Also, if you are a Biochemistry Major and took
BCHM 317 last semester, please take the following 3 question survey before the
start of the next class:
Survey:
OXIDATION/PHOSPHORYLATION REDUNDANCY
Learning Goals/Objectives for Chapter 9A:
After class and this reading, students will be able to
list energy sources used to move ions/molecules from low to high
concentrations across a concentration gradient;
explain how ATP is used to drive the thermodynamically uphill
movement of Na and K ions by the Na?K ATPase
We have previously discussed how chemical potential energy in the
form of reduced organic molecules can be transduced into the chemical potential energy of
ATP. This ATP can be used to drive reductive biosynthesis and movement (from individual
cells to whole organisms). ATP has two other significant uses in the cell.
Active Transport: Molecules must often move
across membranes against a concentration gradient - from low to high chemical potential -
in a process characterized by a positive DG. As protons could be "pumped" across the inner
mitochondrial membrane against a concentration gradient, powered by the DG associated with electron transport (passing
electrons from NADH to dioxygen), other species can cross membranes against a
concentration gradient - a process called active transport - if coupled
to ATP hydrolysis or the collapse of another gradient. This active transport is
differentiated from facilitated diffusion we studied earlier, which
occurred down a concentration gradient across the membrane. Many such species must be transported into the cell or into
intracellular organelles against a concentration gradient!
Signal Transduction: All cells must know how
to respond to their environment. They must be able to divide, grow, secrete, synthesize,
degrade, differentiate, cease growth, and even die when the appropriate signal is given.
This signal invariably is a molecule which binds to a receptor, typically on the cell
surface. (Exceptions include light transduction in retinal cells when the signal is a
photon, and lipophilic hormones which pass through the membrane.) Binding is followed by
shape changes in transmembrane protein receptors which effectively transmits the signal into
the cytoplasm. We will discuss three main types of signal transduction pathways:
nerve conduction, in which a presynaptic neuron releases a
neurotransmitter causing a postsynaptic neuron to "fire";
signaling at the cell surface which leads to activation of kinases
within the cytoplasm;
apotosis or programmed cell death
We will discuss signal transduction in the final three
sections.
Energy Requirements for Active
Transport.
For active transport to occur, a membrane receptor is required which
recognizes the ligand to be transported. Of major interest to us, however, is the energy
source used to drive the transport against a concentration gradient. The biological world
has adapted to use almost any source of energy available.
Energy released by oxidation: We have already
encountered the active transport of protons driven by oxidative processes. In electron
transport in respiring mitochondria, NADH is oxidized as it passes electrons to a series
of mobile electron carriers (ubiquione, cytochrome C, and eventually dioxygen) using
Complex 1, 3 and 4 in the inner membrane of the mitochondria. Somehow the energy lost in
this thermodynamically favored process was coupled to conformational changes in the
complex which caused protons to be ejected from the matrix into the inner membrane space.
One can imagine a series of conformation-sensitive pKa changes in various side chains in
the complexes which lead in concert to the vectorially discharge of protons.
ATP hydrolysis: One would expect that this
ubiquitous
carrier of free energy would by used to drive active transport. In fact, this is one of
the predominant roles of ATP in the biological world. 70% of all ATP turnover in the brain
is used for the creation and maintenance of a Na and K ion gradient across nerve cell
membranes using the membrane protein Na+/K+ ATPase.
Light: Photosynthetic bacteria have a membrane protein
called bacteriorhodopsin which contains retinal, a conjugated polyene derived from
beta-carotene. It is analogous to the visual pigment protein rhodopsin in retinal cells.
Absorption of light by the retinal induces a conformation changes in the retinal and
protein, which leads to vectorial discharge of protons ;
Collapse of an ion gradient: The
favorable collapse of an ion
gradient can be used to drive the transport of a different species
against a concentration gradient. We have already observed that collapse of a proton
gradient across the inner mitochondria membrane (through FoF1ATPase) can drive the thermodynamically unfavored synthesis of ATP.
Collapse of a proton gradient provides a proton-motive force
which can drive the active transport of sugars. Likewise a sodium-motive
force can drive active transport of metal ions. Since the energy to make the
initial ion gradients usually comes from ATP hydrolysis, ATP indirectly powers the
transport of the other species against a gradient.
Often times, transport of one species
is coupled to transport of another. If the species are charged, a net change in
charge across the membrane may occur. Several terms are used to describe various
types of transport:
symport - two species are cotransported in the same
direction by the same transport protein
antiport - two species are cotransported in opposite
directions by the same transport protein
electrogenic - a net electrical imbalance is generated
across the membrane by symport or antiport of charged species
electroneutral - no net electrical imbalance is
generated across the membrane by symport or antiport of charged species
Figure:
Examples of Transport: Metal Ions
Na/K - These ions are both transported by the
Na/K ATPase. This protein keeps the K+in and Na+out
high compared to their respective concentrations on the other side of the
membrane. The protein
exists in two essential conformations, E1 and E2, depending on the phosphorylation state
of the protein. ATP and 3 Na+ bind to the cytoplasmic domain of the enzyme in
the E1 conformation. In the presence of Na ions, the bound ATP is cleaved in a
nucleophilic atack by an Asp side chain of the protein. (Hence, the protein is a Na+-activated
ATPase. The phosphorylated enzyme changes conformation to the E2 form in which Na+ ions are
now on the outside of the cell membrane, from which they dissociate. The phosphorylated
protein in conformation E2 now binds 2 K+ ions on the outside, which activates
hydrolysis of the Asp-PO3 mixed anhydride link. The dephosphorylated protein is
more stable in the E1 conformation to which it changes as it bring K+ ions into the cell.
This is an example of an electrogenic antiporter. Transport proteins that use this
mechanism of transport are designated as P types, since ATP cleavage is
required and PO43- is covalenty transferred to an Asp
residue from the ATP. P-Type transporters are inhibited by vanadate
(VO43-),
a transition state analog of phosphate. Note: Transport mediated
by P type membrane proteins can, in the lab, be used to drive ATP synthesis.
Detailed kinetic analysis of ATP and vanadate interactions show there are a
low affinity and high affinity site for each on Na/K ATPase. The high
affinity vandate site appears to be the same as the low affinity ATP site,
which suggest that vandate binds tightly to the E2 form of the enzyme.
The low affinity vandate site appears to be the same site (based on
competition assays) as the ATP site, which is probably the E1 form.
Hence vandate binds preferentially to the E2 form would inhibit the
transition to the E1 form. Vanadate also inhibits phosphatases, enymes
that cleaves phosphorylated Ser, Thr, and Tyr - phosphoesters in proteins.
This supports the notion that vanadate binds preferentially to the E2 form,
which has a phosphoanhydride link (Asp-O-phosphate) that is hydrolyzed,
promoting the conversion of E2 back to E1. Vanadate is
probably at transition state analog inhibitor in that it can readily adopt a
trigonal bipyramidal structure, mimicking the transition state for cleavage
of the tetrahedral anhydride bonds of ATP and Asp-O-PO4.
K - In addition to the above mechanism, K ions can be
transported with protons in an electroneutral antiport mechanism by a K+/H+-ATPase found in
stomach cells, which gives rise to a low pH in the lumen of the stomach.
Ca - Calcium levels are very low in cells. Transient
increases are more likely to be detected in a signal transduction pathways than a
transient decrease in high basal or constituitive cytoplasmic levels. Ca2+-ATPase,
homologous to the Na/K-ATPase, removes Ca2+ from the cytoplasm to either the
outside of the cells or into internal organelles. In addition a Na+-Ca2+
exchange protein (an antiporter) transports calcium ions out of the cell using a
sodium-motive potential. Transport of calcium ions
There are also other types. F-type are similar to the F0F1ATPases
and can transport protons against a concentration gradient powered by ATP
breakdown. Notice that this is the opposite role for this enzyme that we
discussed in mitochondrial oxidative phosphorylation. V-type (vacuolar)
are found in the membranes of acidic organelles (like lysosomes) and acidic vesicles within neurons, where
neurotransmitters are stored.
Examples of Transport:
Sugars
Lactose - Lactose can be transported into E. Coli
against a concentration gradient using galactoside permease, one of the proteins encoded
by the lac operon. This protein uses a proton-motive force to pump lactose into the cell.
The proton gradient is created by an electron transport complex in the membrane which is
inhibited by cyanide, reminiscent of the cytochrome C oxidase complex in oxphos.
Driven by oxidation - The proton gradient formed during
aerobic oxidation and photosynthesis in mitochondria and chloroplast, respectively, is
paid for by free energy decreases associated with oxidation of organic molecules.
Driven by ATP cleavage - As mentioned above, protons
are transported into the the lumen of the stomach by a K+-H+ ATPase.
Driven by light - Photosynthetic bacteria have
a membrane
protein called bacteriorhodopsin which contains retinal, a conjugated polyene derived from
beta-carotene. The retinal is covalently attached to the protein through a Schiff base
linkage to an epsilon amino group of Lys (much as pyridoxal phosphate is in
PLP-dependent enzymes). Bacteriorhodopsin is analogous to the visual pigment
protein rhodopsin in retinal cells. Absorption of light by the retinal induces a
conformational changes in the all trans-retinal, which causes an associated conformational
change in bacteriorhodopsin. The
initial state (BR) changes through a series of intermediates (K, L, M, N,
and O). Various side chains and the protonated N of the Schiff base
of retinal change their relative positions with respect to each other, which leads to changes in protonation states of the side chains and
ultimately vectorial discharge of protons through the membrane. As the
M state forms, H+ is moved to the extracellular side of the
membrane (as shown below). Later a H+ is taken up on the
cytoplasmic side (at the Schiff base of the retinal link) leading to
reformation of the BR state. Experiments have been done to trap
the protein in some of these intermediate states. In one (Leuke et al,
1999), a mutant (Asp 96 to Asparagine or D96N) trappped the protein in a
state, MN, that occurs after a H+ has been moved to
the extracellular side but before a compensatory H+ has been
taken up on the cytoplasmic face. The mutation hinders the reuptake of
the proton.
Cystic Fibrosis Transmembrane Conductance
Regulator (CFTR) - This is a member of a family of an ATP-Binding
Cassette or ABC
transporter proteins. The membrane protein has 12 transmembrane
helices. In contrast to other ion transporters which transport a
discrete number of ions (3 sodium and 2 potassium ions, for example), this
changes conformation to form an open pore through which chloride ions flow.
This protein is defective in Cystic Fibrosis.
Multidrug Resistance Transporter - MDR -
This is another example of an ATP-Binding Cassette or ABC transporter.
It acts in a somewhat promiscuous fashion in pumping nonpolar toxic
molecules out of the cell. This would seem quite beneficial to the
organism, unless the toxic molecule is a chemotherapeutic drug used to kill
a tumor cell.
Phospholipid Flippase or Transbilayer amphipath
transporter (TAT) - This is a member
of the P-Type ATPase family which instead of moving ions across the
membrane flips amino lipids (like PE) across leaflets in the bilayer. In an
early chapter we noted that
flip-flop diffusion in liposomes was slow
compared to that in cells, suggesting that the flip-flop diffusion was
catalyzed in the cell. Catalysis requires ATP cleavage and produces
two conformations of the protein. During the conformational change of
the protein, a phospholipid appears to bind to the protein and is flipped to
the other side of the membrane.
The disposition of phosphatidylserine, a negatively charged phospholipid,
between membrane leaflets is especially interesting and import. Almost
all the PS is localized in the inner leaflet. Cells in which PS is
found in the outer leaflet are target for program cell death (apoptosis).
PS in the outer leaflet can also promote blood clotting as clotting factors
are recruited to the surface. It appears that a P-type ATPase is
required. Using gene silencing by RNA interference in C. Elegans,
Darland-Ranson found that onespecific P-type ATPase, TAT-1 out of 6 found in
the organisms had PS flippase activity, which would retain PS in the inner
leaflet. Cells with PS in the outer leaflet were often targets of
phagocytosis, suggesting the phagocytes have receptors that recognize PS.
Cells with PS receptors may also bind and internalize virus, which have
membrane leaflets acquired from infected cells as the virus buds off from
the cells. Such cells might have PS in their outer leaflets since the
infected cells may be in the process of dying through apoptosis, which would
increase PS in the outer leaflet.
As mentioned earlier, one of the biggest problems in medical drug
development is the productions of drugs that can diffuse across the cell
membrane. This requires that the drug be sufficiently nonpolar while at
the same time being sufficiently polar to have reasonable aqueous solubility,
allowing blood transport. Another approach to getting drugs across the
membrane is to modify them to bind to transporters whose normal function is to
move solutes against a concentration gradient across a lipid bilyaer. The
extent of modification of the drug depends on how close the structure of the
drug is in comparison to the normal ligand for the transporter. This
approach has been used by the biotech company
XenoPort, to
develop drugs that can be more readily absorbed by the small intestine, which
has many active transporters designed to move nutrients into cells.
Check-in:
Oxidation/Phosphorylation
Chime:
Bacteriorhodopsin Crystallized From Bicelles
(1KME) 
Pre-Class Questions:
Signal
Transduction: 9A. ATP - Question
Moodle
Online Quiz (PASSWORD PROTECTED):
ACTIVE TRANSPORT
