Transcript The MDR ATPase - mustafaaltinisik.org.uk

Biochemistry 2/e - Garrett & Grisham
Chapter 10
Membrane Transport
to accompany
Biochemistry, 2/e
by
Reginald Garrett and Charles Grisham
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Biochemistry 2/e - Garrett & Grisham
Outline
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10.1 Passive Diffusion
10.2 Facilitated Diffusion
10.3 Active Transport
10.4 - 10.6 Transport Driven by ATP, light, etc.
10.7 Group Translocation
10.8 Specialized Membrane Pores
• 10.9 Ionophore Antibiotics
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Passive Diffusion
No special proteins needed
• Transported species simply moves
down its concentration gradient - from
high [c] to low [c]
• Be able to use Eq. 10.1 and 10.2
• High permeability coefficients usually
mean that passive diffusion is not the
whole story
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Facilitated Diffusion
G negative, but proteins assist
• Solutes only move in the
thermodynamically favored direction
• But proteins may "facilitate" transport,
increasing the rates of transport
• Understand plots in Figure 10.3
• Two important distinguising features:
– solute flows only in the favored direction
– transport displays saturation kinetics
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Biochemistry 2/e - Garrett & Grisham
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Biochemistry 2/e - Garrett & Grisham
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Biochemistry 2/e - Garrett & Grisham
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Biochemistry 2/e - Garrett & Grisham
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Active Transport Systems
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Energy input drives transport
Some transport must occur such that
solutes flow against thermodynamic
potential
Energy input drives transport
Energy source and transport machinery
are "coupled"
Energy source may be ATP, light or a
concentration gradient
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The Sodium Pump
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aka Na,K-ATPase
Large protein - 120 kD and 35 kD subunits
Maintains intracellular Na low and K high
Crucial for all organs, but especially for
neural tissue and the brain
ATP hydrolysis drives Na out and K in
Alpha subunit has ten transmembrane
helices with large cytoplasmic domain
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Na,K Transport
• ATP hydrolysis occurs via an E-P
intermediate
• Mechanism involves two enzyme
conformations known as E1 and E2
• Cardiac glycosides inhibit by binding to
outside
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Na,K Transport
• Hypertension involves apparent
inhibition of sodium pump. Inhibition in
cells lining blood
• Vessel walls results in Na,Ca
accumulation
• Studies show this inhibitor to be
ouabain!
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Calcium Transport in Muscle
A process akin to Na,K transport
• Calcium levels in resting muscle cytoplasm are
maintained low by Ca-ATPase - a Ca pump
• Calcium is pumped into the sarcoplasmic
reticulum (SR) by a 110 kD protein that is very
similar to the alpha subunit of Na,K-ATPase
• Aspartyl phosphate E-P intermediate is at Asp351 and Ca-pump also fits the E1-E2 model
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The Gastric H,K-ATPase
The enzyme that keeps the stomach at pH 0.8
• The parietal cells of the gastric mucosa
(lining of the stomach) have an internal
pH of 7.4
• H,K-ATPase pumps protons from these
cells into the stomach to maintain a pH
difference across a single plasma
membrane of 6.6!
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The Gastric H,K-ATPase
• This is the largest concentration
gradient across a membrane in
eukaryotic organisms!
• H,K-ATPase is similar in many respects
to Na,K-ATPase and Ca-ATPase
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Osteoclast Proton Pumps
How your body takes your bones apart!
• Bone material undergoes ongoing remodeling
– osteoclasts tear down bone tissue
– osteoblasts build it back up
• Osteoclasts function by secreting acid into
the space between the osteoclast membrane
and the bone surface - acid dissolves the Caphosphate matrix of the bone
• An ATP-driven proton pump in the membrane
does this!
•
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The MDR ATPase
aka the P-glycoprotein
• Animal cells have a transport system
that is designed to recognize foreign
organic molecules
• This organic molecule pump recognizes
a broad variety of molecules and
transports them out of the cell using the
hydrolytic energy of ATP
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The MDR ATPase
• MDR ATPase is a member of a
"superfamily" of genes/proteins that
appear to have arisen as a "tandem
repeat"
• MDR ATPase defeats efforts of
chemotherapy
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Light-Driven H + Transport
The Bacteriorhodopsin story
• Halobacterium halobium, the salt-loving
bacterium, carries out normal respiration if
O2 and substrates are plentiful
• But when substrates are lacking, it can
survive by using bacteriorhodopsin and
halorhodopsin to capture light energy
• Purple patches of H. halobium are 75% bR
and 25% lipid - a "2D crystal" of bR - ideal
for structural studies
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Bacteriorhodopsin
Protein opsin and retinal chromophore
• Retinal is bound to opsin via a Schiff
base link
• The Schiff base (at Lys-216) can be
protonated, and this site is one of the
sites that participate in H+ transport
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Bacteriorhodopsin
• Lys-216 is buried in the middle of the 7TMS structure of bR, and retinal lies
mostly parallel to the membrane and
between the helices
• Light absorption converts all-trans
retinal to 13-cis configuration - see
Figure 10.22
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Bacteriorhodopsin
The protons visit the aspartates....
• Asp-85 and Asp-96 lie on opposite sides
of a membrane-spanning helix
• These remarkable aspartates have pKa
values around 11! (Why?)
• Protons are driven from Asp-96 to the
Schiff base at Lys-216 to Asp-85 and
out of the cell
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Halorhodopsin
• Halorhodopsin transports Cl - instead of H +
• Halorhodopsin has Lys-242 Schiff base but
no aspartates and no deprotonation of
Schiff base during the transport cycle
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Secondary Active Transport
Transport processes driven by ion
gradients
• Many amino acids and sugars are
accumulated by cells in transport
processes driven by ion gradients
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Secondary Active Transport
• Symport - ion and the amino acid or
sugar are transported in the same
direction across the membrane
• Antiport - ion and transported species
move in opposite directions
• Several examples are described in
Table 10.2
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Group Translocation
The phosphotransferase system (PTS)
• Discovered by Saul Roseman in 1964
• Sugars are phosphorylated from PEP
during transport into E. coli cells
• Four proteins required: EI, HPr, EII, and
EIII
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Group Translocation
• EI and HPr are universal and work for
all sugars
• EII and EIII are specific for each sugar
• Mechanism involves transfer of P from
PEP to EI and then to HPr and then to 2
sites on EIII and then finally
phosphorylation of sugar
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Porins
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Found both in Gram-negative bacteria and in
mitochondrial outer membrane
Porins are pore-forming proteins - 30-50 kD
General or specific - exclusion limits 600-6000
Most arrange in membrane as trimers
High homology between various porins
Porin from Rhodobacter capsulatus has 16stranded beta barrel that traverses the
membrane to form the pore (with eyelet!)
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Why Beta Sheets?
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for membrane proteins??
Genetic economy
Alpha helix requires 21-25 residues per
transmembrane strand
Beta-strand requires only 9-11 residues
per transmembrane strand
Thus, with beta strands , a given amount
of genetic material can make a larger
number of trans-membrane segments
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The Pore-Forming Toxins
• Lethal molecules produced by many
organisms
• They insert themselves into the host cell
plasma membrane
• They kill by collapsing ion gradients,
facilitating entry by toxic agents, or
introducing a harmful catalytic activity
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Colicins
• Produced by E. coli
• Inhibit growth of other bacteria (even
other strains of E. coli)
• Single colicin molecule can kill a host!
• Three domains: translocation (T),
receptor-binding (R), and channelforming (C)
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Clues to Channel Formation
• C-domain: 10-helix bundle, with H8 and
H9 forming a hydrophobic hairpin
• Other helices amphipathic (Fig. 10.30)
• H8 and H9 insert, with others splayed
on the membrane surface
• A transmembrane potential causes the
amphipathic helices to insert!
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Other Pore-Forming Toxins
• Delta endotoxin also possesses a helixbundle and may work the same way
• There are other mechanisms at work in
other toxins
• Hemolysin from Staphylococcus aureus
forms a symmetrical pore
• Aerolysin may form a heptameric pore with each monomer providing 3 beta
strands to a membrane-spanning barrel
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Amphiphilic Helices
form Transmembrane Ion Channels
• Many natural peptides form oligomeric
transmembrane channels
• The peptides form amphiphilic -helices
• Aggregates of these helices form
channels that have a hydrophobic
surface and a polar center
• Melittin (bee venom), magainins (frogs)
and cecropin (from cecropia moths) are
examples
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Amphipathic Helices
• Melittin - bee venom toxin - 26 residues
• Cecropin A - cecropia moths - 37
residues
• Magainin 2 amide - frogs - 23 residues
• See Figure 10.35 to appreciate helical
wheel presentation of the amphipathic
helix
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The Magainin Peptides
• Discovered by Michael Zasloff
• He noticed that incisions on Xenopus
laevis (African clawed frog) healed
without infection, even in bacteria-filled
aquarium water
• He deduced that the frogs produced a
substance that protected them from
infection!
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The Cecropins
• Produced by Hyalophora cecropia (the
cecropia moth - see Figure 10.36)
• Induced when the moth is challenged by
bacterial infections
• These peptides are thought to form helical aggregates in membranes,
creating an ion channel in the center of
the aggregate
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Gap Junctions
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Vital connections for animal cells
Provide metabolic connections
Provide a means of chemical transfer
Provide a means of communication
Permit large number of cells to act in
synchrony
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Gap Junctions
• Hexameric arrays of a single 32 kD
protein
• Subunits are tilted with respect to
central axis
• Pore in center can be opened or closed
by the tilting of the subunits, e.g. as
response to stress
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Ionophore Antibiotics
Mobile carrier or pore (channel)
• How to distinguish? Temperature!
• Pores will not be greatly affected by
temperature, so transport rates are
approximately constant over large
temperature ranges
• Carriers depend on the fluidity of the
membrane, so transport rates are highly
sensitive to temperature, especially near the
phase transition of the membrane lipids
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Valinomycin
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A classic mobile carrier
A depsipeptide - a molecule with both
peptide and ester bonds
Valinomycin is a dodecadepsipeptide
The structure places several carbonyl
oxygens in the center of the ring structure
Potassium and other ions coordinate the
oxygens
Valinomycin-potassium complex diffuses
freely and rapid across membranes
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Selectivity of Valinomycin
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Why?
K + and Rb + bind tightly, but affinities for
Na + and Li + are about a thousand-fold
lower
Radius of the ions is one consideration
Hydration is another - see page 324 for
solvation energies
It "costs more" energetically to desolvate
Na+ and Li+ than K+
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Gramicidin
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A classic channel ionophore
Linear 15-residue peptide - alternating D & L
Structure in organic solvents is double
helical
Structure in water is end-to-end helical
dimer
Unusual helix - 6.3 residues per turn with a
central hole - 0.4 nm or 4 A diameter
• Ions migrate through the central pore
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