1) Extremophiles are microorganisms, usually from the Archeal domain, that grow under

a wide variety of extreme conditions (temperature, pressure, pH, salt, etc.). Some of
these extremophiles can even grow in temperatures up to 121C, the sterilization
temperature of an autoclave. Identifying the adaptations that allow these microorganisms
to thrive at such high temperatures is an area of active research both for improving
industrial processes (i.e biofuels) and exobiology. Recent studies of the membrane
composition of found that the major lipids in these organisms are tetraether phospholipids
and diether phospholipids. In the diagram the R represents a variety of head groups.

A) Diagram what a membrane made up of diether phospholipids would look like.

Compare this to eukaryotic cell membranes.

B) Diagram what a membrane made up of tetraether phospholipids would look like.

Compare this to eukaryotic cell membranes.

C) Diagram what a membrane made up of a mixture of diether and tetraether

phospholipids would look like. Compare this to eukaryotic cell membranes.

D) Would you expect the ratio of tetraether to diether phospholipids in the thermophile's
membrane to increase or decrease with increasing temperature? Give two reasons to
justify your answer.

E) Would you expect either of these phospholipids in the thermophiles's membrane to

become more or less saturated as temperature increases? Explain your answer.

F) It turns out that thermophiles don't modify the saturation state of the lipid in response
to temperature. They do however introduce pentacyclic groups into the lipid structure in
response to temperature (structure below). Studies have shown that increasing the
number of these groups in the tetraether phospholipids allows them to pack more tightly
together. Given this information would you expect more or fewer pentacyclic
modifications as temperature increases? Explain your reasoning.

2) If you measure the phase transition temperature (TT) of lipids isolated from E. coli
grown at 37C you find that it is 22C.
A) Draw the phase transition graph marking TT and the growth temperature (TG).

B) Using your graph, do you expect the lipids to be more gel-like or more liquid-like at
the following temperatures:15C, 30C, 43C.

C) You decide to grow E. coli at a range of temperatures for 24 hours, isolate the lipids,
and then measure the phase transition temperature of the lipids. You obtain the following
results:
TG
TT
15C
0C
30C
15C
37C
22C
43C
28C
Why does the phase transition temperature change with growth temperature? Describe 2
mechanisms that would allow this to happen.

3) (11-34) Ion transporters are often linked together as a consequence of their actions.
For instance, cells can raise their intracellular pH by exchanging external Na+ for internal
H+. The change in internal Na+ is then redressed using the Na+/K+ pump.
A) Can these two transporters working together correct the low pH without altering Na
concentration?

B)How does the linked action of these two pumps affect K+ concentration and membrane
potential?

4) (11-38) A rise in intracellular Ca2+ concentration by causes muscles to contract. In

addition to an ATP driven Ca2+ pump on the sarcoplasmic reticulum (a specialized form
of ER), heart muscle cells have an antiporter on the plasma membrane that exchanges
Na+ for Ca2+ as well as the standard Na+/K+ pump.
A) Draw a muscle cell with these three pumps, the direction of ion transport, whether
ATP is used, and note whether the Na+, K+, and Ca2+ concentrations are high or low.

B) Digitalis is a Na+/K+ pump inhibitor used to treat heart disease by making the heart
pump more strongly. Diagram and explain how this works noting the changes in Na+,
K+, and Ca2+ that occur in various compartments.

C) Digitalis in not often used clinically anymore because of its toxicity. What happens to
cells when too much of the drug is taken? Explain the sequence of events that lead to cell
death.

5) (11-39) When membrane proteins are purified and reconstituted into artificial
membrane vesicles, they can be inserted in either their normal orientation or inverted.
This means that a reconstituted pump is pumping into some vesicles and pumping out of
others depending how in inserted. You have developed a sorting procedure that will
allow you to purify vesicles that only have a Na+/K+ pump oriented so that the part that
normally faces the cytosol is on the outside of the vesicle. Predict what would happen
under the following conditions:
A) Solution inside and outside has Na+ and K+, but no ATP
B) Solution inside the vesicles contains both Na+ and K+, the solution outside has both
ions plus ATP.
C) Solution inside has Na+; solution outside has Na+ and ATP
D) The same solution as in B), but before you purified your oriented vesicles.
6) The gastric parietal cell secretes H+ into the stomach to make stomach acid. If you are
constantly vomiting, secretion is ramped up to refill the stomach with acid. This is the
initiating event that can cause a number of problems for people with bulimia. The
diagram describes the pumps and channels in the gastric parietal cell and their placement.
H+ and HCO3- are generated from H2O and CO2 by carbonic anhydrase.
A) Label all the membrane proteins an ion channel, a pump, a symporter, or an antiporter.
B) Is transport by the Cl-/HCO3 transporter energetically favorable or unfavorable? If it
is unfavorable, explain where the energy is coming from to power transport.
C) Explain what would happen to intracellular pH if you treated gastric parietal cells with
a drug that inhibits each of the following:
i) H+/K+ pump
ii) Cl-/HCO3 transporter
iii) carbonic anhydrase

D) Using this diagram, explain what constant vomiting does to blood pH and how that
change occurs.
E) Using the diagram, does vomiting cause any additional changes in electrolytes in the
bloodstream (Na+, K+, Cl-, etc). Explain your reasoning.

7) GLUT1 is a glucose transporter found in many cells. The Km of GLUT1 for D-glucose
is 1.5mM, and the Vmax for GLUT1 transport of glucose is 500 moles transported per
milliliter of cells.
A) Plot velocity of glucose transport vs. extracellular glucose concentration.
B) Is GLUT1 transport of glucose an example of facilitated or passive diffusion across
the plasma membrane? Draw what the other kind of transport would look like on
your graph.
C) How would this graph change if the cells expressed only a partially functional mutant
version of GLUT1 with a higher Km of 7.5mM for glucose?
D) How would this graph change if the cells expressed wildtype GLUT1, but only had
half as many transporters on their surfaces?

E) There is a high free energy barrier to transporting glucose across the plasma
membrane in the absence of GLUT1 and other transporter molecules. Explain the cause
of this free energy barrier.
F) Transporters lower the energetic barrier and facilitate transport across the membrane in
both directions. If transporters are not unidirectional, what causes glucose to be
transported from outside to inside cells instead of vice-versa?
G) When GLUT1 transporters are reconstituted in liposomes and then incubated with
glucose, the levels of glucose transport into and out of the liposomes reaches a state of
equilibrium. Why do erythrocytes in the body that absorb glucose not achieve
equilibrium between import and export of glucose? [Hint: What happens to glucose once
it enters these cells?]
8) You have developed a fluorescence based flippase/scramblase assay. This assay
utilizes 1) phospholipids that have been labeled with the fluorescent molecule NBD and
2) the ability of dithionite to reduce the NBD labeled phospholipid to non-fluorescent
amine products. Flippase/Scramblase activity is measured by the loss of fluorescence as
lipids are transferred to the outer leaflet. The assay is diagrammed below:

You have decided to study a scramblase that is specific

for phosphatidylcholine by making vesicles that have the
scramblase, NBD-phosphatidylcholine and NBDphosphatidylserine. You make the vesicles and at time
A you add dithionite and at time B you add detergent.
The solid line is the data from vesicles without
scramblase and the dotted line is the data with the
scramblase.
A) Why does the lipid-only vesicle decrease to half its
fluorescence when dithionite is added?
B) Why does addition of detergent cause the
fluorescence to go to zero?

C) Explain why the scramblase vesicles end up at a lower fluorescence than pure lipid
vesicles?
D) Explain why the scramblase vesicles dont completely bleach? How could you test
your idea?
9) You have prepared lipid bilayers containing N-palmitoyl ceramide
phosphoethanolamine (PCPE) and compared its membrane behavior with D-erythro-Npalmitoyl-sphingomyelin (PSM). You have focused on membrane fluidity as measured
by fluorescence anisotropy (More
anisotropy means less membrane
movement/fluidity). You measure the
membrane fluidity of each bilayer with and
without cholesterol under a range of
temperatures and get the following graphs.
A) For pure PSM and PCPE bilayers, label
which parts of the curve the membrane is
liquid and which parts is it gel-like.
B) What is the phase transition temperature
for PSM and PCPE?
C) Which lipid (PSM or PCPE) would you
want more of as temperature drops? Explain
your reasoning.
D) When cholesterol is present, the
temperature behavior of both membranes
changes. What changes do you observe and
explain why cholesterol has these effects?