Biology 263
Cell Biology
Joel Piperberg
March 18, 2011
Second Exam
Page # 1
Name ___________________________________
I. Multiple Choice. Fill in the circle on the test form corresponding to the correct answer.
1. Which technique below is best for determining the precise quantity of radioactive isotope located in a
specimen?
a. c and d
c. liquid scintillation counting
e. ultracentrifugation
b. atomic force microscopy
d. autoradiography
2. Which technique below depends on the ability of a protein like a hormone receptor to bind to its
specific hormone?
a. DEAE-cellulose chromatography
c. gel filtration
e. homogenization
b. selective precipitation
d. affinity chromatography
3. Which technique combines the methods of isoelectric focusing and SDS-PAGE to purify proteins?
a. ion exchange chromatography
d. autoradiography
b. three-dimensional gel electrophoresis
e. two-dimensional gel electrophoresis
c. spectrophotometry
4. The -pleated sheet is stabilized by ________________.
a. H bonds between polar amino acid R groups
b. covalent bonds
c. H bonds between amide (peptide) linkages oriented parallel to the molecule's axis
d. acidobasic combinations
e. H bonds between amide linkages, oriented perpendicular to the molecule's axis
5. Non-denaturing gel electrophoresis has the advantage that it ________________.
a. c and e
b. allows the detection of proteins in a gel by their biological activity
c. separates on the basis of more than one property thus to some degree confusing the results
d. separates solely on the basis of molecular weight
e. separates on the basis of molecular weight and charge
6. A protein applied to a gel moves steadily down the gel and stops before reaching the end while the
current is still on. Why did the protein stop moving?
a. Its charge became positive.
c. It was isometrically affected
e. It is denatured.
b. Its charge became negative. d. Its charge becomes neutral.
7. What kind of polypeptide structure involves interaction within the same chains in the form of H
bonds parallel to the axis of the molecule?
a. primary
b. secondary
c. tertiary
d. -helix
e. b and d
8. Which technique effectively separates proteins on the basis of their size, shape and density and also on
the basis of the medium through which they are moving?
a. ion exchange chromatography
c. non-denaturing gels
e. SDS-PAGE
b. sucrose density gradient centrifugation
d. a and b
9. What bonds are involved in holding together tertiary structure in proteins?
a. glycosidic linkages
c. van der Waals interactions
e. b, c and d
b. hydrophobic interactions
d. ionic bonds
10. The final protein conformation (the 3-D shape) is determined by _________.
Biology 263
Cell Biology
Joel Piperberg
March 18, 2011
Second Exam
Page # 2
Name ___________________________________
a. the interaction between the R groups within the protein's backbone
b. interaction between phospholipid R groups
c. the interaction between adjacent or nearly adjacent regions of the nucleic acid backbone
d. the primary structure
e. all of the above
11. Which property below is typical of helical structure?
a. a high degree of extensibility
b. strength c. flexibility
12. Nucleation is most closely associated with which phenomenon below?
a. renaturation b. protein folding c. transcription d. translation
d. rigidity
e. b and c
e. a and b
13. Which technique could be used for molecular weight determinations in proteins under all
circumstances?
a. gel filtration
c. carboxymethyl (CM) cellulose chromatography
e. SDS-PAGE
b. isoelectric focusing
d. a and e
14. What is the lowest level of polypeptide structure in a protein exhibiting subunit structure?
a. quaternary
b. secondary
c. tertiary
d. -helix
e. primary
15. Some transport proteins convey molecules through a membrane and up a concentration gradient by
binding to the molecules. These transport proteins have some of the properties of enzymes. What
type of transport is coordinated by these proteins?
a. active transport
c. passive diffusion
e. a and d
b. facilitated diffusion
d. carrier-mediated transport
16. Some channel proteins will allow things to pass through at all times. Others allow molecules or
ions to pass through only at certain times. Such a transport protein is called a _____________.
a. rated channel b. gated channel c. gated carrier d. b and e e. integral protein
17. Ouabain is a very specific inhibitor of the active transport of sodium ions out of the cell and is
therefore a valuable tool in studies of membrane transport mechanisms. Which of the following
processes in your own body would you expect to be sensitive to inhibition by ouabain?
a. passive transport of glucose into a muscle cell
b. active transport of dietary phenylalanine across the intestinal mucosa
c. uptake of potassium ions by liver cells
d. active uptake of lactose by the bacteria living in your intestine
e. none of the above
18. _______ is a small, hydrophobic molecule that dissolves in a membrane and increases ion
permeability sometimes with disastrous results for a cell.
a. Penicillin
b. Valinomycin
c. An ionoclast
d. a and c
e. Stillomycin
19. A type of membrane transport that brings molecules into the cell randomly is _________.
a. receptor-mediated endocytosis
c. facilitated diffusion
e. fluid-phase endocytosis
b. a and e
d. a, c and e
20. You are studying the transport of two molecules through the cell membrane. Their transport appears
to be linked and both enter the cell together. If the transport of one is halted so is the other. If an
Biology 263
Cell Biology
Joel Piperberg
March 18, 2011
Second Exam
Page # 3
Name ___________________________________
inhibitor of ATP synthesis is added to the cells containing the transport molecule, there is a cessation
of transport of the two molecules. What words best describe the transport molecule?
a. active antiport b. active symport c. passive symport d. passive antiport e. active uniport
21. One of the most striking features of membranes is exhibited by the distribution of membrane
polysaccharides. This property is called __________.
a. asymmetry
b. diffusion
c. exocytosis
d. asynchrony
e. effilosis
22. Artificial membranes ______________.
a. do not reseal spontaneously if pierced
b. possess many properties in common with natural membranes
c. lack some properties that natural membranes have
d. b and c
e. a, b and c
23. At times, proteins will denature. When they do, they will refold with the aid of special proteins that
provide a "chamber" within which the denatured proteins can refold without interference from the
surrounding cytoplasm. What are these proteins called?
a. ubiquitins b. chaperonins c. molecular chaperones d. b and c e. refoldases
24. Active transport is characterized by _____________.
a. transport against a concentration gradient
b. transport down a concentration gradient
c. the requirement for energy often in the form of ATP hydrolysis
25. What is an electrochemical gradient?
a. a gradient in charge and molecular weight
b. a gradient in molecular weight alone
c. a gradient in concentration
d. a and c
e. b and c
d. a gradient in molecular weight & concentration
e. a gradient in charge and concentration
26. The process by which molecules are secreted from a cell is called _________.
a. exocytosis
b. endocytosis
c. phagocytosis
d. sarcosis
e. meiosis
27. A membrane transport molecule that transports two molecules in the same direction can be called
a(n) _________.
a. uniport
b. antiport
c. symport
d. cotransport system
e. c and d
28. The protein that aids in the formation of the vesicles during receptor-mediated endocytosis is ______.
a. classrin
b. ankyrin
c. trifskelturin
d. clathrin
e. glycophorin
29. The Na+ - K+ pump makes the cell interior ___ because it pumps ____ Na+ out for every ____ K+
pumped in.
a. neutral; 3; 3
b. negative; 2; 3
c. positive; 2; 3
d. negative; 3; 2
e. positive; 3; 2
30. What property of a protein can be determined directly with gel filtration chromatography if the
protein is an oblate spheroid (cigar-shaped)?
a. molecular charge b. molecular weight c. molecular shape d. Stokes' radius
e. b and d
31. You are studying a membrane transport molecule. You have discovered that it transports Cl- and
glucose. You have prepared inside-out and right-side out vesicles. If both of the above substances
are found on the same side of either type of vesicle transport is not seen to occur. If, on the other
Biology 263
Cell Biology
Joel Piperberg
March 18, 2011
Second Exam
Page # 4
Name ___________________________________
hand, glucose is placed inside and Cl- is placed outside the inside-out vesicles, transport takes place.
Which of the following statements about the transport molecule is true?
a. it is an antiport which would work if Cl- were placed inside and glucose were placed outside the
right-side-out vesicles
b. it is a symport that would work if Cl- and glucose were placed outside the right-side-out vesicles
c. it is a symport that would work if Cl- and glucose were placed inside the right-side-out vesicles
d. it is an antiport which would work if Cl- were placed outside and glucose were placed inside the
right-side-out vesicles
e. none of the above
32. What might the cellular basis of a disease called familial hypercholesterolemia (an inherited disease
in which blood cholesterol levels are very high) be?
a. none of the other answers
c. Receptors for LDL bind it too tightly.
e. c and d
b. Receptors for HDL do not bind it.
d. Receptors for LDL are not made.
33. Some membrane transport proteins allow molecules to leave or enter cells by moving down the
concentration gradient by binding to the transport protein. This can occur as long as the molecule to be
transported can bind specifically to the transport molecule. Such a molecule is called a(n) _____ protein.
a. channel
b. a, c and e
c. intermembrane
d. a and e
e. carrier
34. You are purifying a protein. Before you have employed any procedures to purify it, its specific
activity is 300 units/mg protein. After 5 successive purification procedures, the specific activity is
12,000,000 units/mg protein. How much have you purified the protein?
a. 40,000 times
b. 4,000 times
c. 400,000 times
d. 0.00004 times
e. .0004 times
35. If a solution containing the following tripeptides were passed through a column packed with CMcellulose at pH 7, which of them would bind most effectively to the CM-cellulose column? (see drawing
at end of Multiple Choice section for R groups)
a. N - lysine - histidine - arginine - C
d. N - leucine - alanine - glycine - C
b. N - aspartate - glutamate - aspartate - C
e. N - aspartate - phenylalanine - histidine - C
c. N - phenylalanine - aspartate - glycine - C
36. Which technique would be least likely to result in a high degree of purification?
a. differential centrifugation
c. precipitation
e. affinity chromatography
b. ion exchange chromatography
d. homogenization
37. Which technique would be used to detect the differences in conformation seen in DNAs placed in
different chemical environments?
a. X-ray diffraction
d. isoelectric focussing
b. SDS - polyacrylamide gel electrophoresis
e. DEAE- cellulose chromatography
c. spectrophotometry
38. Which molecular component of the lipid bilayer is at least to a small degree amphipathic?
a. extrinsic proteins b. phospholipids c. cerebrosides d. b, c and e e. gangliosides
39. A protein you wish to study is located in the mitochondria of rat liver cells and is readily extractable
from partially purified peroxisomes. It has a highly negative charge at pH 7. You propose to use a series
of techniques to purify the protein. In what order would you use the techniques after you had
homogenized the tissue?
Biology 263
Cell Biology
Joel Piperberg
March 18, 2011
Second Exam
Page # 5
Name ___________________________________
a. sucrose density gradient centrifugation (SDGF)- extraction - isoelectric focussing (IEF) precipitation by ammonium sulfate
b. differential centrifugation - SDGF - extraction - ammonium sulfate precipitation - CM
(carboxymethyl) cellulose chromatography - affinity chromatography
c. ammonium sulfate precipitation - SDGF - extraction - CM cellulose chromatography - IEF - SDGF
d. IEF - affinity chromatography - differential centrifugation - extraction
e. differential centrifugation- SDGF - extraction - ammonium sulfate precipitation - DEAE
(diethylaminoethyl) cellulose chromatography - affinity chromatography
40. Which one of the following molecules will elute last from a gel filtration column? (Assume that all
amino acids have the same molecular weight.)
a. a globular protein composed of 187 amino acids
d. a globular protein with 165 amino acids
b. a very low molecular weight dye
e. a protein consisting of 120 amino acids
c. a cigar-shaped protein of 188 amino acids
41. Which of the techniques below might be used to effect a rapid purification and would be based on
the solubility of the molecule to be purified?
a. sucrose density centrifugation
d. selective precipitation
b. sodium hydroxide precipitation
e. c and d
c. ammonium sulfate precipitation
42. A protein that can be washed off of a cellular membrane by a moderate concentration of a salt
solution is called a(n) ______ protein.
a. transmembrane
b. lipid-anchored
c. a and b
d. peripheral
e. b and d
43. During the summer, some organisms adjust the fluidity of their membranes by __________.
a. increasing phospholipid tail length
d. decreasing phospholipid tail length
b. increasing tail unsaturation
e. increasing tail saturation
c. a and e
44. The effect of ________ on eukaryotic cell membranes is to abolish transition temperature.
a. phospholipids b. cholesterol c. gangliosides d. lipid-anchored proteins e. a and b
45. Red blood cells with a surface area of _______ are lysed and the lipids of their membranes are totally
extracted. These lipids are then spread over the surface of a water tank and the surface area covered is
found to be 900 µ2.
a. 112.5 µ2
b. 1800 µ2
c. 450 µ2
d. 225 µ2
e. 900 µ2
Biology 263
Joel Piperberg
Second Exam
Name ___________________________________
Cell Biology
March 18, 2011
Page # 6
H
CH
2
CH
2
CH
2
N
C
NH
CH
2
NH
arginine
CH 3
CH
2
CH
leucine
CH
O
CH
2
C
O
HO
CH
OH
aspartate
tyrosine
CH
3
CH
valine CH
CH SH
2
cysteine
CH CH S CH
2
methionine
alanine
O
CH
2
CH
2
glutamine
2
NH 2
2
CH
CH OH
2
serine
C
2
OH
O
CH
2
CH
2
CH
2
CH
3
CH 3
OH threonine
2
NH
lysine
H
H
C
C
CH
2
glutamate
3
CH 3
H
glycine
2
phenylalanine
3
C
CH 2 CH 3
CH3 isoleucine
CH
CH
2
2
CH
2
CH
C
2
NH2
asparagine
CH
2
histidine
N proline
H
NH
HN
+
C
H
2
HC
CH
C
CH
tryptophan
2
C
HC
N
H
II. Short Answer. Please be as brief as possible.
1. What is the highest level of structure exhibited by a protein having 12 subunits? (1 point)
2. Why was the Unit Membrane Model of membrane structure eventually rejected? (1 point)
3. Briefly name and define the levels of structure in proteins. What types of bonds are instrumental in
holding each level of structure together? (8 points)
Level of Structure
Definition
Bond(s) Involved
BONUS QUESTIONS - LECTURE-SHORT ANSWER
Biology 263
Cell Biology
Joel Piperberg
March 18, 2011
Second Exam
Page # 7
Name ___________________________________
1. JEOPARDY BONUS QUESTION (Your answer must be in the form of a question). (2 points)
This "giant" ocean dweller has the largest eyes of any creature.
2. What is responsible for breaking disulfide linkages in proteins being subjected to the type of
electrophoresis that allows the direct determination of molecular weight? In that same technique,
another reagent denatures the proteins and coats them with negative charges that will cause them to be
attracted to the positive electrode. What is the name of that reagent? (2 points)
3. You are attempting to isolate mitochondria from liver tissue and the first step is to homogenize the
liver tissue. Why is the homogenization medium kept cold and why is it isotonic? (2 points)
4. You are using a variety of purification techniques to purify a protein and monitoring the specific activity
after each step. After the first purification step, differential centrifugation, the specific activity of your
sample is 50 units of activity/mg of protein. After the second purification technique, selective
precipitation, is applied, you decide not to use that technique again. What result would lead you to make
that decision? (1 point)
5. What is a domain with respect to a protein? (1 point)
6. What procedure allows one to distinguish between an extrinsic membrane protein and an intrinsic
membrane protein? (1 point)
7. Identify the techniques described below? (3 points)
a. separation of proteins on the basis of molecular charge and molecular weight
b. separation of proteins on the basis of their charge alone
c. localization of radiolabel in a cell so that it can be seen in a microscope
8. In isoelectric focusing, what is responsible for changing the charge on proteins as they travel through
the electrophoresis tube? (1 point)
9. Name a human cell type that is known to do phagocytosis. It can ingest 25% of its volume in a very
short time. (1 point)
Biology 263
Joel Piperberg
Second Exam
Name ___________________________________
Cell Biology
March 18, 2011
Page # 8
10. What is the name for the remnants of a red blood cell that has been placed in a hypotonic solution.
(1 point)
11. What two processes can occur at a coated pit? (2 points)
12. What is the name of the enzyme that along with 125I and hydrogen peroxide is used to label the
proteins that are exposed at the surface of a cell? (1 point)
13. You fuse a human and a mouse cell together and then label the resultant cell with fluorescently
labeled antibodies against human and mouse membrane proteins. The anti-mouse antibodies are labeled
with fluorescein and the anti-human antibodies are labeled with rhodamine. Describe the appearance of
the fused cell immediately after fusion and after about an hour. What does this experiment demonstrate?
(3 points)
14. What is the name for a cell formed by the fusion of two cells from a different species? What virus is
usually used to facilitate the fusion? (2 points)
15. What are two ways in which a cell can limit the mobility of proteins in the membrane? (2 points)
16. You are designing a drug that is supposed to work in the cell cytoplasm. What two properties
should it have to ensure that it will cross the membrane into the cell cytoplasm? (2 points)
17. A vesicle surrounded by a lipid bilayer is produced in a lab. It is smaller than a normal cell.
Chemotherapy agents can be placed inside the vesicle or in the lipid bilayer. It can be directed to a
tumor by tumor-specific antibodies embedded in the lipid bilayer. What is it called? (1 point)
18. Look at the following experimental results obtained by a deep-space probe investigating life on other
plants. Membrane proteins were extracted with SDS and subjected to SDS-polyacrylamide gel
Biology 263
Cell Biology
Joel Piperberg
March 18, 2011
Second Exam
Page # 9
Name ___________________________________
electrophoresis on tube gels. The resultant gels were stained with Coomassie Blue which stains proteins
(a) and Periodic Acid-Schiff (PAS) stain which specifically stains carbohydrates (b).
When whole cells or inside-out vesicles were exposed to lactoperoxidase and 125I, the profile in Graph (c)
was obtained when the gels were cut into 1 mm thick slices and the radioactivity was monitored in the
scintillation counter. In Graph (d), we see the results of exposure to galactose oxidase and 3H borohydride, a process which radioactively labels sugar residues to which galactose oxidase and 3H borohydride are exposed. In Graphs (c) and (d), the dotted line indicates labeling of whole cells and the
solid lines indicate labeling of inside-out vesicles. What kind of membrane proteins are A, B, C and D?
(i. e., On which surface are they exposed? Do they have polysaccharides and on which side of the
membrane are the polysaccharides exposed, if present?) Ignore any preconceived notions about
membrane proteins! Just interpret the data!!! (Please see the following page for the graphs.)
Exposure on intracellular or
extracellular surface?
Glycoprotein?
Which leaflet has the sugar
group (if any)?
A
B
C
D
Which protein has the highest molecular weight?
Which protein, if any, may be involved in membrane transport? (5 points)
10. You prepare sealed and leaky red blood cell ghosts and treat them with trypsin. Gel A shows the
proteins in the membranes before treatment with trypsin. On Gel B, draw where the bands would be
Biology 263
Cell Biology
Joel Piperberg
March 18, 2011
Second Exam
Page # 10
Name ___________________________________
after treatment of the sealed red blood cell ghosts with trypsin. On Gel C, draw where the bands
would be after treatment of the leaky red blood cell ghosts with trypsin. (4 points)
2
Extracellular Surface
1
4
3
5
Cytoplasmic Surface
4
3
1
52
A
Top of Gel
Bottom of Gel
B
Top of Gel
Bottom of Gel
C
Top of Gel
Bottom of Gel
18. A vesicle surrounded by a lipid bilayer is produced in a lab. It is smaller than a normal cell.
Chemotherapy agents can be placed inside the vesicle or in the lipid bilayer. It can be directed to a
tumor by tumor-specific antibodies embedded in the lipid bilayer. What is it called? (1 point)
19.
CM-Cellulos e Chromatography of Androgen Receptors
6000
0.25
cpm
5000
[KCl]
0.2
4000
[KCl]
cpm
0.15
3000
0.1
2000
0.05
1000
B
A
50
40
30
20
10
0
0
0
Fraction Number
a. Which peak in the graph above represents the most positive molecule? (1 point)
b. Look at the graph above. At what KCl concentration does peak B wash off the column? (1 point)
Biology 263
Joel Piperberg
Second Exam
Name ___________________________________
Cell Biology
March 18, 2011
Page # 11
DEAE-Cellulose Chromatography of Glucocorticoid Receptors
4000
0.45
cpm
0.4
3500
[KCl}
0.35
3000
0.3
0.25
2000
0.2
1500
[KCl}
cpm
2500
0.15
1000
0.1
500
B
A
0.05
50
45
40
35
30
25
20
15
10
5
0
0
0
Fraction Number
a. Which peak in the graph above represents the most negative molecule? (1 point)
b. Look at the graph above. At what KCl concentration does peak B wash off the column? (1 point)
Biology 263
Joel Piperberg
Second Exam
Name ___________________________________
Cell Biology
March 18, 2011
Page # 12
Biology 263
Joel Piperberg
Second Exam
Name ___________________________________
Cell Biology
March 18, 2011
Page # 13