Chapter 9

Membranes and Membrane Transport

. . . . . . . . . . . . . . . . . . . . . . . .

Chapter Outline

v Membrane functions:
Boundary for cell and organelle
Surface on which reactions can occur
Regulation of material flux through membrane proteins
Signal transduction interface
Specialized properties: Photosynthesis, electron transport, electrical activity
v Membrane components
Lipids: Amphipathic molecules arranged as bilayers with polar groups out and nonpolar
groups in
Proteins: Surface associated or embedded in bilayer
v Plasma membrane
Delimits cell
Excludes and retains certain i ons and molecules
Major role in energy transduction
Cell locomotion
Reproduction
Signal transduction
Interactions with other cells or extracellular matrix
v Lipid interactions
Monolayers: Formation of single-molecule-thick layer at air/water interface with polar
groups in contact with water
Micelles: Lipid spheres with polar groups out and hydrophobic tails in the center:
Critical micelle concentration is the concentration of amphiphilic compound at which
micelles form
Lipid bilayer: Two lipid monolayers with hydrophobic surfaces face to face
Liposomes: Vesicles formed by lipid bilayers
v Fluid mosaic model
Singer and Nicholson, 1972
Phospholipid bilayer forming fluid matrix
Two classes of membrane proteins
- Peripheral (extrinsic) proteins
Associated with bilayer surface via ionic interactions and H bonds
Extractable with high salt or agents that disrupt H bonds (urea)
- Integral (intrinsic) proteins
Associate with hydrophobic bilayer interior via hydrophobic
interactions
Extractable with detergents
v Membrane mobility
Protein
Chapter 9
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127
- Frye and Edidin, 1970: Lateral movement of membrane proteins following
fusion of mouse and human cells
- Lateral movement may be impeded by interactions with cytoskeleton
Lipids
- Rapid lateral movement
- Slow transverse movement
v Membrane asymmetry
Lateral asymmetry arises from clustering of membrane components within the plane
- Lipid clustering: Phase separation induced by divalent cations and influenced
by lipid type
- Protein clustering: Self-associating membrane proteins e.g., bacteriorhodopsin
Transverse asymmetry
- Lipids: Lipid asymmetry due to two processes
Asymmetric synthesis
Energy-dependent transport: Flippases
- Proteins: Asymmetric molecules
- Carbohydrates: Glycoproteins and glycolipids on outer surface
v Membrane phase transitions: Radical change in physical state occurring within narrow range of
transition (or melting) temperature. Below T
m

Lipids close-pack: Lose lateral mobility and rotational mobility of fatty acid chains
Consequences: Membrane thickens and decreases surface area
Characteristics
- T
m
increases with chain length degree of saturation and is influenced by nature
of head group
- Pure phospholipid bilayers show narrow temperature range
- Native membranes show broad transition influenced by protein and lipid
composition
v Membrane Proteins
Functions: Transport, receptors
Two types: Peripheral and integral
v Integral membrane proteins: Two classes
Single transmembrane segment proteins: Hydrophobic alpha helix that spans the lipid
bilayer: Glycophorin: 19-amino acid long alpha helix that spans the membrane with
extracellular domain decorated with oligosaccharides that are ABO and MN blood group
antigens
Multi-transmembrane segment proteins: Essentially globular proteins embedded in
membrane
- Bacteriorhodopsin: Seven alpha helical segments embedded in bilayer:
Segments organized into a channel
- Porins: Beta sheet motifs
v Lipid-anchored membrane proteins: Four types
- Amide-linked myristoylated proteins
Myristic acid (14:0 fatty acid)
Amide linkage to amino group of N-terminal glycine
- Thioester-linked: Fatty acid attached to cysteine as thioester (or Ser or Thr as
ester)
- Thioether-linked prenylated proteins
Prenyl: Long-chain isoprene polymers: Farnesyl or geranylgeranyl
Attachment as thioether to C-terminal cysteine of CAAX (A=
Aliphatic)
AXX cleaved after phrenyl addition
- Amide-linked glycosylphosphotidylinositol (GPI) anchors
Lipid: Oligosaccharide-modified phosphoinositol
Linkage: Carboxy terminus attached via phosphoethanolamine to
mannose residue of oligosaccharide
Chapter 9
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128
v Membrane transport: Three types
Passive diffusion
- Entropically driven process: Molecules move down a concentration gradient
G = RTln([C
2
]/[C
1
]) for uncharged molecule
G = RTln([C
2
]/[C
1
]) +ZF for charged molecules
R = 8.3145 J/Kmol, T = K, Z = charge, F = 96485 J/Vmol, =
electrical potential
- Rate depends on concentration gradient and lipid solubility
Facilitated diffusion
- Entropically driven process as in passive diffusion
- Involves integral membrane protein
Rate depends on concentration but is saturatable
Specificity and affinity due to protein/transported molecule interaction
- Examples:
Glucose transporter: RBC band 4.5: 55 kD protein functions as trimer
Anion transport system: RBC band 3: 95 kD protein: Cl
-
, HCO
3
+

exchange
Active transport: Energy driven process
- Primary active transport: Energy sources
ATP hydrolysis (most common)
Light energy
- Secondary active transport (Energy is ion gradient formed by some other
process)
- Electrogenic transport: Active transport of ions and net charge transport both
occur
v Na
+
,K
+
-ATPase (sodium pump)
120 kD -subunit; 35 kD -subunit
3 Na
+
out, 2 K
+
in per ATP hydrolyzed: Electrogenic
Ouabain: Cardiac glycoside that inhibits sodium pump
v Calcium ATPase
2 Ca
2+
out of cytoplasm per ATP hydrolyzed
Restores/maintains low cytoplasmic calcium
v H
+
,K
+
-ATPase
1 H
+
out, 1 K
+
in per ATP hydrolyzed
Gastric enzyme: pH largest gradient known
v Vacuolar ATPases: Pump H
+
in number of vacuoles and cells
v Multidrug resistance in malignant cells and transport of yeast a factor peptide transported by
ATPases
v Light-energy driven pumps
Bacteriorhodopsin
Halorhodopsin
v Secondary active transport systems
Na
+
or H
+
coupled movement of amino acids or sugars
Symport: Ion and substance move in same direction
Antiport: Ion and substance move in opposite directions
v Light-driven pumps
Bacteriorhodopsin: Proton pump
Halorhodopsin: Chloride pump
v Specialized membrane pores
-Helical pores
- Colicin Ia: C-domain forms -helical bundle
- -Endotoxin: -helical bundle
-Sheet pores
- Hemolysin and aerolysin: pores
Chapter 9
.
Membranes and Membrane Transport

129
Peptide pores
- Melittin and cecropins
Gap junctions: Intracellular connectivity
v Ionophores: Facilitate movement of molecules across lipid bilayer
Mobile carriers
- Sensitive to membrane phase transition
- Forms complex with ion that diffuses across membrane
- Example: Valinomycin: Potassium ionophore
Channel-forming ionophore
- Insensitive to membrane phase transition
- Forms ion-specific channel that spans the membrane
- Example: Gramicidin

Chapter Objectives

Lipids associate to form two- and three-dimensional structures. Understand the forces
responsible for this behavior, including hydrophobic interactions and van der Waals forces.
Know why monolayers of lipids form at an air/water interface, and what a micelle is and how it
forms. Lipids are also capable of forming bilayers, an important structural component of
biological membranes.

Biological membranes are composed of various lipids arranged in a bilayer and, embedded in
the bilayer, integral (or intrinsic) proteins. The fluid mosaic model of membranes suggests that
both lipids and proteins are free to move within a bilayer. The two surfaces of bilayers of
biological membranes are asymmetric with respect to protein, lipid and carbohydrate
composition.

Membrane phase transitions occur when membrane components, in particular lipids, interact in
a manner causing loss of fluidity. The temperature of this transition, a transition from solid to
liquid, is known as the melting temperature (Tm). What are the effects of degree of saturation, of
chain length, of cholesterol on Tm?

Two types of membrane proteins are peripheral and integral proteins. Peripheral proteins
interact through electrostatic bonds and hydrogen bonds with the surfaces of bilayers. Integral
proteins are strongly associated with the bilayer. There are three kinds of protein motifs
responsible for anchoring integral proteins to membranes. Certain integral proteins have a
single transmembrane segment, in the form of an -helix composed of hydrophobic amino acid
residues, anchoring the protein to the lipid bilayer. Another structural motif found is the 7-
helix, transmembrane segment used by integral proteins involved in transport and signaling
activities. Certain proteins have covalently linked lipid molecules that serve as anchors. You
should understand the four kinds of anchors.

Passive Diffusion
Passive diffusion proceeds down a concentration gradient. The driving force is a change in free
energy given by G = RTln([C2]/[C]1) where [C2] < [C1] and the substance moves from side 1 to
side 2. For a charged species, the driving force is an electrochemical potential given by G =
RTln([C2]/[C1]) + ZF where Z is the charge, F is Faraday's constant, and is the membrane
potential.

Facilitated Diffusion
Facilitated diffusion is reminiscent of enzyme kinetics because it is a carrier-mediated process
and as such depends on an interaction between a carrier and a transported molecule. The flux
is still dependent on a difference in concentration and it occurs from high concentration to low
concentration but the dependence is no longer linear. The flux shows saturation at high
concentrations and is critically dependent on stereochemistry of the compound. The glucose
transporter and the anion transporter (both in erythrocytes) are examples of facilitated diffusion.

Chapter 9
.
Membranes and Membrane Transport

130
Active Transport
Unlike passive and facilitated diffusion, active transport can move a substance against a
concentration gradient. However the overall G of the reaction must be favorable and this is
achieved by coupling transport to some other energy-yielding process like ATP hydrolysis,
capture of light energy, and coupling to other gradients. The sodium pump or Na
+
,K
+
-ATPase is
a well characterized active transporter for movement of 3 Na
+
out of the cell and 2 K
+
into the cell
coupled to hydrolysis of ATP. The enzyme is an intrinsic membrane protein that exists in two
conformational states that differ in ion- and ATP-binding properties. Understand how transient
phosphorylation leads to conformational changes and movement of ions. The cardiac glycosides
are important inhibitors of the sodium pump. Understand the consequences of sodium pump
inhibition to calcium ion levels in heart muscle. Finally, because of the difference in charge
transported, (a difference of one positive charge) the sodium pump is electrogenic leading to
formation of a membrane potential. The calcium transporter of sarcoplasmic reticulum is also
an ATP-dependent transporter, but of calcium. It has a similar mechanism of action to the
sodium pump, shuffling between two conformational states with ATP hydrolysis driving calcium
uptake. This transporter is a key player in relaxation of muscle and is also electrogenic. The
H
+
,K
+
-ATPase moves protons out of the cell and potassium back into the cell with ATP
hydrolysis. This nonelectrogenic pump is capable of producing extremely high concentration
gradients of protons.

Light energy-driven active transport systems include bacteriorhodopsin, a H
+
-pump, and
halorhodopsin, a Cl
-
-pump. There are many important examples of transport systems driven by
ion gradients. Proton gradients, produced by electron-transport driven proton pumping or by
proton-ATPases, sodium gradients, produced by the sodium pump, and other cation and anion
gradients are used to move a range of molecules, including sugars and amino acids. You should
know the terms symport and antiport.

Specialized Pores
The porins are a class of membrane proteins that allows diffusion of a range of substances
across membranes. With the exception of size, there is little specificity to porin-mediated
transport. Ionophores are compounds that allow passage of specific ions across a membrane.
There are two general classes of ionophores, mobile carriers and channel formers. Mobile
carriers form a complex with the ion to be transported and this complex diffuses across the
membrane. In effect, a mobile ionophore increases the apparent partition coefficient of the ion.
Channel formers bridge the membrane and provide a hole or channel through which ions pass.

Problems and Solutions

1. In Probl em 1(b) i n Chapter 8 (page 265) you were asked to draw al l the
phosphati dyl seri ne i somers that can be formed from pal mi ti c and l i nol ei c aci ds. Whi ch
of these PS i somers are not l i kel y to be found i n bi ol ogi cal membranes?

Answer: Phosphatidylserine with unsaturated lipids at position 1 are very rare. Unsaturated
fatty acids are usually found at position 2. Glycerophospholipids with two unsaturated chains,
or with a saturated chain at C-1 and an unsaturated chain at C-2, are commonly found in
biomembranes.

2. The purpl e patches of the Halobacterium halobium membrane, whi ch contai n the
protei n bacteri orhodopsi n, are approxi matel y 75% protei n and 25% l ipi d. If the protei n
mol ecul ar wei ght i s 26,000 and an average phosphol i pi d has a mol ecul ar wei ght of
800, cal cul ate the phosphol i pi d to protei n mol e rati o.

Answer:
Chapter 9
.
Membranes and Membrane Transport

131

Let x = the weight of bacteriorhodopsin - lipid complex.
Weight of lipid in the complex = 0.25x
Weight of protein in the complex = 0.75x
Moles lipid =
0.25x
800
g
mole
= 3.1310
4
x, and
Moles protein =
0.75x
26,000
g
mole
= 2.8810
5
x
Molar ratio (lipid : protein) =
3.1310
4
x
2.8810
5
x
=10.8


3. Sucrose gradi ents for separati on of membrane protei ns must be abl e to separate
protei ns and protei n-l i pi d compl exes havi ng a wi de range of densi ti es, typi cal l y 1.00 to
1.35 g/mL.
a. Consul t reference books (such as the CRC Handbook of Biochemistry) and pl ot the
densi ty of sucrose sol uti ons versus percent sucrose by wei ght (g sucrose per 100 g
sol uti on), and versus percent by vol ume ( g sucrose per 100 mL sol uti on). Why i s one
pl ot l i near and the other pl ot curved?
b. What woul d be a sui tabl e range of sucrose concentrati ons for separati on of three
membrane-deri ved protei n-l i pi d compl exes wi th densi ti es of 1.03, 1.07, and 1.08
g/mL?

Answer: The density, at 20

C (, g/mL), of sucrose solutions and their percent by volume (g per
100 mL) are shown in the first two columns. The third column shows the corresponding percent
by weight for values shown in the second column.

% Sucrose % Sucrose
(g /mL) (g per 100mL) (g per 100g)*
0.9988 0 0.00
1.0380 10 9.63
1.0806 20 18.51
1.1268 30 26.62
1.1766 40 34.00
1.2299 50 40.65
1.2867 60 46.63
1.3470 70 51.97
*The values in this column are calculated as follows. First, the weight of a weight per volume
solution is calculated. For example, a 10% weight per volume solution of sucrose has a density
of 1.0380 g/mL. Therefore, 100 mL of this solution has a weight of:

100 mL 1.038
g
mL
=103.8g
The percent weight is determined by calculating the amount of sucrose required to make 100 g
of solution.

10g
103.8g
=
x
100g
x = 9.63g sucrose in 100 mL.

Chapter 9
.
Membranes and Membrane Transport

132
percent
80 60 40 20 0
0.9
1.0
1.1
1.2
1.3
1.4
% (g per 100
mL)
% (g per 100
g)
d
e
n
s
i
t
y

(
g
/
m
L
)

Why is there a difference in the two plots?


For the 10% solution (g per 100 mL), 100 mL of this solution weights :
100 ml 1.0380
g
mL
=103.8g of which 10g was sucrose and
103.8g -10g = 93.8g was water.
The volume of 93.8g of water is
93.8g
0.9988
g
mL
= 93.91mL.
Thus, the 100mL volume of 10% sucrose is composed of 93.91mL water and
100mL-93.91ml = 6.09mL of sucrose.
Therefore, the 10g of sucrose occupied 6.09mL corres ponding to a density of
10g
6.09mL
=1.64
g
mL


To prepare a 10g per 100 g solution, 90g of water is mixed with 10g sucrose.
The final volume is
90g
0.9988
g
mL
+
10g
1.64
g
mL
= 96.21mL
The solution' s density is
100g
96.21mL
=1.0394
g
mL
Thus, the 10% (g per 100g) solution's density (1.0394
g
mL
) is greater than
the 10% (g per 1 00mL) solution's density (1.0380
g
mL
).


4. Phosphol i pi d l ateral moti on i n membranes i s characteri zed by a di ffusi on coeffi ci ent
of about 1 x 10
-8
cm
2
/sec. The di stance travel ed i n two di mensi ons (i n the membrane) i n
a gi ven ti me i s r = (4Dt)
1/2
, where r i s the di stance travel ed i n centi meters, D i s the
di ffusi on coeffi ci ent, and t i s the ti me duri ng whi ch di ffusi on occurs. Cal cul ate the
di stance travel ed by a phosphol i pi d i n a bi l ayer i n 10 msec (mi l l iseconds).

Answer:

For D = 110
-8
cm
2
sec
, t = 10msec = 10 10
-3
sec
r = 4 D t
= 4 (110
-8
cm
2
sec
) (10 10
-3
sec)
= 2.010
-5
cm = 2.010
-7
m = 2.0m


Chapter 9
.
Membranes and Membrane Transport

133
5. Protei n l ateral moti on i s much sl ower than that of l i pi ds because protei ns are l arger
than l i pi ds. Al so, some membrane protei ns can di ffuse freel y through the membrane,
whereas others are bound or anchored to other protei n structures i n the membrane. The
di ffusi on constant for the membrane protei n fi bronecti n i s approxi matel y
0.7 x 10 cm
-12

cm
2
/sec, whereas that for rhodopsi n i s about 3 x 10
-9
cm
2
/sec.
a. Cal cul ate the di stance traversed by each of these protei ns i n 10 msec.
b. What coul d you surmi se about the i nteracti ons of these protei ns wi th other
membrane components?

Answer:

For fibronectin, D = 0.7 10
-12 cm
2
sec
For rhodopsin, D = 3.0 10
-9
cm
2
sec
t = 10msec= 10 10
-3
sec
r = 4 D t
For fibronectin r = 4 (0.7 10
-12
cm
2
sec
) (10 10
-3
sec) = 1.67 10
-7
cm = 1.67nm
For rhodopsin r = 4 (3.0 10
-9
cm
2
sec
) (10 10
-3
sec) =1.10 10
-5
cm =110nm


b. The diffusion coefficient is inversely dependent on size and unless we know the size of each
protein we can surmise very little. The Mr of rhodopsin and fibronectin are 40,000 and 460,000
respectively. For spherical particles D is roughly proportioned to [Mr]
1/3
. We might expect the
ratio of diffusion coefficients (rhodopsin/fibronectin) to be

(40, 000)

1
3
(460, 000)

1
3
= 2.3
The measured ratio is 4286! Clearly the size difference does not explain this large difference in
diffusion coefficients. Fibronectin is a peripheral membrane protein that anchors membrane
proteins to the cytoskeleton. Its movement is severely restricted.

6. Di scuss the effects on the l i pi d phase transi ti on of pure di myri stoyl
phosphati dyl chol i ne vesi cl es of added (a) di val ent cati ons, (b) chol esterol , (c) di stearoyl
phosphati dyl seri ne, (d) di ol eoyl phosphati dyl chol i ne, and (e) i ntegral membrane
protei ns.

Answer: Myristic acid is a 14 carbon saturated fatty acid and as a component of dimyristoyl
phosphatidylcholine is expected to participate in hydrophobic interactions and van der Waals
interactions. At a particular temperature, Tm, these forces are strong enough to produce local
order in a bilayer of this phospholipid.

a. Divalent cations (e.g., Mg
2+
, Ca
2+
) interact with the negatively charged phosphate group and
thus stabilize bilayers and increase the Tm.

b. Cholesterol does not change the Tm; however, it broadens the phase transition. As a lipid, it
can participate in hydrophobic and van der Waals interactions. Above the Tm of dimyristoyl
phosphatidylcholine, cholesterol stabilizes interactions; however, below the Tm, it interferes with
the packing of dimyristoyl phosphatidylcholine.

c. Distearoyl phosphatidylserine contains stearic acid, an 18-carbon, fully saturated fatty acid,
which should participate favorably in van der Waals interactions and hydrophobic bonds. Its
slightly longer chain length may perturb the geometry of vesicles. Also, the longer chain and
negatively-charged head group should raise Tm.
Chapter 9
.
Membranes and Membrane Transport

134

d. Oleic acid is an 18-carbon fatty acid with a single double bond in cis configuration between
carbons 9 and 10. Although capable of hydrophobic interactions, the unsaturated fatty acids
are expected to interfere with van der Waals interactions. The Tm will be decreased.

e. Integral proteins will broaden the phase transition and could either raise or lower the Tm
depending on the nature of the protein.

7. Cal cul ate the free energy di fference at 25C due to a gal actose gradi ent across a
membrane, i f the concentrati on on si de 1 i s 2 mM and the concentrati on on si de 2 i s 10
mM.

Answer:

G = RTln
[ C
2
]
[ C
1
]
,
where [ C
1
] and [ C
2
] are the concentrations of C on opposites of the membrane.
G = 8.314 10
3
kJ
K mol
298K ln
10mM
2mM
G = 4.0
kJ
mol


8. Consi der a phosphol i pi d vesi cl e contai ni ng 10 mM Na
+
i ons. The vesi cl e i s bathed in
a sol uti on that contai ns 52 mM Na
+
i ons, and the el ectri cal potenti al di fference across
the vesi cl e membrane = outsi de - i nsi de = -30 mV. What i s the el ectrochemi cal
potenti al at 25C for Na
+
i ons?

Answer: The electrical potential is given by the following formula:

G = RT ln
[ C
2
]
[C
1
]
+ ZF
where R is the gas constant, T the temperature in degrees Kelvin, F is Faraday's constant (96.49
kJ/Kmol) and Z is the charge on the ion: +1 in this case.

G = 8.31410
3
kJ
K mol
298 K ln
52mM
10mM
+ (+1) 96.49
kJ
V mol
(3010
3
V)
G =1.19
kJ
mol


Chapter 9
.
Membranes and Membrane Transport

135
9. Transport of hi st i di ne across a cel l membrane was measured at several hi sti di ne
concentrati ons:
[ Hi st i di ne] , M Tr anspor t , mol / mi n
2.5 42.5
7 119
16 272
31 527
72 1220
Does thi s transport operate by passi ve di ffusi on or by faci l i tated di ffusi on?

Answer: A characteristic of transport by passive diffusion is that the rate of transport is linearly
dependent on concentration and so a plot of transport rate versus concentration will be linear.
For facilitated diffusion, the transported molecule interacts with a carrier protein in the
membrane. The rate of transport will be dependent on concentration but the dependence is not
linear. Rather the dependence is reminiscent of Michaelis-Menten enzyme kinetics in that it
shows saturation. A plot of rate versus concentration is presented below. The data show a
linear relationship indicating that transport is by passive diffusion. However, a facilitated
transport system with a high Km relative to the concentrations of histidine tested here will also
be approximately linear. The concentrations tested here are high relative to physiologically
reasonable concentrations of histidine and if facilitated diffusion is at work it may not be of
physiological importance. One way to confirm that the transport is passive is to retest transport
using D-histidine. Using a different stereoisomer of histidine will have no affect on passive
diffusion. Facilitated diffusion will show a specificity for one of the stereoisomers.

80 60 40 20 0
0
400
800
1200
[Histidine]
T
r
a
n
s
p
o
r
t

(







m
o
l
/
m
i
n
)

10. Fructose i s present outsi de a cel l at 1 M concentrati on. An acti ve transport system
i n the pl asma membrane transport s fructose i nto thi s cel l , usi ng the free energy of ATP
hydrol ysi s to dri ve fructose uptake. What i s the hi ghest i ntracel l ul ar concentrati on of
fructose that thi s transport system can generate? Assume that one fructose i s
transported per ATP hydrol yzed; that ATP i s hydrol yzed on the i ntracel l ular surface of
the membrane; and that the concentrati ons of ATP, ADP, and Pi are 3 mM, 1 mM, and 0.5
mM, respecti vel y. T=298 K (Hi nt: Ref er to Chapter 3 to recal l the effects of
concentrati on on free energy of ATP hydrol ysi s.)

Answer: The free energy of hydrolysis of ATP is given by

G = G' +RT ln
[ ADP][P
i
]
[ ATP]
G = 30
kJ
mol
+ 8.314 10
3
kJ
K mol
298K ln
1mM 0.5mM
3mM
= -52.05
kJ
mol

The free energy of a gradient of a substance across a membrane is given by
Chapter 9
.
Membranes and Membrane Transport

136

G = RT ln
[C
2
]
[C
1
]

We can set this equal to the free energy of hydrolysis calculated above but with the opposite sign
and solve for C2 given that C1 is equal to 1 mM.

G = RTln
[C
2
]
[C
1
]
= 52.05
kJ
mol
and
[C
2
] =[C
1
]e
52.05
kJ
mol
RT
=1.010
6
Me
52.05
kJ
mol
8.31410
3
kJ
K mol
298K
[C
2
] =1,330 M !


11. In thi s chapter we have exami ned coupl ed transport systems that rel y on ATP
hydrol ysi s, on pri mary gradi ents of Na
+
or H
+
, and on phosphotransferase systems.
Suppose you have just di scovered an unusual strai n of bacteri a that transports
rhamnose across i ts pl asma membrane. Suggest experi ments that woul d test whether i t
was l i nked to any of these other transport systems.

Answer: If uptake is sensitive to ion gradients, ionophores may be used to destroy the
gradients. Uncouplers like dicumarol or dinitrophenol can be used to degrade proton gradients.
Ouabain can be used to inhibit the sodium pump. Dependence on ATP hydrolysis may be
determined by using nonhydrolyzable ATP analogs. PEP dependent mechanisms similar to PTS
in E. coli are sensitive to fluoride.

12. Whi ch of the fol l owi ng pepti des woul d be the most l i kel y to acqui re a N-termi nal
myri stoyl l i pi d anchor?
a. VLIHGLEQN
b. THISISIT
c. RIGHTHERE
d. MEMEME
e. GETREAL

Answer: Myristoylation occurs on N-terminal glycine residues and the only peptide that
qualifies is e.

13. Whi ch of the fol l owi ng pepti des woul d be the most l i kel y to acqui re a prenyl
anchor?
a. RIGHTCALL
b. PICKME
c. ICANTICANT
d. AINTMEPICKA
e. none of the above

Answer: Prenylations typically occur on cysteines in sequences CAAX where the As are
aliphatic amino acids and X is any residue. The only peptide to fit this description is a. This
peptide has a cysteine followed by alanine and two leucines on its C-terminus.

14. Si nger and Ni col sons fl ui d mosai c model of membrane structure presumed al l of
the fol l owi ng statements to be true EXCEPT:
a. The phosphol i pi ds bi l ayer i s a fl ui d matri x.
b. Protei ns can be anchored to the membrane by coval entl y l i nked l i pi d chai ns.
c. Protei ns can move l ateral l y across the membrane.
d. Membrane shoul d be about 5 nm thi ck.
e. Transverse moti on of l i pi d mol ecul es can occur occasi onal l y.

Answer: When Singer and Nicolson proposed the fluid mosaic model membrane proteins
anchored by covalently-linked lipids had not been discovered. Thus, b is the exception.
Lipid-anchored proteins would; however, fit nicely into the model.

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137
15. The rate of K
+
transport across bi l ayer membranes reconsti tuted from
di pal mi toyl phosphati dyl chol i ne (DPPC) and monensi n i s approxi matel y the same as that
observed across membranes reconsti tuted from DPPC and cecropin a at 35C. Based on
your readi ng of secti ons 9.8 and 9.9 of thi s chapter, woul d you expect the transport
rates across these two membranes to al so be si mi l ar at 50C? Expl ai n.

Answer: The two ionophores have quite different modes of action. Monensin is a mobile ion
carrier that forms a complex with K
+
, diffuses across the membrane, and releases K
+
. Cecropin
is a channel-forming ionophore. One might expect that, as the temperature increases, the rate
of movement of ion would increase in both cases. However, monensin transport will show a
dramatic change around approximately 41.4C, the phase transition temperature for DPPC. As
the temperature increases from 35C, the membrane will become much more fluid and the rate
of monensin -mediated uptake will increase dramatically. The rate of uptake for cecropin is
expected to increase slightly with increasing temperature.

Questions for Self Study

1. Match the items in the two columns
a. Singer and Nicolson 1. Peripheral protein.
b. Extrinsic protein 2. Lipid bilayer structure.
c. Integral protein 3. Lipid transfer from outside to inside.
d. Liposome 4. Phase change.
e. Micelle 5. Intrinsic protein.
f. Flippase 6. Lipid monolayer structure.
g. Transition temperature 7. Fluid mosaic model.

2. Explain why, for proteins with a single transmembrane segment, the segment is a
hydrophobic helix. Why a helix? Why hydrophobic residues?

3. Give three examples of lipid anchoring motifs.

4. Explain the term critical micelle concentration.

5. One method of lysing bacteria involves the use of the enzyme lysozyme, which hydrolyzes the
glycosidic bond between N-acetylmuramic acid and N-acetylglucosamine. Why isnt a protease a
good alternate choice to disrupt bacterial cell walls?

6. For each of the statements below state if each applies to one or more of the following: passive
diffusion (P), facilitated diffusion (F), and active transport (A).
a. Can only move down a concentration gradient. .
b. Is expected to transport L-amino acid and D-amino acid at the same rate. .
c. Can be saturated. .
d. Can occur in both directions across a biological membrane. .
e. Can be used to concentrate substances. .
f. Movement is coupled to exergonic process. .
g. Glucose transporter in erythrocytes. .
h. Rate is linearly proportional to concentration difference. .
i. Movement across biological membrane dependent on lipid solubility. .
j. Sodium pump. .

7. Match the active transport system with an appropriate function.
a. Na
+
,K
+
-ATPase 1. Acidifies membrane bound compartments.
b. Ca
2+
-ATPase 2. Transports a host of cytotoxic drugs.
c. H
+
,K
+
-ATPase 3. Resets levels of important second message after stimulation.
d. Vacuolar ATPase 4. Electrogenic pump inhibited by cardiac glycosides.
e. MDR ATPase 5. Responsible for production of the largest concentration gradient
known in eukaryotic cells.

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138
8. What is a symport? Antiport? How can they be used to move a substance against its
concentration gradient?

9. Bacteriorhodopsin and halorhodopsin are active transport proteins for the movement of
protons and chloride ions respectively. What energy source do they use to support ion
pumping?

10. For the phosphoenolpyruvate: glucose phosphotransferase system how is glycolysis linked
to glucose uptake?

11. What is the difference between a carrier ionophore and a channel-forming ionophore?

Answers

1. a./7; b./1; c./5; d./2; e./6; f./3; g./4.

2. The hydrogen bonding groups in the peptide bond are all involved in hydrogen bonds in a
helix. Hydrophobic residues can interact with the hydrophobic interior of membranes through
hydrophobic interactions.

3. Amide-linked myristoyl anchors, thioester-linked fatty acyl anchors, thioether-linked prenyl
anchors, and amide-linked glycosyl phosphatidylinositol anchors.

4. The critical micelle concentration is that concentration of lipid at which micelle formation is
supported. Concentrations of lipid below the critical micelle concentration do not form micelles.
Lipid solutions whose concentration is greater than the critical micelle concentration contain
micelles in equilibrium with free lipid molecules. The concentration of the free lipid is equal to
the critical micelle concentration.

5. The presence of D-amino acids, the unique linkage of amino acid groups, and the defined
amino acid composition all make the peptide portion of bacterial cell walls difficult to hydrolyze
by proteases.

6. a./P and F; b./P; c./F and A; d./P and F; e./A; f./A; g./F; h./P; i./P; j./A.

7. a./4; b./3; c./5; d./1; e./2.

8. A symport is a transport system that couples movement of two substances in the same
direction. An antiport couples the movement of two substances in opposite directions. They can
be used to move substances against a concentration gradient if transport of the coupled
substance is down a concentration gradient. In this case the energy of the concentration
gradient of the co-transported substance is used to drive uptake.

9. Light.

10. One of the intermediates in glycolysis, PEP, is used as a phosphoryl group donor to
phosphorylate glucose. Glucose phosphorylation is coupled to glucose uptake by cells.

11. Ionophores are substances that can transport ions across a biological membrane. Carrier
ionophores form a lipid-soluble complex with the ion and the complex diffuses from one side of a
membrane to the other. A channel-forming ionophore is a lipid soluble compound that can
dissolve in biological membranes and spans the membrane with a channel or pore through
which an ion diffuses.

Additional Problems

1. The transport properties of two potassium ionophores were being studied in synthetic lipid
bilayers with a phase transition temperature of 50C. Ionophore X transports potassium at a
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139
rate proportional to temperature from 20C to 70C. In contrast, ionophore Y transports
potassium very well above 60C; however, from 60C to 40C the rate of transport falls off
precipitously to very low values below 40C. Based on this information, can you suggest modes
of action for these two ionophores?

2. For integral membrane proteins, -helices play an important role in anchoring the protein to
the membrane. Explain why a helix is a thermodynamically stable structural element to embed
in a membrane.

3. How might you expect helical wheel plots of -helical segments from the following proteins to
differ: (a) a typical globular protein, (b) an integral membrane protein with a single
transmembrane segment, and (c) an integral membrane protein with several -helices forming a
channel through which a water-soluble compound is transported?

4. Explain how soaps and detergents help to remove water insoluble substances.

5. Explain how lipid membrane asymmetry might arise in a natural membrane through action
of a flippase that does not couple lipid movement to another thermodynamically favorable
process (like ATP hydrolysis).

6. Compare the expected rate of passive diffusion across a cell membrane for the following list of
compounds: glyceraldehyde, glyceraldehyde-3-phosphate, erythrose, ribulose, ribose-5-
phosphate, D-glucose, L-glucose.

7. Activity of the sodium pump results in the net movement of a positive charge across the
membrane. How does this lead to a change in the electrical potential of the membrane?

8. Would you expect proton pumps to be capable of creating large proton gradients if the
pumps operated by an electrogenic mechanism? Explain.

9. Construct a helical wheel plot of melittin, whose amino acid sequence is:
Gly-Ile-Gly-Ala-Val-Leu-Lys-Val-Leu-Thr-Thr-Gly-Leu-Pro-Ala-Leu-Ile-Ser-Trp-Ile-Lys-Arg-Lys-
Arg-Gln-Gln-NH2. Assume that this peptide forms an -helix and comment on the structure.



Abbreviated Answers

1. Ionophore X may be a channel-forming ionophore, perhaps like the antibiotic gramicidin.
Channel-forming ionophores span the membrane forming a channel through which an ion can
diffuse across the membrane. Ionophore Y may be a carrier-ionophore. Carrier-ionophores
form a complex with the ion to be transported. This complex diffuses across the membrane and
dissociates, releasing the ion on the opposite side of the membrane. Valinomycin is an example
of a potassium ionophore of this type.

2. The hydrophobic interior of membranes excludes polar compounds. The peptide bond is a
polar bond with both hydrogen bond donor and acceptor groups that would normally be
excluded from membranes. However, in a helical conformation the donors and acceptors are
involved in intrachain hydrogen bonds making a helix a stable conformation even in a
hydrophobic environment.

3. In globular proteins containing -helices, the helices often contribute to the hydrophobic core
of the protein with hydrophobic amino acid residues located along one face of the helix. Integral
membrane proteins, with a single stretch of -helix responsible for anchoring the protein into the
membrane, have a helix composed of hydrophobic amino acids. For integral proteins anchored
by several helices, the helices are often amphipathic with both a hydrophobic surface and a
hydrophilic surface. The hydrophobic surfaces contact the fatty acid side chains of the
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.
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140
membrane lipid component, whereas the hydrophilic surfaces face inward and may form a pore
though which hydrophilic substances diffuse.

4. Soaps and detergents form micelles in solution. The interior of the micelles is a hydrophobic
environment into which nonpolar molecules may dissolve.

5. Energy must be expended to produce an asymmetric distribution of lipids. In the case of a
flippase that simple equilibrates lipids in response to a concentration gradient of free lipids, lipid
asymmetry might arise if lipids preferentially interact with a membrane protein.

6. For passive diffusion, the flux across the membrane is linearly dependent on the permeability
coefficient given by P = (KD)/x, where K is the partition coefficient, D the diffusion coefficient,
and x the membrane thickness. All of the compounds are water soluble and are expected to
have relatively low partition coefficients, decreasing in value in going from glyceraldehyde to
glucose. The presence of phosphate groups on two of the compounds will decrease diffusion
relative to the unphosphorylated compounds because of an increase in size (leading to a
decrease in D) and a decrease in K. A difference in diffusion rates is not expected between the
glucose isomers because diffusion does not involve molecular recognition.

7. There are two ways of looking at this. The net movement of a positive charge gives rise to an
imbalance of charge across the membrane, resulting in an electrical potential. Alternatively, the
movement of charge across the membrane represents a current. Current flowing across the
resistance of the membrane will produce a voltage change.

8. No. If the pumps were electrogenic a electrochemical gradient would be formed with an
electrical potential component and a chemical gradient and this would require considerably
more energy than just a chemical gradient.

9. The helical wheel plot of melittin in an -helical conformation is shown below. One face of the
helix is lined with hydrophobic amino acids, and a cluster of basic amino acids is found at the
C-terminus. A proline residue is positioned approximately in the middle of the helix.

60
210
30
Gln 26, Val 8
Gly 1

0
90
120
150
180
240
270
300
330
Ile 2, Ile 20
Gly 3,
Lys 21
Arg 22, Ala 4
Val 5, Lys 23
Leu 6, Arg 24
Gln 25, Lys 7
Leu 9
Thr 10
Thr 11
Gly 12
Leu 13
Pro 14
Ala 15
Leu 16
Ile 17
Ser 18
Trp 19
Polar face



Summary
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141

Lipids form a variety of structures spontaneously in solution, including monolayers, micelles
and bilayer structures. Lipid bilayers have a polar surface, composed of charged or neutral lipid
head groups, and a nonpolar interior, composed of hydrophobic lipid chains. The fluid mosaic
model of membrane structure, proposed by Singer and Nicholson, pictures the lipid bilayer as a
fluid, dynamic matrix, with lipids and proteins able to undergo free, rapid lateral motion. A
variety of experiments has now confirmed the essential features of the fluid mosaic model.
The fatty acid chains in membrane lipids are oriented roughly perpendicular to the bilayer
plane, and this ordering is more pronounced near the bilayer surface. As one proceeds into the
bilayer interior, the ordering of the lipid chains decreases, so that the interior is a highly fluid
environment. Transverse motion of lipids and proteins is very slow. Different lipid classes show
different distributions between the inner and outer monolayers of the membrane bilayer.
Proteins are asymmetrically distributed between the two faces of the bilayer, allowing a variety of
vectorial (i.e., directionally dependent) functions including transport processes. Lateral
asymmetries also exist in membranes, with proteins and lipids able to arrange themselves in
clusters or aggregates important to cell function. Lipids in membranes exhibit dramatic,
cooperative changes of state at characteristic temperatures. Such phase transitions between the
solid, gel-like state at lower temperatures and the fluid, liquid-crystalline state at higher
temperatures, are sensitive to the lipid composition and to the presence of proteins, and may be
important in a host of biological functions.
Membrane proteins are of three fundamental types. Peripheral proteins form ionic
interactions or hydrogen bonds with the surface of the lipid bilayer. Integral proteins intercalate
into the lipid bilayer and are strongly associated with the membrane. The lipid anchored
proteins attach to membranes via covalently linked lipid moieties. Peripheral proteins can be
extracted with high salt, EDTA or urea, while integral proteins can only be removed with organic
solvents or detergents. Detergents are amphipathic molecules, with both polar and nonpolar
moieties, and function by intercalating into the membrane and solubilizing lipids and proteins.
At the critical micelle concentration (CMC), detergents spontaneously form micelles and become
much more effective solubilizing agents.
Integral membrane proteins take on a variety of conformations in membranes. Proteins such
as glycophorin of the erythrocyte membrane have a single hydrophobic -helix extending across
the bilayer, with hydrophilic segments extending on either side of the lipid bilayer. Other
proteins, such as bacteriorhodopsin of the purple patches of Halobacterium halobium, traverse
the bilayer several times, with six or more hydrophobic alpha helices spanning the bilayer.
These latter proteins are often involved in membrane transport activities and other processes
that require a substantial portion of the peptide to be imbedded in the membrane.
Four different types of lipid anchoring motifs for membrane proteins have been found to date,
including amide-linked myristic acid anchors, thioester-linked fatty acyl anchors, thioether-
linked prenyl anchors, and amide-linked glycosyl-phosphatidylinositol anchors. Amide-linked
myristic acid (14:0) proteins the fatty acid is attached in amide linkage to the -amino group of
N-terminal glycine. In thioester- and thioether-linked proteins lipids are attached to cysteine
residues. Ether-linked lipids are long-chain polyisoprenoids. In GPI
(glycosylphosphatidylinositol) anchors the C-terminus of the target protein is linked via
phosphoethanolamine to a mannose residue on an oligosaccharide attached to
phosphatidylinositol.
Transport processes are important to all life forms. The acquisition of nutrients, the
elimination of waste materials and the generation of concentration gradients vital to nerve
impulse transmission and the normal function of brain, heart, kidneys and other organs all
depend on membrane transport systems. All transport processes are mediated by transport
proteins, which may function either as channels or carriers. The three classes of transport are
passive diffusion, facilitated diffusion and active transport. In passive diffusion, the transported
species moves across the membrane in the thermodynamically favored direction without the
assistance of a specific transport system. Analogous to Brownian motion, passive diffusion is in
essence an entropic process. The rate of flow of an uncharged molecule depends upon
concentration and the permeability coefficient of the molecule. For charged species, the charge
of the molecule and the electrical potential difference also affect transport. In facilitated
diffusion, the transported species moves according to its thermodynamic potential, but with the
help of a specific transport system. Facilitated diffusion systems display saturation behavior.
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142
The glucose transporter and the anion transporter of erythrocytes are both facilitated diffusion
systems.
Active transport systems use energy input to drive a transported species against its
thermodynamic potential. The most common energy input is ATP hydrolysis, but light energy
and the energy stored in ion gradients may also be used. All active transport systems are
energy-coupling devices. Na,K-ATPase, which transport Na
+
ions out of cells and transports K
+

ions into the cells, is an active transport system. Na,K-ATPase consists of a 120 kD subunit
and a 35 kD subunit. The enzyme mechanism involves an aspartyl phosphate intermediate.
Na,K-ATPase is strongly and specifically inhibited by cardiac glycosides such as ouabain.
Calcium transport across the sarcoplasmic reticulum membrane is mediated by Ca-ATPase, an
enzyme that is homologous to Na,K-ATPase. The gastric H,K-ATPase likewise transports protons
across the membrane of stomach mucosal cells, generating the high concentrations of acid in
the stomach that are essential to digestion of food. The H,K-ATPase is homologous to Na,K-
ATPase and Ca-ATPase. Proton pumps in osteoclasts enable these cells to degrade the mineral
matrix of bone during the remodeling and reconstruction of bone tissue. ATPases also transport
peptides and drugs. Yeast -factor is transported out of yeast cells by a 1290-residue transport
protein that consists of two identical halves formed from a gene duplication. An analogous
transport protein known as the multidrug resistance (MDR) ATPase actively transports a wide
spectrum of drugs out of human cells. This transport system is induced by the chronic
administration of drugs (in cancer chemotherapy, for example). The yeast -factor transporter
and the MDR ATPase are two members of a superfamily of prokaryotic and eukaryotic transport
proteins.
Bacteriorodopsin (bR) is a light-driven proton transport system from Halobacterium halobium.
The characteristic purple color of this transport protein arises from a molecule of retinal
covalently bound in a Schiff base linkage with the -amino group of Lys-216 on the protein. bR
is a 26 kD transmembrane protein that packs so densely in the membrane that it naturally
forms a two-dimensional crystal. The structure consists of seven transmembrane helical protein
segments, with the retinal moiety lying parallel to the membrane plane, about 10 below the
extracellular surface of the membrane. The mechanism of proton transport involves conversion
of the retinal chromophore from the all-trans configuration to the 13-cis configuration upon light
absorption. An analogous transport protein, halorhodopsin, mediates light-driven anion
transport across the H. halobium membrane.
Secondary active transport systems use the ion and proton gradients established by primary
active transport systems to transport amino acids, sugars and other species in certain cells.
Most of these operate as symport systems, with the ion or proton and the transported amino
acid or sugar moving in the same direction. The lactose permease of E. coli is a lactose/H
+

symport system, which actively transports lactose into E. coli cells, deriving energy from the
proton-motive force across the bacterial membrane.
In addition to the specific systems described above, several rather nonspecific systems also
carry out transport processes. For example, Gram-negative bacteria enable the transport of
small molecule nutrients and certain other molecules through the outer membrane via porins,
which form large, non-specific pores in the outer membrane. Certain porins are specif ic, such
as LamB and Tsx of E. coli and porins P and D1 of P. aeruginosa, which possess specific binding
sites for maltose and related oligosaccharides, nucleosides, anions and glucose, respectively.
Gap junctions permit the passive flow of small molecules between animal cells. Such junctions
connect the cells metabolically, providing a means of chemical transfer and communication.
Gap junctions also provide a means for transport of nutrients to cells disconnected from the
circulatory system, such as the lens cells of the eye. Gap junctions are formed from hexameric
arrays of a single 32 kD protein. The subunits are tilted with respect to the axis of the hexamer
and the plane of the membrane. Gap junctions open and close via a cooperative sliding, twisting
motion, with a localized conformation change at the cytoplasmic end of the channel.
Several small molecules produced by microorganisms and referred to as ionophore antibiotics
facilitate ion transport across membranes. These ionophores may act either as channels or as
mobile carriers. Valinomycin from Streptomyces is a mobile carrier. It is a cyclic structure
containing 12 units from four different residues. In the valinomycin-K
+

complex, polar groups of
the valinomycin structure face the center of the ring structure, coordinating K
+
, and the
nonpolar side chains are directed outward from the ring, where they interact favorably with the
nonpolar interior of the membrane bilayer. Gramicidin from Bacillus brevis is a channel-forming
Chapter 9
.
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143
ionophore. It is a linear peptide formed from both L- and D-amino acids, and it forms a head-to-
head helical dimer in lipid membranes. The helix creates an ion channel through the bilayer
membrane. Many other peptides form transmembrane channels, including melittin from bee
venom and the cecropins from Hyalophora cecropia, the cecropia moth. These transmembrane
helical channels are amphipathic, with polar residues clustered on one face of the helix and
nonpolar residues elsewhere. In the membrane, the polar residues cluster to form an ion
channel, leaving the nonpolar residues to interact with the hydrophobic interior of the bilayer
membrane.