SB-BB

Passage 4

The majority of dietary vitamin A is stored in the liver and is then transported to several target organs as retinol by its carrier protein, retinol binding protein (RBP). In the liver, retinol forms a complex with RBP known as holo-RBP. STRA6 is the membrane receptor for RBP; STRA6-mediated retinol uptake is specific to RBP and uses a transport mechanism distinct from known transport mechanisms. Holo-RBP binds to STRA6-transfected cells with high affinity (Kd = 59 nM). Only one retinol is provided to its receptor at a time, and RBP remains outside of the cell. Endocytosis inhibitors do not prevent STRA6-mediated retinol uptake. Upon uptake from holo-RBP, retinol is converted into retinyl esters by lecithin retinol acyltransferase (LRAT) or stored in the cell by cellular retinol binding protein I (CRBP-I).

Researchers assayed the time-dependent uptake of radiolabeled retinol (3H-retinol) in untransfected cells (control) and cells transfected with STRA6 alone, STRA6 and LRAT, and STRA6 and CRBP-I (Figure 1).

Figure 1 Time-dependent uptake of 3H-retinol in control and transfected cells (Note: The activity of STRA6/LRAT cells is defined as 100%.)

The researchers developed an assay to measure STRA6-mediated retinol release from holo-RBP. In this assay, different concentrations of holo-RBP were added (at time 0) to membranes expressing STRA6 or STRA6/LRAT. Retinol fluorescence, which is enhanced when retinol is bound to RBP, was measured over time (Figure 2). STRA6 and LRAT levels were the same in each reaction.

Figure 2 Retinol fluorescence assay in untransfected cells and cells transfected with STRA6 and STRA6/LRAT (Note: Retinol fluorescence at each time point of each reaction is normalized to its own value at 0 min which is defined as 100%.)

Adapted from R. Kawagushi et al., “A Membrane Receptor for Retinol Binding Protein Mediates Cellular Uptake of Vitamin A,” Science ©2007 American Association for the Advancement of Science; R. Kawaguchi, J. Yu, M. Ter-Stepanian, M. Zhong, G. Cheng, et al. ACS Chemical Biology ©2011 American Chemical Society.

Questions

24. Retinol uptake was measured in untransfected and STRA6-transfected cells in the presence of retinol bound to either RBP or bovine serum albumin (BSA). Uptake of retinol bound to RBP was also measured in cells that were cotransfected with a small inhibitor RNA (siRNA) targeting STRA6. Which graphic shows the expected result of this experiment?

25. Which statement is supported by the data shown in Figure 1?

  1. STRA6 activity is associated with the CRBP-I intracellular protein, but not the LRAT intracellular protein.
  2. STRA6-mediated retinol uptake in cells is only dependent on the ability to bind retinol.
  3. Neither LRAT nor CRBP-I are absolutely required for intracellular retinol uptake.
  4. Release of retinol from holo-RBP is most effective in the STRA6 only condition.

26. What is the dependent variable from the experiment shown in Figure 2?

  1. STRA6 or STRA6/LRAT condition
  2. Retinol fluorescence
  3. Time duration of assay
  4. Untransfected cell membranes

27. Which statement regarding STRA6-mediated retinol uptake is best supported by the passage?

  1. Retinol uptake activity by STRA6 is limited without the presence of intracellular binding proteins.
  2. The expression of STRA6 is likely to be higher in the liver than in target organs.
  3. Retinol uptake activity by STRA6 is accelerated when retinol is stored as intracellular retinol rather than as retinyl esters.
  4. Binding of holo-RBP to STRA6 is likely to have a Hill coefficient greater than 1.

28. From the data shown in Figure 2, which statement best explains the relationship between STRA6 and holo-RBP concentration in retinol release?

  1. STRA6-catalyzed retinol release increases as the holo-RBP to STRA6 molar ratio increases.
  2. Retinol release in the STRA6 alone condition is not affected when the holo-RBP to STRA6 molar ratio changes.
  3. STRA6 becomes more dependent on LRAT for retinol release when the holo-RBP to STRA6 molar ratio increases.
  4. Retinol release is only dependent on the presence of holo-RBP.

29. The organ in which the holo-RBP complex forms also functions to:

  1. store bile.
  2. secrete glucagon.
  3. produce hydrochloric acid.
  4. detoxify drugs.

30. In a species of beetle, red body color is dominant to brown. Two red beetles are crossed and produce 31 red and 9 brown offspring (F1 generation). If two red F1 beetles are crossed, what is the probability that both red and brown beetles will appear in the F2 generation? (Note: Assume Mendelian inheritance patterns.)

  1. 4/9
  2. 1/2
  3. 2/3
  4. 3/4

31. The diagram shows the size and position of the exons (numbered) and introns (lines) of a gene that codes for hypothetical Protein X, which can exist as two isoforms (either 16 or 17 amino acid residues long).

Which technique can be used to determine if a sample of cells expresses both isoforms?

  1. Synthesize cDNA from Protein X mRNA using primers overlapping exons 1 and 4, followed by gel electrophoresis and band visualization.
  2. Separate Protein X mRNA by gel electrophoresis and visualize band pattern using a DNA probe complementary to exon 3.
  3. Perform a PCR of Protein X genomic DNA using primers overlapping exons 1 and 3, followed by gel electrophoresis and band visualization.
  4. Perform a restriction digest of Protein X genomic DNA using an endonuclease that cuts in the middle of exon 2, followed by gel electrophoresis and band visualization.

32. The kinetic properties of two enzymes are shown in the table. Both enzymes have the same substrate.

Enzyme kcat(s-1) Kd(mM) Hill coefficent Isoelectric point
X 1.5 × 106 10.0 2.25 7.9
Y 2.1 × 106 18.0 1.01 3.3

Based on the data, which statement best describes how Enzyme X differs from Enzyme Y?

  1. The maximal velocity of the reaction catalyzed by Enzyme X is higher than that of the reaction catalyzed by Enzyme Y.
  2. Enzyme X has a higher molecular weight than Enzyme Y.
  3. Enzyme X exhibits cooperativity, whereas the activity of Enzyme Y does not.
  4. Enzyme X has a lower binding affinity for the substrate than Enzyme Y.

33. Rate experiments were performed to determine the effect of an inhibitor on the kinetics of a reaction catalyzed by an enzyme. The results were plotted, as shown.

Based on the data, which statement best describes the interaction between the inhibitor and the enzyme? The inhibitor:

  1. irreversibly binds the enzyme.
  2. can only bind the enzyme after the enzyme has already bound substrate.
  3. binds the enzyme and the enzyme–substrate complex with the same affinity.
  4. prevents the binding of the enzyme to its substrate by occupying the enzyme’s active site.

Answers

Question 24 Solution: The correct answer is D. This is a Biology question that falls under content category “Transmission of genetic information from the gene to the protein.” The answer to this question is D because 3H-retinol uptake should be highest in STRA6-transfected cells when retinol is bound to RBP. The passage states that STRA6-mediated retinol uptake is specific to RBP, so retinol uptake when retinol is bound to BSA should be at control levels. siRNA targeted against STRA6 will reduce STRA6 levels so uptake of retinol bound to RBP should be lower than in the absence of the siRNA. It is a Scientific Reasoning and Problem Solving question because you are asked to predict the results of the experiment detailed in the question.
Question 25 Solution: The correct answer is C. This is a Biochemistry question that falls under content category “Assemblies of molecules, cells, and groups of cells within single cellular and multicellular organisms.” The answer to this question is C because retinol uptake occurred in the STRA6 alone condition above control levels; therefore neither LRAT nor CRBP-I was absolutely required for retinol uptake. It is a Data-based and Statistical Reasoning question because you are asked to interpret the graphs showing how STRA6 affects cellular retinol uptake.
Question 26 Solution: The correct answer is B. This is a Biology question that falls under content category “Assemblies of molecules, cells, and groups of cells within single cellular and multicellular organisms.” The answer to this question is B because the dependent variable is the variable that is being measured in the experiment. It is a Reasoning about the Design and Execution of Research question because it asks you to identify a design component of an experiment.
Question 27 Solution: The correct answer is A. This is a Biochemistry question that falls under content category “Assemblies of molecules, cells, and groups of cells within single cellular and multicellular organisms.” The answer to this question is A because Figure 1 shows that the STRA6 alone condition had lower levels of retinol uptake compared to STRA6 coupled to LRAT or CRBP-I. It is a Data-based and Statistical Reasoning question because you are asked to interpret the figures that show how STRA6 affects cellular retinol uptake and accumulation.
Question 28 Solution: The correct answer is C. This is a Biochemistry question that falls under content category “Assemblies of molecules, cells, and groups of cells within single cellular and multicellular organisms.” The answer to this question is C because retinol fluorescence was similar in STRA6 alone and STRA6/LRAT conditions when the holo-RBP to STRA6 molar ratio was low (0.5 µM holo-RBP). When the membranes were incubated with 4 µM holo-RBP, the retinol fluorescence in the STRA6 alone condition was higher than that in the STRA6/LRAT condition, indicating that as the molar ratio of holo-RBP to STRA6 is increased, STRA6 becomes more dependent on LRAT for retinol release. It is a Data-based and Statistical Reasoning question because you are asked to interpret the figures showing how the concentration of holo-RBP affected retinol release in STRA6- and STRA6/LRAT-expressing membranes.
Question 29 Solution: The correct answer is D. This is a Biology question that falls under content category “Structure and integrative functions of the main organ system.” The answer to this question is D because according to the passage the holo-RBP complex forms in the liver and one of the function of the liver is drug detoxification. It is a Knowledge of Scientific Concepts and Principles question because it asks references functions of an organ of the digestive system.
Question 30 Solution: The correct answer is A. This is a Biology question that falls under the content category “Transmission of heritable information from generation to generation and the processes that increase genetic diversity.” The answer to this question is A because given Mendelian inheritance patterns, a 3:1 ratio of F1 offspring means that the original crossed beetles are both heterozygotes, and the F1 offspring are 25% red (homozygous dominant), 50% red (heterozygous), and 25% brown (homozygous recessive). If two red F1 beetles are crossed and both red and brown beetles appear in the F2 generation, the F1 red beetles that were crossed must both be heterozygotes. The probability that, of the red F1 beetles, both were heterozygous is 2/3 × 2/3, or 4/9 (only red beetles were selected from and 2/3 of the red F1 beetles were heterozygous). It is a Scientific Reasoning and Problem Solving question because you are asked to apply your knowledge of Mendelian genetics to solve a problem.
Question 31 Solution: The correct answer is A. This is a Biology question that falls under the content category “Transmission of genetic information from the gene to the protein.” The answer to this question is A because isoforms are produced through alternative splicing of pre-mRNA. Based upon the lengths of the two isoforms, each isoform must contain at least exons 1 and 4. From that, it can be determined that one consists of exons 1, 2, and 4 (17 residues) and the other of exons 1, 3, and 4 (16 residues). Generating cDNA from mRNA will generate a sequence for each isoform, as splicing will have occurred. Since both isoforms must contain exons 1 and 4, primers overlapping exons 1 and 4 will produce cDNA for both of the isoforms. The band pattern of the gel will identify whether both or only one of the isoforms is expressed. It is a Reasoning about the Design and Execution of Research question because you are asked to identify an experimental design that will provide the necessary data to answer a specific question.
Question 32 Solution: The correct answer is C. This is a Biochemistry question that falls under the content category “Structure and function of proteins and their constituent amino acids.” The answer to this question is C because the data in Table 1 show that Enzyme X has a Hill coefficient greater than 1, which means it exhibits cooperativity. In contrast, Enzyme Y has a Hill coefficient that is essentially 1, which means it does not. It is a Data-based and Statistical Reasoning question because you are asked to interpret experimental data in order to draw a conclusion.
Question 33 Solution: The correct answer is C. This is a Biochemistry question that falls under the content category “Structure and function of proteins and their constituent amino acids.” The answer to this question is C because the data is presented as a Lineweaver–Burk plot, which indicates that the inhibitor is a noncompetitive inhibitor. A characteristic of noncompetitive inhibitors is that they bind the enzyme and the enzyme–substrate complex with the same affinity; Vmax is affected, but KM does not change. It is a Data-based and Statistical Reasoning question because you are asked to interpret experimental data in order to draw a conclusion.