Biology 4410 Spring 2006 Name First Examination Version B This examination consists of two parts, a multiple-choice section and an essay section. Be sure to put your name on both the mark-sense sheet and on the exam booklets. Be sure to mark the exam version in the key ID box in the upper left corner of the mark sense sheet. The exams must be turned in before you leave the room. On the multiple-choice test: Each question has only a single correct answer. When a group of choices is used for more than one question, a choice may be correct for more than one of the questions. Multiple choice. (30 questions; 2 pt each) 1. The following choices describe liposomes composed of glycerol-based phospholipids. Which of the following liposomes will have the lowest melting temperature, as determined by differential scanning calorimetry? (You can assume that the head group and fatty acid chain length are the same for each choice.) Liposomes in which the fatty acid chains are fully saturated. Liposomes with one cis double bond per fatty acid chain. Liposomes with one trans double bond per fatty acid chain. Liposomes with two cis double bonds per fatty acid chain. Liposomes with two trans double bonds per fatty acid chain. 2. A liposome is a phospholipid bilayer formed in a small hole in the partition between two chambers. a sealed vesicle formed by annealing the fragments of an erythrocyte membrane. an erythrocyte ghost. an artificial bilayer in the form of of a spherical vesicle, formed by mixing pure phospholipids in water. a carbohydrate body that is covalently attached to the polar head groups in the glycocalyx of the plasma membrane. 3. In which part of a membrane would cholesterol be found? Covalently attached to the polar head groups of the phospholipids. Embedded in the fatty acid layer in the center of the lipid bilayer. Bound to peripheral protein molecules. Attached to the cytoplasmic domain of integral membrane proteins. Attached to carbohydrate groups in the glycocalyx. Page 1
The following information pertains to questions 4 6. To determine the effect of polar head group composition on the fluidity of a phospholipid bilayer, a biologist performed the following experiment. She made liposomes containing varying proportions of phosphatidyl choline (PC) and phosphatidyl ethanolamine (PE). The PC and PE used in the experiment had identical fatty acid chains (14 carbons long, completely saturated). She analyzed the liposomes using differential scanning calorimetry. The results of the experiments are shown in the graph below. The percent values at the right of each curve represent the molar percent of PC in the liposomes. The graph shows the results of seven different experiments, using liposomes with 100% PC, 95% PC, 90% PC, 80% PC, 50% PC, 20% PC, and 0% PC. (There is a small peak at about 18 C in the 100% PC curve. It is an artifact. Ignore it.) 100%PC:0%PE Rate of Heat Flow 95%PC:5%PE 90%PC:10%PE 80%PC: 20%PE 50%PC: 50%PE 20%PC: 80%PE 0%PC: 100%PE Temperature (C) 4. What effect does varying the proportion of PC and PE have on the lipid bilayer? The proportion of PC and PE has no effect on the bilayer fluidity. Increasing the percentage of PC increases the fluidity (and decreases the melting temperature) of the bilayer. Increasing the percentage of PE increases the fluidity (and decreases the melting temperature) of the bilayer. Increasing the percentage of PC increases the amount of cholesterol in the bilayer. Increasing the percentage of PC increases the amount of sphingomyelin in the bilayer. Page 2
5. Which of the following best explains the effect of varying the PC:PE ratio? The PC has a greater number of double bonds in its fatty acid structure than PE. The PC has a greater number of fatty acid chains in its structure than PE. The PC has a larger polar head group structure than PE. The PC has a greater amount of cholesterol in its structure than PE. The PC has a greater amount of sphingomyelin in its structure than PE. 6. The peak that is seen in the 50% PC curve is wider than the peaks seen at 100% PC and 0% PC. Which of the following is the most likely explanation for this? The packing of the phospholipid molecules is more irregular in the 50% PC liposomes. Therefore, the structure is less crystalline, more amorphous, and has a broader phase transition temperature. In the 50% PC liposomes, all of the PC is located in the outer leaflet of the bilayer, and all of the PE is located in the inner leaflet. Therefore, the outer leaflet melts first, giving an overall broader phase transition temperature. The packing of the phospholipid molecules is more regular in the 50% PC liposomes. Therefore, the structure is more crystalline, less amorphous, and has a broader phase transition temperature. In the 50% PC liposomes, all of the PE is located in the outer leaflet of the bilayer, and all of the PC is located in the inner leaflet. Therefore, the inner leaflet melts first, giving an overall broader phase transition temperature. 7. What role does cholesterol play in membrane structure and function? It acts as a translocator to thread proteins through the membrane. It acts as a channel to mediate ion transport. It broadens the thermal transition temperature, thereby preventing rapid phase transitions in the membrane. It serves as a signal for protein sorting in the trans face of the Golgi apparatus. It is a major component of the glycocalyx. Page 3
The following information pertains to questions 8 12. It is possible to make erythrocyte plasma membrane ghosts by lysing erythrocytes in a hypotonic buffer. Under appropriate conditions, the ghosts can be resealed to form closed vesicles. The resealed ghosts retain the original orientation ( right-side-out ) of the original plasma membrane, so that the outer leaflet of the resealed ghost membrane is the same as the outer leaflet of the erythrocyte plasma membrane. Before the ghosts are resealed, they can be suspended in buffer containing different dissolved substances; therefore, the experimenter can control precisely the composition of the buffer inside the resealed ghost and outside the resealed ghost. The Na + -K + pump is an integral membrane transport protein that simultaneously transports Na + out of the cell and K + into the cell, hydrolyzing ATP in the process. Therefore, this protein must have a Na + binding site, a K + binding site, and an ATP binding site. In a series of ghastly experiments on the erythrocyte Na + -K + pump, the following results were obtained. The ghosts were made in a buffer with appropriate concentrations of Na + and K + to permit the measurement of Na + -K + pump activity. In the table, the ATP and oubain columns tell whether or not the substance was present in the buffer inside the ghosts or outside the ghosts. The last two columns tell whether or not the transport of Na + and K + was observed. Expt. # ATP present inside ghosts? ATP present outside ghosts? Oubain present inside ghosts? Oubain present outside ghosts? Results: Was Na + transported? Results: Was K + transported? 1 Yes Yes No No Yes Yes 2 Yes No No No Yes Yes 3 No Yes No No No No 4 No No No No No No 5 Yes No Yes Yes Yes No 6 Yes No Yes No Yes Yes 7 Yes No No Yes Yes No 8. Which of the following conclusions do the data support? ATP is not required for the transport of Na + and K +. The ATP binding site is located on the cytoplasmic domain of the Na + -K + pump. The ATP binding site is located on the exterior domain of the Na + -K + pump. There are two ATP binding sites, one on the cytoplasmic domain and the other on the exterior domain. Page 4
9. Which set of experiments gives the greatest insight into the location (exterior vs. cytoplasmic side) of the ATP binding site? #6 and #7 #4 and #5 #2 and #3 #1 and #2 #1 and #4 10. Based on these data, what conclusion can you reach about the action of oubain? Oubain interferes with the binding of ATP. Oubain interferes with the binding of Na +. Oubain interferes with the binding of K +. More than one of the above. 11. Where is the oubain binding site located? On the cytoplasmic domain of the Na + -K + pump On the exterior domain of the Na + -K + pump On both the cytoplasmic and interior domains of the Na + -K + pump 12. It is possible to alter the conditions during resealing to produce inverted ( insideout ) ghosts, in which the outside of the ghost is the equivalent of the cytoplasmic face of the erythrocyte membrane. If these experiments were repeated using inverted ghosts, what results would you predict? Since the same type of membrane is used, the results would be identical to the table shown. ATP would have to be present outside the inverted ghosts for Na + and K + transport to occur. If oubain were present inside the inverted ghosts (but not on the outside), then it would exhibit its inhibitory effect. Both and are true statements. The following choices are used for questions 13 17. In each question, identify the structure shown. phosphatidyl choline phosphatidyl ethanolamine phosphatidyl serine sphingomyelin cholesterol Page 5
13. 14. 15. 16. Page 6
17. 18. A black membrane is a phospholipid bilayer formed in a small hole in the partition between two chambers. a sealed vesicle formed by annealing the fragments of an erythrocyte membrane. an erythrocyte ghost. an artificial bilayer in the form of of a spherical vesicle, formed by mixing pure phospholipids in water. a carbohydrate body that is covalently attached to the polar head groups in the glycocalyx of the plasma membrane. 19. Which of the following statements best describes the action of the Na + -K + -ATPase active transport pump? The protein transports three Na + into the cell and two K + out of the cell, hydrolyzing ATP in the process. The protein transports three Na + into the cell, two K + out of the cell, and two glucose molecules out of the cell, hydrolyzing ATP in the process. The protein transports three Na + out of the cell, and two K + into the cell, hydrolyzing ATP in the process. The protein transports three Na + out of the cell, two K + into the cell, and two glucose molecules into the cell, hydrolyzing ATP in the process. Page 7
20. In an experiment that we discussed in class, B-lymphocytes (isolated from mouse spleen) were stained with fluorescein-labeled antibodies specific for a mouse B- lymphocyte protein. What phenomenon or process was demonstrated by these experiments? Fluorescent light-driven pumping (active transport) of H + across the membrane Antibody-mediated rejection of the mouse protein Coupled transport of the fluorescent labels from the exterior to the cytoplasmic side of the plasma membrane. Glycosylation of the plasma membrane proteins Lateral mobility (lateral diffusion) of the plasma membrane proteins. The following choices are used for questions 21 24. Simple diffusion directly across a phospholipid bilayer Facilitated diffusion across a membrane Active transport across a membrane Both and Both and 21. Small hydrophobic and polar molecules (such as O 2, CO 2, N 2, H 2 O, and glycerol) can pass through a membrane by this mechanism, but larger uncharged polar molecules and ions (such as glucose, Na +, and K + ) cannot pass through a membrane by this mechanism. 22. A membrane protein must mediate this process. 23. This process requires the expenditure of the cell s energy, usually in the form of ATP hydrolysis. 24. In this process, the substance being transported moves from a high concentration to a low concentration until equilibrium is reached. 25. Membrane transport proteins that bind to the specific solute being transported and undergo a series of conformational changes to transfer the bound solute are not found in neurons. are carrier proteins. always hydrolyze ATP in the process of transporting the solute. can be voltage-gated, ligand-gated, or mechanically-gated. are peripheral membrane proteins. Page 8
26. An antiport is a channel protein. is a carrier protein that transports only a single type of solute. is a carrier protein that transports two different solutes, each from the same side of the membrane. is a carrier protein that transports two different solutes, one from one side of the membrane and one from the other side of the membrane. is a synthetic carrier of ions (also known as an ionophore) useful as a model for ion transport studies. 27. Which of the following best describes the events of a nerve impulse in a neuron? An electrical signal, in the form of a stream of electrons, is transmitted across conductor molecules bound to carbohydrates in the glycocalyx of the membrane. The action of the Na + -K + -ATPase is reversed, causing it to pump Na + into the cell and K + out of the cell. This causes a small section of the neuron membrane to be briefly depolarized, an effect which is propagated along the length of the neuron in a wave of depolarization. A voltage-gated K + -channel is opened, resulting in a rush of K + from the outside of the cell to the inside of the cell. This causes a small section of the neuron membrane to be briefly depolarized, an effect which is propagated along the length of the neuron in a wave of depolarization. A voltage-gated Na + -channel is opened, resulting in a rush of Na + from the outside of the cell to the inside of the cell. This causes a small section of the neuron membrane to be briefly depolarized, an effect which is propagated along the length of the neuron in a wave of depolarization. A ligand-gated (neurotransmitter-gated) K + -channel is opened, resulting in a rush of K + from the outside of the cell to the inside of the cell. This causes a small section of the neuron membrane to be briefly depolarized, an effect which is propagated along the length of the neuron in a wave of depolarization. The following choices are used for questions 29 31. Band 3 Bacteriorhodopsin Spectrin Na + -K + ATPase Glycophorin 28. This protein consists of two subunits, α and β, that are twisted together. It is attached to microfilaments (actin filaments) on the cytoplasmic side of the erythrocyte membrane. Page 9
29. This protein is an integral protein in the erythrocyte plasma membrane, with a single α-helix forming the transmembrane domain. Its exact function is unclear, although it may help to keep red blood cells from clumping together during circulation. 30. This protein is a multipass membrane protein that catalyzes the coupled transport of Cl - and HCO 3-. Essay 1. Membrane protein mobility and distribution may be restricted to specific domains (regions) of the cell s plasma membrane. For example, the intestinal epithelial cells of mammals have plasma membranes with at least three distinct regions (apical, lateral, basal). The distribution of different proteins in the PM is believed to be of importance in the functions of the cells, such as the transport of glucose from the lumen of the intestine into the bloodstream. There are three integral membrane proteins in mammalian intestinal epithelial cells that play key roles in the glucose transport process (from the lumen of the intestine into the bloodstream). List these proteins, and briefly state what each protein does. (9 pt) Page 10
The model for glucose transport (from the lumen of the intestine into the bloodstream), given in class and in the textbook, requires that the proteins (that you listed in part a.) be located in specific regions of the epithelial cell s PM (apical, lateral, or basal). The alternate hypothesis is that the proteins are distributed uniformly throughout all regions of the PM. Design an experiment to distinguish between these two possibilities. The experiment must be do-able with equipment in the cell biology labs of Kennesaw State University (e.g., no electron microscopes, cyclotrons, deep space probes, phaser pistols, etc.). Be sure to state the specific results that you expect to see in your experiment, with explicit reference to each protein (i.e. be sure to state where each protein is located). (9 pt) Page 11
Some integral membrane proteins exhibit lateral mobility within the membrane, but other proteins have restricted or no mobility. Design an experiment to determine mobility of the intestinal epithelial proteins (same proteins from part a). Be sure to explicitly state the different results you would get if the proteins were mobile or non-mobile. (6 pt) 2. How do integral membrane proteins, peripheral proteins, and lipid-anchored membrane proteins differ from each other? (6 pt) Page 12
3. F ab fragments are antibody fragments containing the antigen binding sites from the original antibody molecules. They are made by treating antibody molecules with the proteolytic enzyme papain. Each F ab fragment contains only one antigen binding site per molecule. In class, we talked about an experiment to demonstrate the lateral mobility of integral membrane proteins by observing patching and capping after treating cells with fluorescently labeled antibody. What results do you predict should be seen if fluorescently-labeled F ab fragments were used instead of whole antibody molecules in the experiment? (2 pt) Would this result (using F ab instead of whole antibody molecules) support, contradict, or have no bearing on the conclusions that we reached in class regarding lateral mobility in the patching and capping experiment? Briefly explain. (2 pt) Page 13
4. Porin (found in the outer membrane of gram-negative bacteria, such as Escherichia coli) and bacteriorhodopsin (found in the purple patch membranes of Halobacterium salinarum) are both integral membrane proteins, yet the secondary structures of their transmembrane domains are very different. Describe, in detail, the structures of the transmembrane domains of porin and bacteriorhodopsin. (6 pt) Page 14