = fatty acid.
CH2O- f.a.#3
The two basic elements in this structure are the glycerol skeleton and the fatty acids. Glycerol (also called
glycerin) is:
IX-1
C1
C2
C3
CH2OH
proS = C1
HOCH
CH2OH
P goes here
Although glycerol is a symmetric molecule it is prochiral and most derivatives of glycerol are asymmetrically
substituted. We apply the CIP rules and call the proS alcohol group #1. Reference to derivatives of glycerol are then
made by writing e.g. sn-glycerol-3-phosphate, the prefix sn signifying stereochemical numbering system; glycerol-3phosphate is a component of phospholipids. With the central OH and H pointing out of the page we get the assignment
shown above.
In the neutral lipids each of the 3 alcohol functions of glycerol are esterified with long-chain fatty acids-the
(much) higher homologues of acetic acid, e.g.:
CH3CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2COOH
β
COOH
1
H3 C
2
α
ω
The number of methylene group is denoted n; n ranges from 10-22. The overall chain length is thus 12-24. n is almost
always even (most commonly 14 or 16), odd-length chains are very rare. Thus C16 and C18 fatty acids are the most
common.
The carbon atoms are numbered starting at the carboxyl (=1) and proceeding toward the methyl. The first
methylene (C2) is also called alpha, followed by beta, etc. However the terminal methyl is always called omega. A
straight-chain fatty acid of this type is represented n+2:0, the "n+2" denoting total chain length and the "0" the number
of double bonds e.g. 18:0, octadecanoic acid or stearic acid.
As many as three double bonds may be present in the hydrocarbon chain; they are not conjugated. The first
double-bond is usually present between C9 and C10 (e.g. 18:1, octadecenoic acid, oleic acid). If a second double bond is
present it will be found between C12 and C13 (18:2, octadecadienoic acid, linoleic acid) and the third double bond is
usually present between C15 and C16 (18:3, octadecatrienoic acid, linolenic acid); 18:3's are fairly uncommon. When
we do not specify the location of the double bonds then the above set of locations is implied. An important special
case is arachidonic acid, which is 20:4 with double bonds at 5, 8, 11 and 14. If, as in this example, the double bonds
occur in a non-standard location, then the nomenclature is extended by following the 20:4 with a superscripted delta e.g.
20:4 ∆ 5,8,11,14.
As examples of the kind of distributions that exist, the most common fatty acids found in liver are shown in
the next table.
Mammals are unable to synthesize fatty acids containing multiple double bonds (mammals can introduce the
double bond at position 9 but not at higher numbered carbon atoms). Fatty acids containing multiple double bonds
must be provided in the diet and hence are called the essential fatty acids; they are obtained from plants. The unusual and
important fatty acid arachidonic acid mentioned above is synthesized from linolenic acid and is an intermediate in the
pathway to the prostaglandins, an extremely important family of compounds with hormone-like action which reduce
blood pressure and cause smooth muscle to contract (Fig 23-54 V&V; see p. 704 et seq. of V&V for general
information on prostaglandins).
IX-2
Present in Neutral Fats
Fatty Acid
16:0
16:1
18:1
18:2
18:3
Others
Trivial Name
% Composition
Palmitic
Palmitoleic
Oleic (octadecenoic)
Linoleic (octadecadienoic)
Linolenic (octadecatrienoic)
24%
6%
43%
20%
1%
6%
Present in Phospholipids
16:0
18:0
18:1
18:2
20:4
Other
28%
20%
17%
12%
18% ∆ 5,8,11,14
5%
Stearic
Arachidonic
Bacteria tend to have fatty acids with shorter chains which may be branched. They never have more than 1
double bond; this is fixed between C7 and C8 counting from the methyl terminus. Thus it is at a variable location
relative to the carboxyl group e.g. vaccenic acid is 18:1 (∆ 11) counting in the conventional way but 18:1 (ω 7) using
the alternative method. Note that it is the omega notation that is used in the ω 3 family of fatty acids that are supposed
to have particularly benign effects in combating inflammation.
The backbone of the saturated fatty acid is zigzag because of the tetrahedral character of the carbon backbone.
It is very flexible and roughly linear, resembling a bratwurst in a space filing model.
However the double bonds in fatty acids are usually cis; fatty acids with trans double bonds are very rare,
though are found in hydrogenated fats-courtesy of the food industry.
H
c c
H
c c
H
cis
H
trans
Consequently the introduction of a cis double bond introduces a 30° kink (an elbow) into the hydrocarbon chain so
that the sausage becomes a boomerang (V&V: Fig. 11-4). Each arm of this boomerang is independently flexible but
the presence of the kink interferes with the close packing of fatty acids in the quasi-crystalline array found in the pure
compound and in membranes. This interference destabilizes the cooperative assembly making the pure compound more
fluid; this fluidity is crucial in the role of lipids in membrane structure.
Introduction of a second and third double bond tends to undo the effect of the elbow induced by the first, so
that the chain of a fatty acid containing multiple double bonds is closer in linearity to the saturated acid. As the double
bonds are not conjugated an unsaturated fatty acid retains much of its flexibility.
Saturated fatty acids with more than 12 carbon atoms are solids at body temperature. The melting points of
fatty acids are sensitive to chain length and to the degree of unsaturation.
Modification to chain length
Effect on Melting temperature (°C)
Add two carbons (e.g. C 14 ⇒ C 16)
Add 1 double bond
Add second
Add third
increases by 8° C
decreases by 60°C.
additional decrease of 20° C
additional decrease of 5° C
IX-3
An important but highly undesirable reaction is oxidation of these double bonds, typically by free radical
chain reactions; it is a major mechanism for the inactivation of membranes.
H
c c
H
+O2
CH2
cis
CH
OOH
This process is called peroxidation; it results in fats acquiring an unpleasant taste (rancidity); more importantly,
elimination of the double bond stiffens the membrane. These peroxidation reactions are minimized by anti-oxidants.
These are radical scavengers and can terminate free radical chain reactions and hence preserve membrane integrity.
Vitamin E is a naturally occurring antioxidant. BHA (butylated hydroxyanisole) and BHT (butylated hydroxytoluene)
are widely used by the food industry; you will see these names on the labels on the cartons of various foods e.g. corn
flakes.
Fatty acids are AMPHIPATHIC (dual sympathy) in character. They have a polar head-group (the COOH) and
a hydrophobic sidechain (R-). Because of this dual character they are very effective detergents; hence concentrations of
free fatty acids must be kept low in vivo. For example the transport of fatty acids in the serum occurs after binding of
the fatty acid to the protein serum albumen.
The analysis of the composition and quantification of a mixture of fatty acids is very straightforward; gasliquid chromatography of the methyl esters of fatty acids both separate and quantitate the components.
Triglycerides
Esters between fatty acids and glycerol are called glycerides. In neutral fats all three hydroxyl groups of the
glycerol are esterified. These are called triacylglycerols or triglycerides; monoacyl and diacyl-glycerols are much less
common in nature (though they are present in trace amounts as intermediates in metabolism) but are used extensively
by the food industry as emulsifiers for food such as your nice, smooth peanut butter. As the mono- and di-glycerides
are completely digestible they are quite harmless (other than being a source of calories).
Because of the variable character of the fatty acids the physical state of triacylglycerols can vary between liquid
to solid at room temperature, hence OILS and FATS.
If all three fatty acids are identical e.g. tripalmitoylglycerol, then the fat is said to be simple. However mixed
triglycerides are much more common. As a very simple case imagine that a fat had the composition of 50% each of
fatty acids A and B. Then a given triglyceride molecule could have any one of the following 8 alternative forms: AAA,
AAB, ABA, BAA, ABB, BBA, BAB and BBB with relative abundance’s determined statistically. The natural fat will
obviously have a very complex composition. There is no obvious rationale for this complexity.
Phospholipids
In contrast to the electrically neutral and only weakly bipolar triglycerides, membrane lipids are strongly
bipolar-they are even more amphipathic than are free fatty acids. They contain a large hydrophobic region provided by
the fatty acid sidechains and they also contain a highly polar region; this is often composed of a phosphate group
esterified to a polar alcohol, typically at C3 of glycerol.
There are two major classes of membrane phospholipids; the first are derivatives of glycerol, the second
class are derivatives of sphingosine.
IX-4
Glycerol
H
CH
CH
CH2
OH
OH
OH
Sphingosine: -a complex amino-alcohol (actually a family of which this is the most important)
R
CH
CH
CH2
OH
NH2
OH
R is a 15-carbon alkyl sidechain.
Phospholipids derived from glycerol are called phosphoglycerides and they can be summarized as:
O
CH2-O-Fatty Acid #1
16:0. 18:0
CH-O-Fatty Acid #2
16:1, 18:1, 18:2, 18:3
R-O-P-O-CH2
OThe structure upto but not including R is the phosphatidyl moiety. Relative to neutral fat, the third fatty acid
has been replaced by phosphate or a phosphorylated alcohol.
R
Product
pK's and Net Charge
-H
Phosphatidic Acid
1,7 (-1.5)
-CH2-CH2-NH2 (ethanolamine)
P-ethanolamine (PE)
(a.k.a. cephalin) (PC)
1,10 (0,Z)
-CH2-CH2-N+ (CH3)3 (choline)
P-choline
(a.k.a. lecithin)
1 (0,Z)
P-serine
1,10,3 (-1)
P-inositol
1 (-1)
-CH 2-CH-NH2
COOH
myo-inositol
(a cyclitol)
OH
OH
OH
OH
OH
Z = Zwitterion, P- = phosphatidyl
IX-5
When R = H we have phosphatidic acid. The generalized structure leads to the prefix "phosphatidyl" (P in the previous
table). Note that fatty acid # 1 is usually saturated while fatty acid # 2 is usually unsaturated. (Think of choline and
serine as derivatives of ethanolamine with the trimethyl-N in the former and the α−COOH in the latter.) PC and PE
are the most abundant.
Another lipid of some importance is a "dimer" of phosphatidic acid with the structure:
This is diphosphatidyl glycerol, also called cardiolipin. It has a charge of -2. Cardiolipin is a very important
component of the inner membrane of the mitochondrion.
As was the case with the triglycerides the fatty acid composition of the phospholipids is mixed, with the
constraint that the fatty acid on C 1 is almost always saturated, typically a mixture of 16:0 and 18:0, while that on C2
is almost always unsaturated (a mixture of 16:1, 18:1, 18:2 and 18:3). The precise proportions depend upon a number
of factors, including diet.
IX-6
There is one class of lipid in membranes that is not derived from glycerol. This phospholipid is built around the amino
alcohol sphingosine; it is an important constituent of membranes associated with nervous tissue.
IX-7
NH 2
CH3-(CH2)12-CH=CH -CH-CH-CH2-OH
OH
Replaces fatty acid 1.
(note this OH is not esterified)
The parent saturated species is sphinganine, the unsaturated species shown above is 4-sphingenine and has the
trivial name of sphingosine. It can be thought of as glycerol with a long alkyl chain attached to C1 and the OH at C 2
replaced by an amino function. (Note that I am maintaining a numbering system consistent with glycerol. The official
numbering system is inverted, which provides confusion with no good rationale).
The amino function is attached to a fatty acid by an amide bond. The product is called a ceramide. The fatty
acid is typically C 16-C24, either saturated or mono-unsaturated.
The terminal hydroxyl (C3) can be linked to phosphoryl choline.
O-CH2-O-P-O-CH2-CH2-N+-(CH3)3
O
and the final product is called sphingomyelin. It is an important component of brain and nerve tissue, being the
insulating sheath on nerve fibers.
In addition to these compounds there are two important, minor classes of lipids present in membranes. Both
are analogs of sphingomyelin but do not contain phosphoryl choline. Rather this base is replaced by a sugar. If the
sugar is simply glucose or galactose the compound is called a cerebroside. The link is e.g. gal β(1 ⇒ C 1 of ceramide).
(As mentioned above the numbering is unfortunately opposite of glycerol). The sugar chain may be extended with halfa-dozen sugars (mainly more glu and gal, or N-acetyl glu and N-acetyl gal) in either a linear or a branched chain. One of
these extra sugars is often N-acetyl neuraminic acid (sialic acid, NANA); the lipid is called a ganglioside. (Note that in
NANA our typical position 1 is numbered 2, because of the COOH).
Cerebrosides and gangliosides belong to the family of glyco(sphingo)lipids. Although they are only minor
constituents they are important in surface phenomena including nerve signal transmission across a synapse and cell
recognition such as in blood-group specificity and organ and tissue specificity.
Saponification
Fats that can be hydrolyzed by dilute base to yield fatty acids are said to be saponifiable. Both neutral lipids
and phospholipids are thus saponifiable.
IX-8
MEMBRANES
The cell membrane defines the volume of space occupied by a cell and ensures that the cell contents remain
confined within that space. But in addition membranes confer a number of benefits upon cells:
1) An important function of a cell membranes is that of PROTECTION allowing the cell to maintain a
constant local environment irrespective of any changes that might occur in the external phase. However a membrane
must not be absolutely protective for it must allow selective COMMUNICATION with the exterior so that
nutrients can enter and waste products leave-it is not totally impermeable. Macroscopic material can enter via
phagocytosis (solids) or pinocytosis (liquids)-remember Amoeba-while material in solution passes through the
membrane via some transport device. It is this protection requirement of the membrane that explains why it is made of
lipids for most biomolecules are polar and will not spontaneously cross a lipid barrier.
2) In higher cells the internal volume of the cell is compartmentalized into domains each of which is
separated from its neighbor by a membrane. These have a number of functions: (i) separation of enzymes; (ii)
separation of metabolic pathways; and (iii) separation of metabolites. Recall that glycolysis occurs in the cytoplasm
and the citric acid cycle in the mitochondrion.
3) A foundation (scaffold) for the organized arrangements of enzyme systems (e.g. the electron transport and
oxidative phosphorylation systems of the mitochondrial inner membrane)
4) In higher organisms membranes provide a surface that is important in cell-cell recognition (e.g. the blood
group antigens).
We are now going to consider the important attributes of membranes-their chemical composition, structure
and some of the more striking biological processes that they support.
The major components of a cell membrane are phospholipid and protein. The approximate composition is:
Lipid + Protein 70-80%
Water
20%
Carbohydrate
1-10%
The relative proportion of lipid-to-protein is highly variable and ranges from about 80% lipid in myelin to
50% lipid in the inner membrane of the mitochondrion.
There are a large variety of phospholipids found in biological membranes. However most of the functionality
of these molecules can by understood by recognizing a common structural element--the existence of polar and nonpolar
domains in the same molecule (they are amphipathic and hence detergents, just like fatty acids). This can be
schematically represented in several alternative representations as:
These are somewhat misleading in that they obscure that the length of the polar head group (the circle) is actually about
two-thirds that of the hydrocarbon chain.
When a phospholipid is added to water you should recognize that two forces come into play. The polar head
group enjoys a favorable coulombic interaction with the polar water molecules (and also can repel each other via their
mutual coulombic repulsion); these effects tend to disperse the lipid molecules. However the individual non-polar
hydrocarbon chains tend to lock up the water molecules into clathrate-like domains which are energetically unfavorable
and the hydrophobic effect "squeezes" the tails of individual phospholipids into large aggregates. Thus provided the
IX-9
concentration of the detergent exceeds a critical value (the critical micellar concentration or cmc) the detergent
molecules spontaneously assemble into one of a variety of structures. The actual value of the cmc depends on the
specific detergent: when the ratio of the hydrophobic to the hydrophilic volume is large the cmc is around uM, when
the ratio is small the cmc is approximately mM. To be accurate the cmc is the ratio k-1/k1 where k1 is the rate
constant for aggregation (about 109 M -1 sec-1) and k-1 is the rate constant for a molecule leaving the aggregate
(ranging from 104 - 10 9 sec-1). Thus these aggregates form when the energy gained by sequestering the hydrocarbon
chains exceeds the unfavorable energy from the repulsion of the head groups.
The structure of these aggregates depends upon the environment in which they are created. The simplest kind
is found when a drop of a phospholipid is placed upon the surface of water, i.e. at an air-water interface:
The Air-Water Interface
AIR
(Non-polar)
WATER (Polar)
In this situation the polar head enters the aqueous domain but the apolar tail is pushed up into the air. In this
case you might guess that the orientation of the apolar tail is a consequence of being squeezed out of the water This is
partially true but in addition it is because air itself is apolar and dissolves the apolar tail; air is hydrophobic because
dinitrogen and dioxygen are homonuclear diatomics and have no dipole moment.
When the phospholipid is totally immersed in water more complicated structures arise; these are all designed
to avoid contact between the aqueous phase and the hydrocarbon tails. The simplest is the micelle. This is a small
spherical structure with a diameter of about 100Å:
Note that this is also a MONOLAYER and that all of the solvent is excluded from the internal space. Thus
the fatty acids are removed from, and do not affect, the solvent. Micelles are most commonly found with monoacyl
derivatives. The presence of the second fatty acid chain, as is found in a normal phospholipid, interferes with the close
packing and the expanded micelle would allow the entry of water, and so we get the second class of structures; these are
called BILAYERS.
IX-10
An Idealized Vesicle (Liposome)
Leaflets
Note that each layer of the bilayer is referred to as a leaflet. The bilayer is about 50Å thick with about 30Å
due to the hydrocarbon chains. The bilayer may be an "infinite" sheet but is usually folded back upon itself to form a
large sac (or VESICLE) which may be so large that it can almost be seen by eye (Finite sheets are unstable due to
destabilizing interactions at the periphery). This sac has solvent on both sides of the membrane but because of the
bilayer character of the membrane the polar solvent and nonpolar fatty acyl side-chains are not in contact. There are
often sacs within sacs so that the vesicle has the structure of an onion. In all of these structures one gets the maximum
advantage from both the hydrophobic effect and from the ionic interaction.
Both bilayer structures are stable. When phospholipids are introduced slowly into water (e.g. by infiltrating
water into a pure phospholipid) lipid bilayer sheets are the predominant species. However if this suspension of bilayers
is treated roughly, e.g. by ultrasound, then formation of the vesicles (500 Å) and ultimately micelles is promoted.
Exercise: As an exercise redraw the above structures in the solvent benzene.
Evidence that the Lipid Bilayer is present in Biological Membranes
In 1925 GORTER and GRENDEL extracted the lipids from the erythrocyte membrane with acetone, applied
the acetone extract to an air-water interface and measured the area of the lipid monolayer that was formed. They
discovered that the lipids occupied an area twice the area of the surface of the erythrocyte and they postulated the
existence of a lipid bilayer in the erythrocyte membrane. (Actually their experiments had two canceling errors: The
lipids were incompletely extracted and the area of the film was underestimated.)
Later more careful experiments confirmed this basic observation but the amount of lipid measured could only
form a bilayer if the phospholipids were substantially expanded, i.e. there was not quite enough lipid to construct a
lipid-only bilayer.
At about the same time (1935) Danielli and Davson also proposed the presence of a lipid bilayer in
membranes. This proposal of D&D has gained the most attention but was, for them, the most trivial aspect of their
model. Danielli was a physical chemist and was very knowledgeable about the behavior of surfactants (soaps) in
aqueous media. To him the concept of a bilayer was "no big deal" and his main interest was in providing an
explanation for the observation that biological membranes were so much more stable than ordinary films, e.g. a soap
bubble. He thus proposed that biological membranes were a sandwich of proteins and lipids:
IX-11
In this model the protein was used to stabilize the lipid bilayer; it was the "bread around the peanut butter". This
picture remains with us today though, as we shall see, somewhat modified.
Early support for this proposal of D&D came from electron microscope studies which revealed a triple-decker
structure:
10 A
30 A
10 A
In biological membranes each surface is negatively charged while the interior is relatively positive; this may
be the explanation why positive ions have more difficulty than negative ions in penetrating the membrane.
The current picture of a biological membrane (next page) draws heavily on these early ideas but introduces a
major refinement that helps us understand many of the important properties of the biological membrane. We now
speak of the "Fluid Mosaic Model". In this viewpoint the membrane is again represented as a planar lipid bilayer but
the disposition of the proteins is more complex than was suggested by D&D.
Membrane proteins are divided into two classes:
1) Peripheral or extrinsic proteins; these are found on the surface of the membrane and are only weakly
attached to the membrane via polar interactions with the surface of the membrane. They are easily removed by washing
the membrane with a salt solution (e.g. 1M KCl).
2) Intrinsic proteins which are integrated into the body of the membrane. These intrinsic proteins may be either partly
or totally inserted into the membrane bilayer and can only be removed by "dissolving" the membrane. They either stick
out of one-or-other side of the membrane or they stick out of both sides. They are never completely buried within the
lipid bilayer, the proportion sticking out ranging from 25% to 90% (prostaglandin H synthase (see below)).
The most dramatic evidence that globular proteins are embedded in the membrane comes from a technique
called FREEZE-ETCHING or FREEZE-FRACTURE electron microscopy.
The membrane sample is placed upon a copper disc and treated with glycerol/DMSO to avoid ice formation.
The sample is chilled to 77°K with liquid freon, transferred to a vacuum chamber, and then stressed mechanically with a
microtome knife. The sample fractures along a cleavage plane which is presumed to be the hydrophobic interior of the
membrane i.e. between the phospholipid leaflets. The water of the exposed surface is sublimed away by application of a
vacuum thus exposing any underlying structure. A replica of the surface is made by applying a thin deposit of
IX-12
platinum followed by carbon (for strength). The lipid membrane is then dissolved leaving the replica intact; this is
then examined in the electron microscope.
Intrinsic or Integral
Extrinsic
The micrographs so obtained show a smooth background bearing small particles. The number and distribution of these
particles vary with the character of the membrane i.e. they correlate with the protein content
Membrane
No of Pits
Synthetic (PC only)
Myelin
RBC/Mitochondria
None
Very Few
2500/uM 2
The protein content of membranes varies considerably with the source and particularly the biological function
of the membrane. Myelin, the membrane that surrounds the nerve fibers (axons), functions as an electrical insulator
and contains only 20% protein. Conversely the mitochondrial inner membrane, which contains an elaborate array of
enzymatic activities, is about 80% protein, about half of which is embedded in the core of the lipid bilayer.
Categories of Membrane Proteins
Many membrane proteins have enzymatic activities. However others are required for facilitating the
movement of polar metabolites across the membrane (transport). We also find that receptors for hormone and
neurotransmitter are proteins. Furthermore some proteins have a structural role-the protein SPECTRIN, a major
component of the RBC, is a case in point-while others play a role in surface recognition phenomena (though in this
case the exposed surface of the protein is "decorated" with carbohydrate).
EXTRINSIC PROTEINS are not obviously different from conventional proteins. Indeed, many of the
proteins that we have dealt with so-far in these lectures may well be peripheral proteins. They are readily soluble, they
do not aggregate and do not readily bind lipid.
INTRINSIC (INTEGRAL) PROTEINS generally require drastic treatment before they can be liberated from
the membrane. The most common reagents for solubilization are detergents and the products of such solubilization
procedures usually exist in quasi-solution by virtue of the continued association of detergent with the protein
preparation. As a consequence. the isolation, chemical and physical characterization of intrinsic proteins has lagged
behind that of both the peripheral proteins and the membrane lipids.
IX-13
Because the intrinsic proteins penetrate or even cross the hydrophobic region of the membrane one might
expect that they do not conform to our usual picture of protein structure
Traditional
Intrinsic Membrane
P P
P
NN P
P N
N
N P
PN
P
N
PN
N P
N
P
N
N
N
N
P
P
N
P
P
P
P
P
P
P
P N
N P
P N
N P
P N
N P
P
N N
P
P N
P
P
P = polar, N = nonpolar.
By extension we expect these proteins to have an unusually high proportion of NONPOLAR amino acids. In fact the
amino acid composition of intrinsic proteins is not abnormal; overall the proteins are not unduly hydrophobic.
If it isn't the composition of the polypeptide then it must be sequence; this is revealed by simple
mathematical analysis as follows. Every amino acid can be assigned a value, its hydropathy, which is a measure of
how easily it dissolves in a non-polar solvent (see Table below).
The Kyte-Doolittle Hydropathy Analysis
Recipe (Illustrated for a 7-point average (19 is more typical)):
1) Write down the amino acid sequence e.g.
Ala-Thr-Trp-Lys-Glu-Ala-Gly-Phe-Gly................
2) Set i (the index into the sequence of residues) to 1.
3) Starting at residue i add up the Hydropathy Values for the first i+6 residues e.g. 1.8 + (-0.7) + (-0.9) + (-3.9) + (-3.5) +
1.8 + (-0.4) = - 5.2.
4) Divide by 7 and save the result (-0.74) in HA(i+3), position i+3 of the hydropathy array (position i+3 is the center
residue of the block of 7 beginning at location i).
5) Increase i by 1.
6) Is i + 6 > No. of residues in the sequence. If so go to line 7, otherwise go to line 3.
7) Plot HA(i) versus i. Note that the first three and the last three entries are undefined and can be set equal to the values of
the actual amino acids rather than the averaged values.
The Hydropathy Values for the common amino acids are:
Ile
Val
Leu
Phe
Cys(SH/SS)
Met
Ala
Gly
Thr
4.5
4.2
3.8
2.8
2.5
1.9
1.8
-0.4
-0.7
Ser
Trp
Tyr
Pro
Glu
Gln
Asp
Asn
Lys
-0.8
-0.9
-1.3
-1.6
-3.5
-3.5
-3.5
-3.5
-3.9
IX-14
Arg
-4.5
Units are arbitrary but are related to Free Energy of Transfer of the amino acid from a non-polar solvent to a polar solvent.
Notice that the values decrease with increasing polarity.
Hydropathy Plot of largest sub-unit of cytochrome oxidase from Paracoccus denitrificans.
Computer analysis of a protein sequence in terms of these hydropathy values can identify nonpolar stretches as
clearly defined peaks; it has been found empirically that membrane proteins contain several such stretches that are 2025 amino acids long. This number of residues is sufficient to yield a helix of length 30 Å, the thickness of the
hydrocarbon part of the phospholipid bilayer (each amino acid advances the helix 1.5 Å), and thus these hydrophobic
stretches have been identified as transmembrane helices. The formation of these helices allows the polypeptide to
maximally satisfies its hydrogen bonding capabilities by internal H-bond formation and hence maximize its stabilitythere are no groups in the hydrocarbon lipid phase that can make hydrogen bonds with the polypeptide. As a
consequence helices that are buried within the membrane are 5-10 x more stable than are helices in conventional
proteins. Curiously although proline is normally considered to be a helix breaker it is quite common in transmembrane
helices; perhaps the enhanced stability of the helix can overcome the presence of proline. The predictions of the
hydropathy plot has been verified by the x-ray structures of the photosynthetic reaction center of Rhodopseudomonas
viridis which contains 8 transmembrane helices and the various subunits of cytochrome oxidase which contain 1-12
helices depending on the subunit; these are located precisely where they are predicted by the hydropathy plot. The only
qualifications are that the actual helices tend to be a couple of residues longer than predicted and helix 7 of subunit I of
oxidase is actually shifted 10 residues towards the amino terminus relative to the prediction. The picture that exists is
shown on the left with the rods representing the helices and the small circles the individual amino acids.
These transmembrane helices are often found to have polar residues which are clustered to one side of the
helix. The left side above now represents the "outer face" of each helix exhibiting only non-polar residues while the
right side shows the "inner face" which exhibits a high proportion of polar residues. These amphipathic helices are
IX-15
believed to aggregate into "barrels" with the polar side of each helix on the inside of the barrel and the non-polar side
facing the membrane lipid. This is an easy way of visualizing how a polar trans-membrane pore might be formed.
Membrane proteins are classified as monotopic, bitopic or polytopic depending upon whether they interact
with 1 leaflet, cross the membrane once and thus interact with both leaflets or cross the membrane several times.
It has recently been established that intrinsic proteins can have structures that consist almost exclusively of βstrands. Such is the case for the protein called porin (above left). This is found in the outer membrane of Gram-negative
bacteria where it forms a pore (above right is a top view); this pore penetrates the membrane and provides a channel
through which small polar molecules (< 600 Da) can pass. The protein consists of three identical subunits each
forming a β-barrel comprised of 16-18 β-strands in an antiparallel arrangement (see end and side views above).. The
three β-barrels assemble to form a pore with a diameter of about 8 Å and a length of 20 Å. The polar amino acids line
the pore and the faces exposed to the lipid are lined with non-polar residues. Positive residues lie on one face of the
pore, negative residues on the opposing face. (Science Vol. 254, p1627)
IX-16
Maltoporin
Individual
Monomer
Pore
(18 strand β-barrel)
Hydrophobic Sheath
Some membrane proteins are only
anchored to the membrane surface. This is
conventionally illustrated by cytochrome b5.
Cytochrome b5 is a hemeprotein present in the
endoplasmic reticulum of rabbit liver; it is a
polypeptide of 137 residues. It was originally
obtained by treatment of rabbit liver
microsomes with trypsin that reduced its size to
93 residues. This product was crystallized and its
X-ray structure has been determined. Truncated
cytochrome b5 is a very polar protein.
Detergent extract of the same
microsomes gives the completely unmodified
polypeptide of 137 residues; this polypeptide
has been sequenced but not crystallized. The 26
C-terminal mostly hydrophobic residues anchor
the protein to the membrane and this has led to
the picture on the left; it is that of a monotopic
protein. But the picture is wrong. It is based on
two premises: (i) the hydrophobic segment is
too short to fully cross the membrane twice; and
(ii) the amino and carboxyl termini are on the
same side. Taken together these facts imply that
the two segments of helix only partially
penetrate the lipid bilayer. However fact #2 has
recently been shown to be in error; the two
termini are on opposite sides of the membrane
so one only needs a single helical span that
fully traverses the membrane. Does this mean
that monotopic proteins do not exist? Probably
not. Prostaglandin H synthase is a membrane protein whose X-ray structure has recently been solved. It contains several
domains including a number of helices that lie on the surface (on right of ribbon cartoon below). It seems that these
helices burrow partially in the membrane and form the anchor. This kind of structure will not be obvious from a
hydropathy plot.
IX-17
Some proteins are attached to membranes by hydrocarbon anchors. So far there are 4 types:
(i) The N-terminus forms a peptide bond with myristic acid (14:0) (α subunit of G-proteins).
(ii) The COOH group C-terminus is attached to ethanolamine which in turn is attached to an oligosaccharide
which in turn is attached to phosphatidyl inositol (e.g. acetylcholinesterase).
(iii). A cysteine residue internal to the polypeptide is attached to palmitic acid (16:0) as a thioester (e.g. Gprotein receptors).
(iv) An internal cysteine is attached to a polyisoprene (e.g. Fig 11-48 V&V) via a thioether link (e.g. the ras
protein).
These lipid modifications serve either to (i) anchor the protein to the membrane or (ii) to modify the catalytic activity.
Mobility of Membrane Components
Some relevant diffusion equations.
D, the diffusion constant, is defined by Fick's first law:
J = -D dc
A
dx
J/A is the flux-the mass (e.g. moles) that flows through a 1 cm. square "window" in 1 sec. (For the 2D case the flow is
across a 1 cm line segment) D, the diffusion constant, quantifies the intrinsic mobility of the species and the derivative
term is the concentration gradient that drives the flow. The sign is present so that a decreasing concentration gradient
gives positive flow.
D=
k BT
6πηR
Stokes Einstein equation; R = radius (cm)
η = viscosity (poise)
IX-18
where 6πηR is the frictional coefficient for translation (assuming a solid sphere of radius R). k B = Plank's constant.
Because the gradients are different in the 2D and 3D cases the dimensions of D are independent of dimensionality .
Values of D range from about 1 x 10-5 cm2 sec-1 for a small molecule such as glucose to 1 x 10-6 cm2 sec-1 for a
small protein (RNAase, 14 kDa). Membrane proteins move laterally with D's in the range 4 x 10-9 cm2 sec-1 to 1 x
10-12 cm2 sec-1.
The collision frequency of a pair of molecules depends upon the sum of their individual diffusion coefficients
(Smoluchowski's equation).
kD = 4π RDN/1000
where kD is the second-order rate constant for the diffusion controlled encounter, D is the sum of the diffusion
coefficients (dominated by the smaller molecule) and R is the sum of the radii (dominated by the larger molecule). N is
Avagadro's No.
A measure of the ability of a particle to move is its mean free path, s = (2nDt) 1/2 where n = no. of
dimensions. n = 2 for a 2D membrane.
Mobility of Proteins
The first convincing demonstration that the proteins of the membrane might be rather mobile was provided by
FRYE and EDIDIN, who exploited the surface antigens present on mouse and human cells.
The Mobility of Proteins
Classical: Frye & Edidin
Mouse Cell
Human Cell
Sendai Virus
Heterocaryon
~ 40 min.
_
s = 1 micron/minute
-10 -1
D = 2 x 10 sec cm-1 .
Samples of the heterocaryon were treated immediately and after various times with mouse alloantibodies &
rabbit antihuman antibodies. The localization of the antibody complexes was then established by reaction with:
Fluorescein labeled goat anti-mouse ⇒ GREEN emission
Rhodamine labeled goat anti-rabbit ⇒ RED emission
IX-19
and the disposition of the color established by observing the distribution of the fluorescence over the cell surface using
a microscope. This technique is called INDIRECT IMMUNOFLUORESCENCE.
Randomization took about 40 min. at 37 °C. This yields a diffusion constant (D) of 2 x 10-10 cm2 sec-1 for
the original antigen present in the membrane; this D is equivalent to a mean free path of about 1 micron per minute.
Notice an inconsistency here; s = a distance but I have just quoted a velocity. The one minute is the time I inserted into
the formula. If we were to agree on a standard time to use in this formula there would be no need for this convention.
Diffusion Constant (10 -9 cm2s-1)
Protein
Mass
RNAase
14 kDa
1000
Rhodopsin
40 kDa
3
Band III (anion carrier)
93 kDa
0.002
Why is there such a large range of values?
(i)
(ii)
Viscosity ca 1-5 poise.
Interaction with the cytoskeleton.
Mobility of Lipids
In contrast to proteins which only move laterally, lipids move laterally and longitudinally
i.e. sideways or from one leaflet to the next.
The quantitation of the rate of transverse motion of both
phospholipids and proteins in a membrane is commonly measured by a
technique called FRAP (Fluorescence Recovery After Phototobleaching).
In this method an analog of e.g. phosphatidyl-choline (PC) is prepared in
which one of the choline methyl groups is replaced with a fluorescent
group. This PC is then inserted into the membrane and eventually it will
distribute itself throughout the surface of the membrane.
A small area of the membrane (diameter ca 2-4 µm) is then
subjected to a very short and intense flash of light from a laser; this has the
consequence of destroying the fluorescent property of the label in this spot
(photobleaching). With time the PC molecules surrounding this spot
exchange places with the bleached molecules within the spot and the
fluorescence is restored. By analyzing the kinetics of this fluorescence
recovery (using s = (4Dt)1/2 where s = the diameter of the spot and t the
half-time for recovery) the diffusion constant for the PC can be calculated; a
typical value is 2.0 x 10-8 cm2 sec–1. This is equal to a lateral mobility of
about 1 micron/second–is roughly equivalent to a phospholipid molecule
moving from one end of E. coli to the other in 1 second.
IX-20
In a second experiment PC was labeled with a colored dye in place of the fluorescent label and vesicles were
prepared from this new derivative. The color of a sample of vesicles was measured and then the dye was decolorized by
the addition of sodium ascorbate, a chemical reductant:
Ascorbate
Absorbance
t 1 ~ 7 hrs.
2
time (hours)
It was observed that precisely one-half of the color was eliminated instantaneously but that the remainder of
the color was lost at a very slow rate, with a half-time of about 7 hours. As membranes are not permeable to sodium
ascorbate the explanation of the experiment is that the rapid, instantaneous loss in color is a result of decolorizing the
dye present in the outer PC leaflet while the slow loss in color reflects the rate at which a labeled PC from the inner
leaflet can exchange places with one from the outer leaflet.
t 1/2 = 10-5 sec.
t 1/2
> 104 sec.
These numbers are for pure phospholipid membranes.
We thus see that membrane phospholipids have considerable in-plane (lateral, transverse) mobility while gross
movements perpendicular to the plane of the membrane are prohibited. This latter conclusion is in fact necessary to
explain the well known observation that the composition of the lipids in the two leaflets are frequently quite different.
If the flip-flop motion were easy then the lipids in the two planes would equilibrate and the composition of both layers
would be identical.
From recent studies on the movement of proteins by techniques such as FRAP and "laser tweezers" it appears
that proteins actually exhibit a hierarchy of motions. Thus lipids and some proteins do indeed move freely as advertised,
others are attached to the cytoskeleton and move hardly at all while a third group can move freely in restricted areas but
only occasionally jump from one restricted area to another.
IX-21
MEMBRANE FUNCTIONS
These are passive and active:
Passive: ⇒ structural role of the membrane.
Active:
a) Transport of metabolites
(i) Porters
(ii) Pumps
(iii) Channels
b) Information Handling
(i)
(ii)
Transmembrane signaling.
Cell surface labeling
c) Specialized Catalysis
TRANSPORT
A major function of biological membranes is that of a barrier. This barrier both prevents the loss of valuable
constituents from the cell and also hinders the entry of unpleasant agents from the outside. At the same time the
membrane must provide a means whereby desirable material can be accumulated or moved from one internal
compartment to another while undesirable material can be eliminated.
For a compound to be transported across a membrane there must be both a driving force and a pathway must
exist. Driving forces can be:
1) Concentration gradients.
2) Electrical Potentials.
3) Metabolic Energy.
4) Combinations of (1), (2) and (3).
(1), (2) and (3) are alternative forms of free energy.
Relevant questions regarding the pathway are:
1) How does the solute move? Does the compound move across the membrane on its own or is its
transport coupled to the movement of another species?
a) The movement of a single species is called uniport. The diffusion of oxygen across a
membrane is an example.
b) The movement of two species in the same direction is called symport or
co-transport. An example in the uptake of lactose that is accompanied by H+ .
c) The movement of species in opposite directions is called antiport or exchangediffusion . An example would be the movement of potassium into a cell with the
obligatory movement of protons out. Note that proton antiport and hydroxide symport (or
vice-versa) cannot be distinguished.
2) Is the movement electroneutral or electrogenic? i.e. Is there net transfer of charge
accompanying the movement of the solute? Electrogenic transfer will arise when charge movement
is not balanced. No net charge transfer implies
(a) the uniport of a neutral solute or
(b) the symport of two species of opposite charge or
(c) the antiport of two species with the same charge.
3) Is transport coupled to metabolism (via ion pumps or group translocation) -is it active
transport ?
IX-22
4) Is transfer through a lipid domain or a protein? This question is necessitated by the
fluid mosaic model for the membrane! Except for those species that move on their own, all transport
systems so far characterized use a protein.
The passage of a solute from one side of a membrane to the other can be divided into 3 events, as cartooned
below:
1) The solute leaves side 1 and enters the membrane.
2) The solute traverses the bilayer.
3) The solute leaves the bilayer and enters side 2.
1.
2.
3.
Nonpolar molecules, e.g. dioxygen, have no difficulty in crossing the lipid membrane for the main barrier to
the first step in the sequence above is the elimination of the hydration layer; nonpolar molecules are unhydrated.
However, for most polar molecules, step 1 is very difficult.
It is well known that the rate of entry of many compounds into a cell is related to each compound's polarity,
with the more polar molecules entering more slowly and vice-versa. In general the ease with which a molecule can
pass between nonpolar and polar solvents is proportional to that molecule's oil-water partition coefficient. Before polar
molecules can effect step 1 they must lose their water of hydration and this is the major barrier to their passage. It
results in a high activation energy for the process which leads to a kinetic barrier to the entry of the material, even
though the thermodynamic pressure (i.e. the concentration gradient) might be favorable. For example, for glucose to
penetrate a membrane it must first break the 5 hydrogen bonds between its -OH groups and the water molecules of the
solvent; this costs about 5(4) = 20 kcal/mole.
Although the transport of most polar molecules is a kinetically unfavorable event there are a number of small,
polar molecules that seem to cross biological membranes readily by simple diffusion. These include water (as
evidenced by the osmotic swelling of cells), methanol, formamide and ethylene glycol (antifreeze). To account for this
observation it is generally assumed that the phospholipid membrane is interrupted by polar channels -PORES- through
which certain small hydrophilic molecules are able to pass. Some selectivity is afforded by the number and size of
these pores and most polar compounds remain in the aqueous phase. These pores are not discrete, permanent structures
but probably arise through the transient random motion of the individual membrane phospholipids. Consequently they
are known as flickering pores.
IX-23
Thus nonpolar molecules and a select group of polar molecule cross the
membrane by simple diffusion with their net movement from regions of high
to low concentration proportional to the concentration gradient. This is expressed
formally in Fick's First Law which we saw earlier; it is a strictly linear
relationship. The value of the diffusion constant is characteristic of both the
solute and the membrane being studied.
J
A
The majority of compounds that cross the membranes do so by processes
other than simple diffusion. The transport invariably involves the interaction of
the transported molecule with a specific transport or carrier system and the
quantitative kinetic properties of transport are different from those of simple diffusion. These transport processes are of
two kinds, facilitated diffusion and active transport.
Conc. Gradient
Facilitated Diffusion.
Facilitated diffusion is a carrier-mediated, non-energy requiring transfer; like passive diffusion it is a process
occurring down a concentration gradient. (Obviously it is driven by thermal energy but does not use metabolic
energy in any of its forms). An
example is the entry of glucose into
the red blood cell. The RBC
membrane contains a carrier protein
that is able to equilibrate the
M
M
metabolite (M) between the two
M
M
phases.
M M
M
M
MM
M
M
M M M
M
M
M
M
M
Phase 1
Phase 2
As long as [M] phase 1 >
[M]phase 2 there is a net flow of
material from phase 1 to phase 2. At
equilibrium, material is still moving
between the two phases but the
amount moving in the two directions
is equal. We have a conventional
equilibrium.
Experimentally, saturation kinetics are observed:
J
=
Jmax
1 + K/[S]
There is a clear parallel to enzyme kinetics with Jmax equivalent to V max (because the rate is limited by the
available sites on the carrier/enzyme) and K to Km Indeed many authors use the same symbols for both enzyme and
transport kinetics; I chose different symbols to reinforce the reality that the carrier is not an enzyme. K = [S] giving a
value of J equal to one-half of Jmax. J max = k t [carrier], analogous to Vmax = k 2 E o. kt is called the transfer
constant. The various data analyses of enzyme kinetics (Bios 301) are relevant in quantifying transport processes.
Just like enzymes these carriers are frequently very selective. For example the D-glucose carrier is much less
efficient with D-galactose (which differs only in the orientation of the OH at C4) and very much less efficient with Lglucose.
It is believed that the carrier proteins have a conformation that presents a hydrophobic exterior while
maintaining a hydrophilic domain to hold the solute (e.g. D-glucose). Thus hydropathy analysis of the glucose carrier
from the erythrocyte (55 kDa, Band 4.5, 2% of membrane protein) shows 12 strong peaks implying 12 transmembrane
helices which may well be organized in a "barrel like" arrangement thus yielding a "gated pore". This pore replaces the
hydration shell of the molecule to be transported by providing an alternative set of H-bonds (consequently ∆G for the
IX-24
solute entering the carrier from the external medium approaches zero). The hydrophobic exterior of the protein renders
it compatible with the hydrophobic bilayer of the membrane while the internal H-bonds facilitate the transfer of the
solute through the lipid phase. This picture accounts for the fact that the activation energy for facilitated diffusion is
much less than that for simple diffusion. The activation energy for glucose transport is only 5 kcal/mole, some 15 kcal
less than the unmediated rate, which results in a (20 - 5)/ 1.37 = 1010 enhancement in rate. (Note that every 1.37 kcal
decrease in the activation energy increases the rate by 10x, and vice-versa.) This is a consequence of the facilitation of
step 1.
IONOPHORES
The role of carriers in facilitated diffusion is supported by the discovery of the IONOPHORIC ANTIBIOTICS.
Certain compounds are able to mediate the transfer of alkali metal ions across artificial and natural membranes. These
ionophores are frequently antibiotics although there are also synthetic ionophores. They do not occur naturally in the
membrane but are important experimental tools in studying the properties of membranes. Like the glucose carrier their
basic structures consists of a hydrophobic exterior which renders them lipid soluble and a hydrophilic interior used to
bind the ion. There are two broad classes of ionophores, the CARRIER-LIKE and the CHANNEL-LIKE. Some of the
relevant structures are shown on the next page.
CARRIER-LIKE.
1) Carriers of charge but not protons-the VALINOMYCIN group. Valinomycin is a mobile carrier that
catalyses the electrogenic UNIPORT of fairly large monovalent cations such as Cs+ , Rb + , K + , or NH 4; it is
moderately specific The ability to move sodium is about 0.01% of that for potassium. Valinomycin is a natural
antibiotic from Streptomyces; it is a cyclic "peptide" of 12 acids and consists of alternating hydroxy- and amino acids.
The polymer bonds are thus alternating peptide and ester bonds. The molecule is folded into a cage-like structure with
several carbonyl oxygen atoms projecting inward forming a polar cavity that contains the cation in an unhydrated form.
Thus we can represent valinomycin as a doughnut-shaped molecule with the outer margin consisting of nonpolar
methyl and isopropyl sidechains and the inner margin presents the C=O to the inner cavity. Ions lose their waters of
hydration when they bind to these C=O groups.
It is the free energy of coordination of the cation with the oxygen atoms that provides the driving
force for removing the waters of hydration. When loaded with the cation the valinomycin bears a single positive charge.
This charge is dispersed over much of the structure and hence does not restrict the mobility of the valinomycin in the
membrane. Na+ is not transported because unhydrated Na+ is too small to be coordinated efficiently by the inwardly
pointing carbonyl groups. Mobile carriers such as valinomycin move about 1000 ions/sec.
2) Carriers of ions and protons but not charge-the NIGERICIN group. Nigericin is a linear molecule with
heterocyclic oxygen-containing rings together with a carboxyl group In the membrane these molecules cyclize to form
a structure similar to that of valinomycin with the oxygen atoms forming a hydrophilic interior. Nigericin loses a
proton when it binds a monovalent cation forming a neutral complex that can diffuse across the membrane. The
protonated, metal-free nigericin can also diffuse across the membrane so that this compound catalyses the electrically
neutral exchange of potassium ions and protons: potassium-proton antiport.
3) Carriers of charge and protons--proton translocators or UNCOUPLERS (usually synthetic). These compounds
have dissociable protons and are permeable across the bilayers both as the protonated acid and as the conjugate base.
These are carbonyl cyanide-p-trifluoromethoxyhydrazone (FCCP) and dinitrophenol. This is possible because these
compounds possess extensive π-orbital systems which delocalize the negative charge of the anion sufficiently to
maintain the lipid solubility of the molecule. They are thus able to collapse a pre-existing proton gradient.
Dinitrophenol is a famous example of an uncoupler. It has been used as a dietary aid, often with fatal results!
The next category are of particular importance to us!
4) Lipophilic Ions. These are not ionophores but are included in this list simply for completeness. There are two
kinds. The first are lipid soluble and charged, and will distribute across a membrane according to the sign and
magnitude of any membrane potential that may be present. The examples are tetraphenyl-phosphonium (TPP, good:
doesn't appear to bind) and tetraphenyl-borate (poor, as binds to membranes; useful catalytically to facilitate movement
of TPP). The second class (e.g. DMO) equilibrates across the membrane only in the neutral form (protonated for
DMO) ; it will distribute across the membrane according to any differences in proton concentration (pH) across the
membrane.
IX-25
CHANNEL-LIKE
Gramicidin is a channel-forming ionophore. It is a peptide of 15 amino acids that is folded into a helix 3 turns
long. A dimer composed of two molecules arranged head-to-head forms a tube-like structure long enough to cross a
membrane (orientation is amino-terminus to amino-terminus is blocked and hence uncharged). Thus it is believed to
form transient conducting dimers in the membrane. It has a poor discriminating ability between ions but is extremely
efficient, moving about 10 7 ions/sec.
TRANSPORT BY AN ELECTRIC FIELD.
Transport can also be effected against a potassium gradient by the application of an electric field. We have
seen (Ch. IV) that the movement of potassium from a region of high concentration to one of low concentration can
produce an electric potential, with the medium of lower concentration positive with respect to that of higher
concentration. Using parallel logic you should be able to convince yourself that application of an electric field across a
membrane which separates e.g. identical solutions of potassium should cause K+ to move down the electric field i.e.
towards the cathode! (we use Faraday's nomenclature, cations⇒ cathode.) This will still require the presence of a
carrier if the membrane is otherwise impermeable to the ion. From Ch IV:
E1 - E 2 =
- RT ln C 1
nF
C2
60 mV of applied potential should build up a concentration difference of about 10x. (Net transfer of potassium occurs
because the applied electrical field is derived from a power supply that can provide the necessary current to absorb the
field which the potassium is trying to build up.) Alternatively an applied potential of the appropriate sign can stop
potassium moving down its concentration gradient e.g. 60 mV of potential, positive in the low K+ phase, will block
movement down a 10:1 differential in potassium concentration.
IX-26
PRINT THIS PAGE FROM CANVAS........... MEMBRANE PROBES.CV5.
If you download this pdf file from the web you will also need to download “Chapter 9
Probes”
IX-27
ACTIVE TRANSPORT
The transport of neutral solutes against a concentration gradient or charged species against an electrochemical
gradient without the net chemical modification of the solute is a common phenomenon. Such a process requires energy
and this is provided by the metabolic activity of the cell.
Because active transport also exhibits saturation kinetics it is frequently described as a modification of
facilitated diffusion, the new feature being the provision of a device which allows an energy input to drive the system.
However in both facilitated diffusion and active transport the molecular events are only partly understood.
Schemes for Active Transport
1) Chemical Modification of the Transported Solute. (This scheme is included for completeness
but is not normally considered to be genuine active transport.)
In this scheme the transported solute is converted to a modified form once it has traversed the membrane. The
most thoroughly characterized example is provided by the bacterial sugar transport system that serves to import a
variety of sugars (e.g. glucose, fructose, sucrose, mannose) and sugar derivatives (e.g. mannitol). The overall scheme
illustrated for glucose, is:
Glucose (out) + PEP (in) ⇔ Glucose-6-P (in) + pyruvate (in)
In this system the sugar is converted to a sugar phosphate once it has entered the cell.
The system is comprised of three proteins. The first, EII, is an integral membrane protein which is specific
for each sugar and is the carrier of the sugar through the bilayer. The remaining two proteins, EI and HPr, are
cytoplasmic, non-sugar specific, energy coupling proteins. Free glucose binds to EII at the outside surface of the
membrane and is presented to the inner surface where the following series of reactions occur:
"histidine relay"
PEP + E I
⇔
Pyruvate + E I.P
(his, N-3-P)
EI.P + HPr
⇔
EI + HPr-P
(his, N-1-P)
HPr-P + EII.G
⇔
HPr + P.EII.G
(his, N-3-P)
P.E II.G
⇔
EII.P.G
(second his, N-1-P)
EII.P.G
⇔
EII + G-6-P
2) Chemical Modification of the Carrier.
The transport of lactose in E. coli is believed to require a different kind of integral membrane
protein-the PERMEASE-which facilitates the accumulation of lactose by E. coli by some 2-300 fold with respect to
the extracellular concentration. Addition of inhibitors of energy production (e.g. uncouplers) eliminates the transport.
As originally formulated the permease model requires:
a) ATP or some other tangible energy species as the energy source.
b) That the permease exist in two conformations, one with a high affinity for lactose and the other
with a low affinity. Conversion of the protein from the high affinity form (EH) to the low affinity form (EL ) is produced
by phosphorylation of the protein by ATP.
IX-28
We now know that this mechanism is not relevant for lactose. However it does apply to proteins that pump cations
such as Na+ , K + and Ca++ (the so-called P-type pumps). The sequence of reactions for a pump is
EH-M
EH-M
+ ATP
+M
E L-P -M
EH
+M
E L-P
E L-P
The sequence requires 2 activities for E (i) a kinase activity which is only available at the inner surface; (ii) a
phosphatase activity only available at the outer surface; and (iii) a site for the metal ion.
It is now recognized that most metabolites are actively transported by exploiting pre-existing gradients of
ions. This process is described by
The Chemiosmotic Theory (formerly the Chemiosmotic Hypothesis).
The chemiosmotic theory states that the essential driving force for transport is not ATP per se but is to be
found in the electrochemical potential difference that exist across a membrane because of the concentration gradients of
ions that are present.
In the lectures on thermodynamics we saw that the electrochemical potential of a solution of an ion is given
by
µ = µ o + RT ln [ion] + zFφ
where φ is the electric potential that might exist in the solution. Thus for two solutions of e.g. potassium that differ
in concentration and which are separated by a permeable membrane the difference in electrochemical potential is (z = 1
for univalent cations)
µ 1 - µ 2 = zF(φ 1 - φ 2 ) + RT ln [side 1]
[side 2]
z = +1
assuming u o is the same in the two solutions. (This is the equation we used earlier (Ch. IV) to establish the
relationship between the electric potential and a concentration differential-recall that we previously set the left hand side
to 0 and obtained the Nernst equation). In this latter form we obtain the work available from a concentration differential
IX-29
of a charged species. The available work consists of two components, an electric component-the first term on the
right-and an osmotic component, the second term on the right.
MITCHELL proposed that many transport processes are driven by the electrochemical potential difference
created by a difference in proton concentration on the two sides of the membrane i.e. by a pH gradient. Transforming
the above relationship to volts by dividing by F and inserting protons we get (z = +1):
∆ µ = ∆φ + RT ln [H+ ]1
F
F
[H + ]2
which Mitchell abbreviated to
or
∆P
= ∆φ - 0.06 ∆pH (in volts)
∆P
= ∆φ - 60 ∆pH (in millivolts)
Mitchell called ∆P the PROTON-MOTIVE FORCE; it is the difference in the proton electrochemical
potential expressed in volts. The first term on the right reflects the membrane potential and the last term the osmotic
component due to the proton gradient (expressed in volts). (Note that with z=1, RT/zF ln = 0.06 log, pH = -log and ∆
because it is the difference in hydrogen ion concentration across the membrane.)
The accepted convention assigns compartment 1 to the inside of an organelle. This makes ∆P negative if the
proton is pumped out, and vice versa.
Mitchell proposed that active transport consisted of two parts. The first part he called primary transport. In this phase
metabolic energy (electron transfer, ATP hydrolysis, light absorption) is used to create an ion gradient. The ions involved are
the proton (driven by electron transfer yielding the proton-motive force), calcium (sarcoplasmic reticulum, via ATP) and
sodium (bacteria, via H+ or ATP). The second part is called secondary transport; in secondary transport these preformed ion
gradients are exploited to actively transport other metabolites.
IX-30
The proton motive force is exploited in several different ways. Thus
H+
H+
D+
A+
d
S
SH2
D+
H+
C
H+
a
A+
B -H+
c
H+
b
ATP
ADP
B -H+
C
H+
The above figure can be interpreted as follows. For a metabolite M, that is accumulated by a ∆P-driven system in
negatively charged compartment (e.g. the mitochondrial matrix or bacterial cytoplasm) we use the following
expression:
RTln [M in]/[Mout] = -(n + Z) F∆φ - nRTln[H + ]in/[H+ ]out.
where n is the number or protons that accompany M and Z is the charge on M.
Case 1: Only ∆φ is important. This may be the case for a cation/anion which moves without the co-transport of H+.
Then the equation becomes:
ln [M in]/[Mout] = - n F ∆ φ
RT
This is the Nernst equation. We have electrophoresis; the movement of a charged particle in an electric field.
IX-31
Case 2: Only ∆pH is important. typically this applies to weak acids or bases. The equation becomes
[Min]/[Mout] = n[H + ]out/[H+ ]in
Weak acids are taken in by co-transport of a proton (e.g. acetate as acetic acid); weak bases move out as the free base
consumes a proton outside (ammonium is transported as ammonia (lipid soluble) which is converted to ammonium in
the acidic external phase). If the weak acid or base are lipid soluble then no carrier is needed; this happens to be true for
acetic acid and ammonia. Note however that formally charged species can be moved by ∆pH provided that protons move
in the opposite direction so that the process is electroneutral e.g. Ca2+ can be extruded if it proceeds with the
simultaneous uptake of 2 protons.
Case 3: ∆P is the driving force. Typically the compound to be transported does not have a charged form or "mobile"
protons. Such a compound is accumulated by proton symport or extruded with proton antiport.
To consider the examples in the figure:
(a) The uptake of some cations (K+ , lysine) is driven by ∆φ (also anion ejection). Uniport.
(b) The uptake of weak acids (succinate, lactate) is as the neutral species by co-transport of a proton; this is
electrically neutral and due to ∆pH exclusively. Symport.
(c) The uptake of neutral sugars is also with the co-transport of a proton (probably by some group on the
carrier) but the net species moved is now cationic; ∆φ and ∆pH contribute. Symport
(d) The movement of other cations (Na+ , Ca ++ ) is via proton anti-port. Again its electrically neutral (the
number of protons transported equals the charge on the cation) and so ∆pH is the important component.
Thus the potential free energy of the proton gradient can be used to drive lactose transport. The movement of
lactose across the membrane is obligatorily coupled to the movement of a proton; this is lactose-proton symport.
There is a 1:1 stoichiometry for lactose and protons entering E. coli and artificial proton gradients can stimulate lactose
uptake into the cell (e.g. by placing E. coli into mildly acidic solution of lactose).
Equilibration occurs when the chemical potential being created via the lactose concentrated inside the cell
equals the electrochemical potential of the proton gradient. We would normally expect this electrochemical potential to
decrease as the protons move into the cell and reduce the difference in proton concentration across the membrane.
However we shall see that the cell is continuously pumping protons out into the medium so that the proton gradient is
maintained at a fixed value.
System
H+ Flux
Mitochondria
Out
Chloroplast
In
E. coli
Depends on pH
The experimental evidence for the presence of proton gradients and the ability of electron transport to create
proton gradients is well accepted (reviewed in lectures on electron transport, to follow).
In summary, the chemiosmotic theory proposes that metabolic processes, principally electron transfer
reactions, proceed in such a way as to consume H+ on one side of a membrane and to extrude H+ on the other. The net
process consists of a transfer of protons across the membrane with the concomitant formation of the proton
electrochemical gradient-the protonmotive force. This can subsequently be exploited in reactions requiring a source of
free energy.
Permeability Properties of the Mitochondrial Membrane
As an example of the complexity of a membrane we consider the transport activities of the inner membrane of the
mitochondrion whose major biochemical activity we will be covering in Ch XI. This membrane contains a rich
assortment of porters, reflecting in large part the differences in metabolism that occur inside and outside the
mitochondrial space.
IX-32
The following list documents the large number of transport systems present in the inner membrane of the
mitochondrion; this is one of the metabolic machines and chemical material is continually flowing across its frontiers.
For this course you should be especially familiar with the systems required to move citrate and malate (Ch. 8)
ADP/ATP and Pi (Ch 11) and acyl carnitine (Dr. Rudolph's lectures) a
The outer membrane is very permeable and we usually assume that it is fully permeable. The inner
membrane is very impermeable but includes a number of very important transport systems. There are no carrier
systems needed for oxygen or carbon dioxide. In the following list note that one cannot distinguish between
movement of hydroxide or counter movement of a proton! There are specific carrier systems for:
1) ADP/ATP antiport: This is electrogenic; the carrier is called the ADENINE NUCLEOTIDE
TRANSLOCASE.
Out
In
ADP
ATP
2) The phosphate- OH- antiporter: electroneutral. Lehninger shows this as an electroneutral H2PO4symport. Either way it moves down the proton gradient.
Pi
OH
-
3) Dicarboxylate-phosphate: electroneutral antiport. (Malate/succinate)
malate
Pi
4) a-ketoglutarate-malate: electroneutral antiport.
a-Kg
malate
5) Tricarboxylate (Citrate/Isocitrate-malate): electroneutral.
citrate
malate
6) Glutamate-aspartate: electrogenic.
glu
asp
7) Glutamate- OH-: electroneutral antiport
glu
OH
8) Pyruvate-OH-:
electroneutral antiport
pyr
OH
-
9) The acylcarnitine/carnitine system: electroneutral antiporter
10) Ca2+ : electrophoretic uniport
IX-33
<=>
H+
Entries (8) and (9) are recent and deduced from the observation of saturation kinetics and/or specific inhibitors
Recall some metabolites never cross the membrane. Thus reducing equivalents present in NADH are moved as malate
while the long-chain fatty acid CoAs are moved across the membrane as acyl carnitine derivatives while acetyl CoA is
moved as citrate. In the case of malate there are complex counter movements which leads to the "glutamate-aspartate
cycle" for the oxidation of cytosolic NADH. Some textbooks describe this cycle incorrectly; Voet & Voet have it
right.
The formal origin of the Membrane Potential is detailed in Ch. IV.
Note that charges on the surface of the membrane are distinct from the bulk charges discussed in Chapter IV. Because
of the presence of fixed negative charges on the surface of the membrane the concentration of protons is higher at the
surface than in the bulk phase. This leads to an acidification of the surface layer relative to the bulk phase. However
these surface protons are "electrically neutral" (the negative and positive charges compensate) and thus the electric
potential due to surface protons is less than the bulk protons. These two effects exactly cancel so that the
protonmotive force of surface and bulk phase protons is the same.
How can one measure the protonmotive force.
The essence of the approach is to measure the pH and membrane potential contributions independently and
add up the two values. Both techniques use permeant compounds, one responds to ∆φ and the other to ∆pH. It is
crucial that such compounds don't bind to cell surfaces, are not metabolized and are not toxic. Furthermore the
concentrations employed must be sufficiently low so as not to perturb ∆φ or ∆pH
The experimental approach is similar in the two cases. A sample of mitochondria is incubated with the radiolabeled permeant compound. After equilibration is reached the amount of radio-label inside and outside the
mitochondrion is measured and the relevant parameter calculated, as follows.
Measurement of the Membrane Potential.
The permeant probe is TPP + . A typical experimental result is that the TPP+ outside the mitochondrion is
1/900'th that inside. These values are inserted into the Nernst equation.
∆φ = RT ln [out]
zF
[in]
With z = +1:
∆φ = 60 log [1/900] = - 177 millivolts.
Measurement of
pH.
The permeant probe might be the weak acid DMO (pK = 6). It will concentrate in the alkaline compartment
(weak bases concentrate in the acid compartment). The experimental result is that DMO (out) = 1/3 DMO(in)
+
A
+
H
out
out
HA
out
HA in
+
A- + H in
in
Note that it is the uncharged form that crosses the membrane. At equilibrium [HA]in = [HA]out and we
assume that the pK' for the weak acid is the same on the two sides of membrane. Then
IX-34
K=
HA
H ⋅A
+
out
out
−
out
=
HA
H ⋅A
+
in
in
−
in
+
in
+
out
;∴H
H
=
−
out
−
in
A
A
i.e. for ([H+ ] [A-])/ [HA] to be a constant [A-] must be high where H + is low and vice-versa; A- concentrates in the
alkaline phase. ∆pH = -log(H+ in]/[H+ out] = -log(1/3) = 0.5 and 60 ∆pH = 30 millivolts and ∆P = -177-30 = -207
millivolts.
E. coli strives to maintain an internal pH of about 7.7; thus when the external pH is above 7.7 the bacterium
actually pumps protons in, and the interior is acid relative to the exterior. In this situation a weak base will be
accumulated. Methylamine is commonly used. Note that both DMO and methylamine are not metabolized, an
important technical requirement.
Mitochondria (mV)
∆P
∆φ
60 ∆pΗ
-200
−170
-30
Bacteria (mV)
-(150-180)
−150
-30 (varies with
pH of external medium)
IX-35
Recognition Processes on the Membrane Surface
The surface of the cell membrane also functions as an informational device. There are two broad classes of
behavior:
1) Receptors (lower level recognition, typically for small molecules).
2) Higher Recognition (immune response, defense, organ recognition)
Receptors are devices for information translation, typically of external stimuli into intracellular events. The
membrane surface possess sites that recognize molecules (mostly small, but c.f. polypeptide hormones) or an electrical
signal and initiates some appropriate chemical response that is unique to the effector molecule. Two obvious examples
are:
1) Hormone Receptors (e.g. adrenergic receptor)-response may be over rather large area, usually leads
to activation of enzymes on other side of membrane.
2) Neurotransmitters-response typically confined to limited area. Usually cause opening of protein
channels in the membrane.
Cyclic AMP: An example of a Hormonal control systems.
The best understood example of the mechanism of hormone action is provided by the cyclic AMP system.
(The cyclic AMP control system is responsive to a variety of hormones. These hormones and their physiological
consequences include:
Adrenaline which stimulates glucose production from glycogen.
Corticotropin from the anterior pituitary which stimulates production of the sex hormones by the adrenal
glands.
Lipotropin which causes activation of the lipase present in the adipose cells.
Parathyroid hormone.
Thyroid-stimulating Hormone.
Vasopressin from the posterior pituitary; increases blood pressure and enhances the ability of the kidneys
to reabsorb water.
Glucagon; a polypeptide from the pancreas which stimulates glycogen breakdown when
sugar is low. It opposes insulin.)
The mechanism of a typical system can be nicely illustrated by describing the sequence of events that occur in
response to adrenaline.
Adrenaline is synthesized in the medulla (core) of the adrenal glands; these are located just above the kidneys.
Adrenaline is synthesized from tyrosine via 3,4 dihydroxyphenylalanine (dopa) and dopamine.
The synthesized compound is not released into the environment but is concentrated to about a 20% solution
and stored in vesicles in the medulla; these vesicles are called the chromaffin granules.
Normally the adrenaline concentration in the blood is very low (ca. 10-10M). However in response to some
event in the environment the nervous system sends an electrical signal to the medulla. The cell membrane of the
medulla contains a voltage-gated calcium channel and the influx of calcium from the extracellular fluid into the
medullary cell stimulates these chromaffin granules to fuse with the cell walls and releases the adrenaline into the
extracellular fluid and thus to the blood system (c.f. acetyl choline, next). The blood adrenaline increases about 1000fold in less than a minute. The adrenaline then finds its way to two principal organs, the heart and the liver. Its effect
upon the heart is to increase the pumping rate and the blood pressure. However we are principally interested in the
events that occur when the adrenaline reaches the liver. In this latter organ the ultimate response is the mobilization of
liver glycogen and a rapid elevation in blood glucose (via G-1-P and G-6-P).
IX-36
H
HO
CH2
H
C COOH
HO
CH2
C COOH
NH 2
NH 2
OH
H
H
HO
CH2
C
H
HO
NH 2
CH
C
H
OH
NH 2
OH
OH
H
HO
OH
CH
C
OH
NH
H
CH3
The outer membrane of liver cells contain three proteins relevant to this process. In order of action they are
1) The adrenaline receptor (β), present at the exterior face.
2) The G-protein, an integral membrane protein. There are in fact a family of G-proteins each made
of three subunits, designated α, β, γ. The β and γ subunits are common to all G-proteins whereas the α subunit is
unique in each case. The G-protein that is relevant in this example is Gs, having the α s subunit (s = stimulatory;
other G-proteins are Gi which reacts with the opiate receptor and GT (or transducin) which reacts with rhodopsin).
3) The enzyme adenyl cyclase, present at the interior face.
Each of these proteins is embedded in the membrane and each of them is believed to have considerable
mobility in the plane of the membrane. The following sequence ensues:
1) Adrenaline finds its way to the outer surface of the cell and binds to its receptor with extremely
high affinity. It does not enter the cell. The combination of the hormone with its receptor produces a structural change
in the receptor protein.
2) Because of this structural change, an encounter between the hormone-receptor complex and the Gprotein leads to a complex of both proteins The G-protein is normally present as a complex with GDP which is
attached to α s. The formation of a complex between the G-protein and the adrenaline receptor diminishes the relative
affinity of α s for GDP while the affinity of α s for intracellular GTP is enhanced; thus the G-protein exchanges bound
GDP for GTP. βγ now dissociates from the receptor-α s -GTP complex and then receptor and α s-GTP dissociate from
each other.
IX-37
Now the enhanced affinity of G-protein with the adrenaline receptor occurs at the cost of a decreased affinity of
adrenaline for the adrenaline receptor; consequently the equilibrium between the receptor and adrenaline is
displaced in the direction of the individual components and, over a period of time, the hormone is released
into the extracellular fluid. This free adrenaline is degraded enzymically in the liver (by methylation of amino
and -OH groups). As long as the medulla is discharging adrenaline, blood adrenaline remains high; however
when the medulla is no longer receiving stimuli from the nervous system blood adrenaline rapidly falls and
the receptor no longer binds adrenaline.
IX-38
3) The α s-GTP encounters the enzyme ADENYLYL CYCLASE and interacts with the catalytic
subunit of the adenyl cyclase complex. Adenylyl cyclase is normally inactive. However the complex of adenylyl
cyclase with α s -GTP is enzymically active and can catalyze the conversion of ATP to 3',5'-cyclic AMP (cAMP).
NH2
N
O-O
O-
O-
P
O P
O P
O
O
O
N
O CH2
NH2
N
N
N
N
O CH2
O
O P
O-
OH
N
N
O
O-
O
OH
The α s -GTP subunit has intrinsic GTP-ase activity and, over time, the bound GTP is hydrolyzed to GDP +
Pi. The α s -GDP complex has only weak binding to adenylyl cyclase and will dissociate and then recombines with βγ.
Thus the production of cAMP occurs only as long as adrenaline is available so that the adrenaline-receptor complex can
activate the G-protein which in turn activates adenylyl cyclase.
The cAMP produced has hormone-like activity and affects a large variety of biochemical systems. A
particularly well understood system is its activation of protein kinase. What are the consequences of activation of the
protein kinase?
The following (#'s 4-6) is for background only. It will be discussed in detail in Dr. Rudolph's lectures.
4). Cyclic AMP binds to PROTEIN KINASE. Protein kinase is a tetramer of structure R2C 2 (R and C
referring to regulatory and catalytic subunits). This tetramer is catalytically inactive. However c-AMP binds to the
regulatory subunit causing dissociation of the tetramer and liberating the free C subunits. These subunits can catalyze
the transfer of Pi from ATP to a variety of enzyme molecules, causing either activation or inactivation in specific
cases.
5) Cyclic AMP is continually hydrolyzed to AMP by a phosphodiesterase (inhibited by caffeine and
theophylline). AMP does not bind to the R-subunit; the free R-subunit is then available to combine with the Csubunit to reform the catalytically inactive R2C 2 tetramer.
6) As long as adrenaline is produced, adenyl cyclase is turned on and cAMP production is vigorous. However
when adrenaline release is turned off both adrenaline and cAMP are metabolized and the status quo is re-established.
Cholera toxin exerts its affect by chemically modifying G-protein. One of the subunits of cholera toxin has
enzymic activity and catalyzes the ADP-ribosylation of G-protein (α-subunit):
AMP-P-ribose-N + G ⇒ AMP-P-ribose-G + N
NAD
nicotinamide
The reaction is modification of an arginine. The modified G-protein has no GTPase activity and thus the
adenyl cyclase is permanently turned on--with undesirable metabolic consequences i.e. death.
(End of background material).
IX-39
Gated Channels
The propagation of an electric pulse along the axon (see figures, next page) is due to (i) an increase in the
permeability of the membrane to Na in a small region due to activation of a voltage-gated Na channel. This
occurs when the membrane potential rises from -60 to -40 mV. This increase leads to an associated current as a small
fraction of the Na rushes into the axon with the result that the membrane potential rises to + 40 mV causing a voltage
spike called the action potential. At a critical threshold of membrane potential the Na channels close, the voltage-gated
K channels open , K flows out, and the original -60 mV membrane potential is eventually restored. (ii) Some of the Na
diffuses to the right and lowers the Na concentration differential across the axon sufficiently that the associated current
exceeds the threshold potential. This then triggers a second voltage-gated channel leading to a new zone of increased
permeability; (i) and (ii) are repeated ad nauseam (Note that "leftwards" diffusing Na has no effect because the membrane
will not have recovered from the increase in permeability that was just complete.) Thus the depolarization of the
membrane, the action potential, propagates as a wave from left-to-right.
Arrival of this wave of electrical activity at the pre synaptic terminal leads to an increase in the permeability
of the pre synaptic membrane for calcium via a voltage-gated Ca channel. The sudden inrush of calcium ions
causes vesicles containing acetylcholine to migrate to the surface, fuse with the membrane and release the acetylcholine
into the synaptic cleft. The acetylcholine diffuses about 150 Å to the post-synaptic surface and binds to the acetyl
choline receptor (2 binding sites per receptor). This receptor is a ligand-gated Na/K channel. When the acetyl
choline site on each subunit of this dimeric protein is occupied the channel opens and there is a momentary increase in
the permeability of the post-synaptic membrane to Na which rushes into the cell down its concentration gradient,
destroying the post-synaptic membrane potential and initiating another electrical pulse. (For voltage-gated Na channels
about 107 Na cross the membrane each second). Potassium also diffuses out.
About 100-200 vesicles are released per pre-synaptic voltage pulse. In the absence of the calcium effect there
is a small spontaneous rate of vesicle fusion but the amount of acetyl choline that arrives at the post-synaptic
membrane is insufficient to produce currents large enough to exceed the threshold voltage (two acetylcholines need to be
bound and the binding curve is allosteric).
An esterase enzyme rapidly hydrolyzes the acetyl-choline to acetate and choline and the original membrane
impermeability to Na is restored. Acetyl choline is synthesized from choline and acetyl CoA by means of a transacetylase enzyme.
(CH3)3N+ CH2 CH2 OH + CH3COSCoA
(CH3)3N+ CH2 CH2 O CO CH3
+ CoA
Synthesis of acetyl choline.
(CH3)3N+ CH2 CH2 O CO CH3
(CH3)3N+ CH2 CH2 OH + CH3 COOH
Hydrolysis of acetyl choline
IX-40
Vesicle
containing
acetyl choline
Voltage Gated
Ca Channel
IX-41
Ligand Gated
Na Channel
Higher Recognition
By higher recognition I mean those processes whereby a cell identifies itself or an invader. This recognition
is obtained by means of the carbohydrate residues located on the outer surface of the membrane.
Cell Surface Carbohydrates
These come in two varieties:
1) Lipid Bound ⇒ Glycolipids
2) Protein bound ⇒ Glycoproteins; most important.
These seem to be the major determinant of cell surface specificity. A particularly well understood example is
the blood group system.
The ABO blood group system is based upon the occurrence of glycolipid and glycoprotein antigens on the red
blood cell membrane; these present the carbohydrate components to the outside world and can interact with specific,
complementary antibodies in the serum. In this instance it is the glycolipid component that is the most important.
The surface of the red blood cell is covered
with carbohydrate much of which is provided by
glycophorin, a transmembrane glycoprotein. This
protein provides a prototype of such structures.
Glycophorin has 40 amino acid residues at the
carboxyl terminus which are inside the RBC, 20 which
form a transmembrane helix and the remaining 70
residues are outside the erythrocyte. These 70 residues
bear about 100 sugars as 16 separate oligosaccharides.
There are about a million glycophorin
molecules/RBC. This protein is about 60% sugar by
weight and bears the MN antigens (not clinically
important).
Some of these sugars are part of the ABO
system but this system is mainly contributed to by
glycosphyngolipids and minority glycoproteins. The
clinically important ABO system is in fact present on
many plasma membranes.
Blood Group
A
B
AB
O
Antigen on host's RBC
A
B
A and B
None (actually H)
Antibody in host's Serum
anti-B
anti-A
None
anti-A and anti-B
[O, actually H] is a precursor to A and B
Individuals in the O category carry anti-A and anti-B antibodies in their serum but their RBC do not bear
either A or B antigens.
Our knowledge of the structure of the ABO antigenic determinant is obtained from related glycoproteins found,
for example, in saliva, which bear the same antigens but can be obtained in soluble form. The A,B,O antigens of
soluble glycoproteins begin with a sugar attached to a serine or threonine residue of the polypeptide chain. Note that
the residual oligosaccharide present in Type O individuals is called the H antigen so this is also called the ABH
IX-42
system. The differences are found at the location of the solid arrow. Nothing gives H, A has a substituted galactose
while B has a galactose at this position.
Present in everyone
Gal-1-β
3(4)GlcNAc
serine-polypeptide
2
3(4) = 3 or 4
1α
Fucose
Gal = D-galactose
GlcNAc = N-AcetylD-gluosamine
B = Gal 1α
GalNAc = N-acetylD-galactosamine
(NH2 is at C2 in both)
3
A = GalNAc 1α
3
(Fucose is a 6-deoxy L aldohexose)
The basis for the ABO blood groups is genetic by specifying the synthesis of an enzyme, a glycosyl
transferase. An individual with e.g. blood group A has a version of the enzyme which transfers a
N-acetylgalactosamine onto the H antigen ; in B individuals the related enzyme transfers a galactose, while in O
individuals the enzyme appears to be inactivated.
THE REMAINING EXAMPLES ARE FOR BACKGROUND ONLY.
Receptor Sites for Viruses.
Influenza virus (RNA) can bind to glycophorin and can link several RBC's together. The coupled RBC's then
agglutinate.
Lectin Receptor Sites
Lectins are proteins extracted from plants which recognize specific carbohydrates structures, both glycoprotein
and glycolipid. As they are multivalent (like antibodies) they can bind and bridge carbohydrates bearing structures and
cause agglutination. They are a powerful experimental tool for establishing the carbohydrate characteristics of a given
surface and for purifying selected carbohydrate derivatives via affinity chromatography.
Cell Recognition and Adhesion
When an invader enters our body specific cells are activated to repel this invasion. This phenomenon is called
a cell-mediated immune response, as distinguished from an antibody response. The cells which provide this defense
system are called lymphocytes, they are members of the family of leukocytes (WBC).
For example, one class of lymphocyte matures in the thymus and is called the T lymphocyte. T lymphocytes
expect all cells to have a particular pattern of carbohydrate on their surface-the "self" pattern. Such cells are tolerated by
the T cells. But when, for example, a host cell is invaded by a virus, viral antigens become incorporated in the
membrane of the host cell and the characteristic surface pattern is modified. The killer-T cell recognizes this change,
binds to the modified host-cell and, by some unknown process, destroys the infected cell before the virus has had a
chance to multiply. The killer-T then returns to its surveillance mode i.e. it acts "catalytically".
IX-43
Killer T's do not invade those cells legitimately present in the blood stream. On the other hand, the
macrophage cells produced by the spleen can recognize tired RBC and selectively remove them from the blood by
phagocytosis.
The homing of lymphocytes from the blood stream to the lymph depends upon specific carbohydrate present
on the lymphocyte membrane. If the sugars are removed (enzymically) they home to the liver returning to the lymph
nodes within 24 hr. suggesting a regeneration of the determinants.
Cell-cell recognition must also be important in morphogenesis, the formation of tissues and of organs etc. It
appears that a cell surface must contain a "conjugate pair"-its characteristic carbohydrate label and the complementary
carbohydrate receptor site. These receptor sites are speculated to be glycosyl transferase so that a specific cell can make
an ES complex with an adjacent cell, transfer a sugar residue to the second cell which is then primed for some
subsequent event
Transplantation (Histocompatibility) Antigens.
Transplants from genetically different donors are routinely rejected. This is principally due to the lack of
compatibility just described between the hosts immune system, the T lymphocytes, and glycoproteins (similar to
glycophorin) on the plasma membrane of the cells of the transplanted organ. (Actually this is a mutual effect with the
organ also rejecting the host by the same mechanism.)
IX-44