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Membrane Transport / Permeability Reading
Membrane proteins & Transport
Ch 7: Membranes / Transport
•Pores/channels, Carriers, Pumps Ch 8: Energy
•Endocytosis & Exocytosis
Passive vs Active
Metabolism
Thermodynamics
• Free Energy
• Activation Energy
Enzymes
ATP coupling
Factors on Enzymatic activity
• Optimal activity graphs
• Michealis Mentin Kinetics
Regulation
Homework
Ch 9 Prequiz
Cell Respiration prequiz
Check your clicker grades
Extra Credit Science Seminar
Estimate tonicity
Before:
Given permeability & 2 solution concentrations
 Determine movement
Now:
given 1 solution concentration and movement
 Determine concentration of 2nd solution
Impermeable to Na+
permeable to Ethanol
The osmotic pressure is:
300 mOsm
a)Inward
b)Outward
c)Neither
300 mOsm
d)Not enough info
RBCs have a tonicity equivalent to 0.9% NaCl
or 0.3 M Na+
(impermeable particle concentration)
Cell crenates when placed in
an unknown mystery solution,
The unknown solution is:
a)0% NaCl
0.9% NaCl
or
300 mM Na+
b)0.9 % NaCl
c)10 % NaCl
d) 300 mM NaCl
Unknown solution
e)0.3 M NaCl
A cell of unknown solute concentration (potato)
Cell loses weight when put in 500 mM Na+.
The cytosol could possibly be:
a)0 mM NaCl equivalent
Unknown
b)500 mM NaCl equivalent
c)1000 mM NaCl equivalent
500 mM NaCl
d)1 M NaCl equivalent
Determine permeability
Given 2 concentrations and movement
 Determine permeability
RBCs - 0.3 M Na+ - impermeable
Cell placed in 300 mM urea
Outside solution is
300 mM Na+
a)Hyper-osmotic
b)Hypo-osmotic
c)Iso-osmotic
300 mM urea
RBCs - 0.3 M Na+ - impermeable
Cell placed in 300 mM urea  Cell explodes
Water moved:
300 mM Na+
a)In
b)Out
c)Neither
300 mM urea
d)unknown
RBCs - 0.3 M Na+ - impermeable
Cell placed in 300 mM urea  Cell explodes
Outside solution must be:
300 mM Na+
a)Hypertonic
b)Hypotonic
c)Isotonic
300 mM urea
d)unknown
RBCs - 0.3 M Na+ - impermeable
Cell placed in 300 mM urea  Cell explodes
Urea is:
300 mM Na+
a)Impermeable
b)Permeable
c)unknown
300 mM urea
What Determines Permeability?
Based on Plasma Membrane
• Separates intracellular fluid (ICF) from extracellular
fluid (ECF)
– Interstitial fluid (IF) = ECF that surrounds cells
Compartmentalization
Permeability: Simple vs Facilitated
Simple: Through the lipid bilayer
Facilitated: Through a membrane protein
Ex: aquaporins = water channels
Simple Diffusion is Passive
= no energy used
Lipids
Water
molecules
CO2 & O2
Simple diffusion: directly through the membrane
What? Simple Based on lipid bilayer – lipids, H2O, O2, CO2
Direction? diffusion Based on Concentration Gradient
Facilitated movement (through protein)
can be passive or active (requires energy)
• Exhibit specificity (selectivity)
• Are saturable; rate is determined by number of
carriers or channels
• Can be regulated in terms of activity and quantity
Facilitated
• Certain lipophobic molecules (e.g., glucose,
amino acids, and ions) use carrier proteins or
channel proteins, both of which:
Specificity
Only K fits
Confromational change
of a K+-carrier
Facilitated Diffusion Using
Channel Proteins
• Aqueous channels formed by transmembrane
proteins selectively transport ions or water
• Two types:
– Leakage channels
• Always open
– Gated channels
• Controlled by chemical or electrical signals
Open channels are free flowing
Facilitated Diffusion
Facilitated = uses membrane protein
diffusion  Conc Gradient
Channel – Free Diffusion
Ex: Na+-channel (leakage)
Or
Acetylcholine-gated Na+-channel
Facilitated Diffusion - Channels
mostly ions
selected on basis of size and charge
500 mM Na+
10 mM Cl-
Na+
Na+/Cl- Channel
50 mM Na+
100 mM ClFree flow - Na doesn’t affect Cl
but depends on conc. gradient
Cl-
A cell has Mg2+-channels. Which of
the following is NOT permeable?
a)Estrogen
b)CO2
c) Ca2+
d)Mg2+
A cell has Na+/K+-channels.
150mM Na+ outside, 10 mM Inside
20mM K+ outside, 80mM Inside
What happens to these ions?
150 Na+
20mM K+
Na+
/K+
a)Na+ influx, K+ influx
b)Na+ outflux, K+ influx
c) Na+ influx, K+ outflux
10 Na+
80mM K+
d)Na+ outflux, K+ outflux
e)None of the above
Facilitated Diffusion: Carrier Proteins
• transport specific polar
molecules (e.g., sugars
and amino acids)
• Binding causes shape
change in carrier
Carrier – set ratios
Ex: Na+-carrier
Na+/Glucose-symport
3Na+/2K+-antiport
Symport or antiports
Glucose
Symport – carrier that
transports substrates in the
same direction
Antiport – carrier that
transports substrates in
opposite directions
Can be sequential or simultaneous
X # of molecules each time
Facilitated Diffusion - Carriers
500 mM Na+
10 mM Cl-
Na+
Na+/Cl- antiport
50 mM Na+
100 mM Cl-
Fixed Ratio: For every Na In one Cl out
& vice versa For every Na Out one Cl In
Depends on conc. gradient
Cl-
Facilitated Diffusion - Carriers
500 mM Na+
10 mM ClNa+/Cl- symport
Na+
Cl-
No movement
Or impaired
Na wants In
Cl wants out
50 mM Na+
100 mM ClFixed Ratio: For every Na In one Cl In
& vice versa For every Na Out one Cl Out
Depends on conc. gradient
A cell has Na+/K+-symports.
150mM Na+ outside, 10 mM Inside
80mM K+ outside, 20mM Inside
What happens in the ABSENCE of ACh?
150 Na+
80mM K+
a)Na+ influx, K+ influx
Na+/K+
symport
b)Na+ outflux, K+ influx
c) Na+ influx, K+ outflux
10 Na+
20mM K+
d)Na+ outflux, K+ outflux
e)None of the above
A cell has Na+/K+-antiports.
150mM Na+ outside, 10 mM Inside
80mM K+ outside, 20mM Inside
150 Na+
80mM K+
a)Na+ influx, K+ influx
Na+/K+
antiport
b)Na+ outflux, K+ influx
c) Na+ influx, K+ outflux
10 Na+
20mM K+
d)Na+ outflux, K+ outflux
e)None of the above
Gated Channels
Receptor
closed
ligand
open
Receptor – anything that can bind another molecule
Can refer to protein as a whole or only a specific region of the protein
Ligand – something that binds to a receptor
Usually a small molecule (ie- not a protein)
A cell has Ach-gated Ca2+-channels.
Which of the following is
permeable in the presence of ACh
a)Na+
Ca2+
b)Clc) Ca2+
d)Mg2+
e) None of the above
A cell has Ach-gated Ca2+-channels.
Which of the following is permeable
in the absence of ACh
a)Na+
Ca2+
b)Clc) Ca2+
d)Mg2+
e) None of the above
A cell has Ach-gated Na+-channels.
150mM Na+ outside, 10 mM Inside
What happens in the presence of ACh?
150 Na+
Na+
a)Na+ influx
b)Na+ outflux
10 Na+
c)No movement
A cell has Ach-gated Na+/K+-channels.
150mM Na+ outside, 10 mM Inside
20mM K+ outside, 80mM Inside
What happens in the presence of ACh?
150 Na+
20mM K+
Na+
/K+
a)Na+ influx, K+ influx
b)Na+ outflux, K+ influx
c) Na+ influx, K+ outflux
10 Na+
80mM K+
d)Na+ outflux, K+ outflux
e)None of the above
A cell has Ach-gated Na+/K+-channels.
150mM Na+ outside, 10 mM Inside
80mM K+ outside, 20mM Inside
What happens in the presence of ACh?
150 Na+
80mM K+
Na+
/K+
a)Na+ influx, K+ influx
b)Na+ outflux, K+ influx
c) Na+ influx, K+ outflux
10 Na+
20mM K+
d)Na+ outflux, K+ outflux
e)None of the above
Active Transport- Primary
PUMP – Like Carrier but uses ATP
Independent of conc. gradient
Active Transport- Primary
Na+-K+ pump
= ATPase = enzyme
Uses ATP to drive
Ion movement
Antiport
Transported substances move in
the opposite direction
Adenosine Triphosphate (ATP)
source of usuable energy = “molecular currency”
The cell’s rechargable battery
Adenine RNA nucleotide
with two additional
phosphate groups
Active Transport- Primary
500 mM Na+
10 mM Cl-
50 mM Na+
100 mM Cl-
Na pump that pumps Na out
Na+
Na pump that pumps Na In
Independent of conc. gradient
Na+
A cell has 3Na+/2K+-pump.
150mM Na+ outside, 10 mM Inside
80mM K+ outside, 20mM Inside
a)Na+ influx, K+ influx
b)Na+ outflux, K+ influx
c)Na+ influx, K+ outflux
d)Na+ outflux, K+ outflux
e)not enough info
A cell has 3Na+/2K+-pump.
Given time, the concentration of
Na and K will be:
a)Na+ high outside, K+ High Outside
b)Na+ high out, K+ High In
c)Na+ high In, K+ High out
d)Na+ high In, K+ High In
Membrane Potential
Typical conc’s – Electrochemical gradient
Secondary active transport use of an exchange
pump (such as the Na+/K+-pump) indirectly to drive the
transport of other solutes
Extracellular fluid
Glucose
Na+-K+
pump
Na+-glucose
symport
transporter
loading
glucose from
ECF
Na+-glucose
symport transporter
releasing glucose
into the cytoplasm
Symport
Transported substances
move in the same direction
Endocytosis – enables large particles and
macromolecules to enter the cell
Receptor mediated Endocytosis – specific macromolecules
Pinocytosis – nonspecific small
Phagocytosis – whole cells / solid particles
PHAGOCYTOSIS
• In phagocytosis a cell
engulfs a particle
• fuses with a lysosome to
digest the particle
1
Extracellular fluid
Plasma
membrane
Cytoplasm
Transport
vesicle
Uncoated
endocytic vesicle
Lysosome
(a)
(b)
Figure 3.12
PINOCYTOSIS
• In pinocytosis, molecules are
taken up when extracellular fluid
is “gulped” into tiny vesicles
RECEPTOR-MEDIATED
ENDOCYTOSIS
• In receptor-mediated endocytosis, binding of
ligands to receptors triggers vesicle formation
• A ligand is any molecule that binds specifically
to a receptor site of another molecule
Exocytosis – moves substance from the cell
interior to the extracellular space
An Introduction to Metabolism
•the totality of an organism’s chemical reactions
The living cell is a miniature chemical factory
where thousands of reactions occur
The cell extracts energy and applies energy to
perform work
All metabolic pathways interconnect
Metabolic Pathways
• A metabolic pathway begins with a specific
molecule and ends with a product
• Each step is catalyzed by a specific enzyme
Enzyme 1
Enzyme 2
B
A
Reaction 1
Starting
molecule
Enzyme 3
C
Reaction 2
D
Reaction 3
Product
The beginning and end are arbitrary and only a tool for
conceptualization
•Requirements for chemical reaction
•Factors affecting enzyme reaction rate
Can this reaction happen?
How fast is the reaction?
We will use:
Cell respiration / photosynthesis as models
Looking at Particular pathways as examples
Energy requirement.
Many reactions need energy to allow it to proceed
Thermodynamics - study of energy transformations
ALL chemical reactions result in a change in
the amount of energy
What is Energy?
• Energy is the capacity to cause change (WORK)
• Energy exists in various forms
Can’t see it – can’t define it “some blob of”
So often measured as ability to do something “work”
Magic!
Physics: work = movement / mechanical
chemistry: work = mechanical + other forms
- energy & work are intertwined
- the energy released from 1 reaction can be used in
another reaction. So is said to “drive” that reaction =
work
Forms of Energy: BONDS HAVE ENERGY
Essentially: Things that can do work
• Kinetic energy is energy associated with motion
• Heat (thermal energy) is kinetic energy associated
with random movement of atoms or molecules
• Potential energy is energy that matter possesses
because of its location or structure
- Gravity, electrical (charge)
• Chemical energy is potential energy available for
release in a chemical reaction (in bonds)
Measured in terms of work
calorie (cal) = energy to raise 1 g water 1°C
1 Calorie (Cal or kcal)
Thermodynamics
The Laws of Energy Transformation
Laws = phenomenon: observations that
have never been observed to be wrong
first law of thermodynamics, the energy of the
universe is constant:
• Energy can be transferred and transformed,
but it cannot be created or destroyed
• The first law is also called the principle of
conservation of energy
The Second Law of Thermodynamics
Every energy transfer or transformation increases
the entropy (= disorder) of the universe
• Heat and entropy related (heat causes molecules to spread)
• During every energy transfer / transformation,
some energy is unusable (often lost as heat)
Everything is falling apart – will happen spontaneously
losing energy / order
(like diffusion – why molecules spread)
A reaction must be losing energy in order for it
to occur
Spontaneity determines if a reaction will occur
∆G, Free-energy (useful work)
• A living system’s free energy is energy that can
be extracted to do work
• Some energy can never be extracted or
recovered (2nd law of thermodynamics)
All reactions that occur
have a negative ∆G
Said to be spontaneous
We conceptually categorize reactions
separately though
Exergonic reaction
release of free energy
spontaneous
Endergonic reaction
absorbs free energy
from its surroundings
 not spontaneous
Always driven by another
reaction (even if only heat)
Catabolic pathways release energy by breaking
down (degredation) complex molecules into
simpler compounds (DISORDER)
• Cellular respiration
breakdown of glucose  CO2 & Energy
Anabolic pathways consume energy to build
complex (synthesis) molecules from simpler ones
(MORE ORDERED)
• The synthesis of protein from amino acids is an
example of anabolism
All reactions that occur
have a negative ∆G
Said to be spontaneous
Measured for each reaction as ∆G°
naught
Standard conditions
Empirically determined
(ie- must be given)
T = 25°C
P = 1 atm
[C]=1 M, all reactants
Water 55.6M
H+ conc = 10-7M (pH=7.0)
Standard conditions are
convenient, but unrealistic
Equilibrium and Metabolism
Under these
conditions
Reactions in a closed
system eventually reach
equilibrium and then do
no work
If you alter the
concentrations
∆G changes
At Equilibrium
Possible ΔG’s as concentration changes
A  B
standard
1M
0M
ΔG° = -5 kcal/mol
More
realistic
0.1M
0.01M
ΔG = -1 kcal/mol
equilibrium
0.1M
0.1M
ΔG = 0 kcal/mol
reversed
0M
1M
ΔG = 2 kcal/mol
No longer ΔG°
Equilibrium and Metabolism
• Cells are not in equilibrium;
cells are open systems
experiencing a constant flow
of materials
open hydroelectric system
∆G < 0
• A defining feature of life is
that metabolism is never at
equilibrium
• A catabolic pathway in a cell
releases free energy in a series
• Closed and open
of reactions
hydroelectric systems
can serve as analogies
Fig. 8-7c
• A catabolic pathway in a cell releases free energy in a
series of reactions
multistep open hydroelectric system
∆G < 0
∆G < 0
∆G < 0
0
∆G
Under standard
conditions
How does cell build up products then?
How can you get an endergonic reaction?
endergonic
A + B
AB
Reaction is coupled to an energy releasing reaction
ATP powers cellular work by
coupling exergonic reactions
to endergonic reactions
• three main kinds of work:
– Chemical
– Transport
– Mechanical
• To do work, cells manage energy resources by
energy coupling, the use of an exergonic
process to drive an endergonic one
• Most energy coupling in cells is mediated by ATP
• Overall, the coupled reactions are exergonic
ATP  ADP + Pi + Energy is exergonic
ΔG° = - 7.3kcal/mol
P
P
P
Adenosine triphosphate (ATP)
H2O
P
i
Inorganic phosphate
+
P
P
Adenosine diphosphate (ADP)
Energy for cellular
work (endergonic,
energy-consuming
processes)
+
Energy
Fig. 8-9
Fig. 8-10
Often we talk
about a reaction as
the overall result
Couple it to ATP
hydrolysis
NH2
Glu
Really 2 reactions
Glutamic
acid
NH3
+
Glu
∆G = +3.4 kcal/mol
Glutamine
Ammonia
P
∆G = -2.5 kcal/mol
+
Glu
ATP
Glu
+ ADP
NH2
P
∆G = -1.4 kcal/mol
Glu
Different reactions but
energy exchanged is
equivalent to adding
them together
Where does rest go?
+
NH3
Glu
+ P
i
Thermodynamics summary
• ALL chemical reactions result energy change
• Measured for each reaction as ∆G°
Changes with concentration, temp, etc.
• ∆G must be negative
for it to… be spontaneous
Standard
conditions
T = 25°C
P = 1 atm
[C]=1 M
pH=7.0
/ happens
as a reaction reaches equilibrium ∆G approaches 0
and can be reversed if concentrations are altered
(pos) +∆G reactions must be coupled to -∆G reactions
• biology - often ATP hydrolysis
• in reality there is a new reaction
ATP  ADP + Pi
ΔG° = -7.3kcal/mol
• Concentration (and other conditions)
affect ∆G
 Reactions must never reach
equilibrium in a living organism
 Must always replenish substrates and
remove products
Which of the following will
occur (spontaneously)?
a)ΔG = +6.5kcal/mol
b)ΔG = +1.5kcal/mol
c)ΔG = +0.1kcal/mol
d)ΔG = -6.5kcal/mol
Which of the following would NOT
work if coupled to ATP hydrolysis?
a)ΔG = -6.5kcal/mol
b)ΔG = +1.5kcal/mol
c)ΔG = +6.1kcal/mol
d)ΔG = +33kcal/mol
Probably
not coupled
Coupling
C  D
ΔG° = 5 kcal/mol
ATP  ADP + Pi
ΔG° = -7.3kcal/mol
C + ATP  D + ADP + Pi ΔG° = -2.3kcal/mol
E  F
ΔG° = 14 kcal/mol
Cannot be coupled to ATP, but maybe another reaction
G  H
ΔG° = -3 kcal/mol
Can be coupled but probably isn’t because it is already spontaneous
Just because it is -ΔG doesn’t mean
it will occur by itself
• Glucose  CO2 + H2O
ΔG = -686 kcal/mol
• Requires activation energy
– enzymes
ΔG calculations are more to assess if a reaction is
feasible under cellular conditions (not standard) or
if another component is likely required (ie- ATP)
Enzymes speed up metabolic
reactions by lowering energy barriers
• A catalyst is a chemical agent that speeds up a
reaction without being consumed by the
reaction
• An enzyme is a catalytic protein
Enzymes
-ase
Hydrolase – breaks apart molecules using H2O
Nuclease – breaks down nucleotides
Protease – breaks down nucleotides
Kinase – adds phosphate group
Notable exception: ribosome
The Activation Energy Barrier
• Every chemical reaction between molecules involves
bond breaking and bond forming
• The initial energy needed to start a chemical reaction is
called the free energy of activation, or activation
energy (EA)
• Activation energy is often supplied in the form of heat
from the surroundings
A
B
C
D
Transition state
A
B
C
D
EA
Reactants
A
B
∆G < O
C
Fig. 8-14
D
Products
Enzymes – by reducing activation energy
Course of
reaction
without
enzyme
EA
without
enzyme
EA with
enzyme
is lower
Reactants
Course of
reaction
with enzyme
∆G is unaffected
by enzyme
Products
Progress of the reaction
Fig. 8-15
Enzyme
Holds the 2
Places them in perfect orientation
Enzymes – by reducing activation energy
• substrate binds to the
active site of the enzyme
• The active site can lower
an EA barrier by
– Orienting substrates
– Straining substrate bonds
– Providing a favorable
microenvironment
– Covalently bonding to the
substrate
active site - Small portion of overall protein
Other parts for shape, supporting role, or regulatory
Carbonic anhydrase
Active site – where substrates bind and
where electron transfer occurs
Carbonic anhydrase
Structural biology
H2O + CO2  H2CO3
biochemistry
High Specificity - Lock & Key Fit Model
substrate binds to the active
site of the enzyme
Lock and Key Fit
Enzyme is rigid structure
vs induced fit
Induced Fit Model
Substrate
Enzyme
Enzyme-substrate
complex
Active site
• Substrate causes a change in shape of enzyme new
• shape brings groups in active site into positions
that enhance their ability to catalyze the reaction
Fig. 8-16
• More accepted model