Exam 2: Biochemistry

Passage Many lysosomal proteins are transported to the lysosome by the cation-independent mannose 6-phosphate receptor (CI-MPR).  CI-MPR is a transmembrane protein found in the Golgi apparatus and vesicles.  It contains 15 globular domains folded similarly on the luminal side of the membrane, each of which has up to 3 glycosylation sites and 6−12 cysteine residues that aid in folding.The ability of CI-MPR to recognize multiple different ligands was studied by fluorescent microarray analysis.  Microarray analysis was performed by cross-linking candidate ligands such as plasminogen, insulin-like growth factor 2 (IGF-II), and mannose 6-phosphate (M6P), to glass slides.  The slides were then incubated with full-length CI-MPR or recombinantly expressed CI-MPR domains, followed by incubation with an anti−CI-MPR antibody.  A fluorescently labeled antibody that binds the first antibody was then added, and the relative fluorescence levels of each candidate ligand were compared.  Higher fluorescence levels correspond to greater binding.Experiment 1Microarray analysis was carried out on slides containing varying levels of candidate ligands, and binding affinities of each CI-MPR domain for its primary ligand were estimated.  Table 1 shows the results.Experiment 2Domains 3, 5, and 9 bind carbohydrates.  Amino acids in these domains were mutated to determine which are required for binding.  Based on this experiment, scientists concluded that Arg, Glu, and Tyr are required for carbohydrate binding by domains 3, 5, and 9 of CI-MPR.Table 1 Adapted from Bohnsack RN, Song X, Olson LJ, et al. Cation-independent mannose 6-phosphate receptor: a composite of distinct phosphomannosyl binding sites. J Biol Chem. 2009;284(50):35215-26. -Which of the following is a covalent bond that could stabilize the tertiary structure of CI-MPR domains?

Passage Myalgic encephalopathy (ME), or chronic fatigue syndrome (CFS), is a physiological disease that affects 0.15% of the human population.  Compared with healthy individuals, persons with ME experience reduced phospholipid levels, muscular degeneration, urinary urgency, and other symptoms that contribute to a lower quality of life.  Studies in resting subjects have yielded inconsistent data and failed to identify a unique metabolic defect in patients with ME.Analyzing the blood plasma of male and female patients with ME, physicians have observed that male patients display elevated levels of 3-methylhistidine, a marker of protein catabolism.  Researchers expanded this finding by measuring the serum concentration of amino acids in blood samples obtained from nonfasting ME patients and healthy controls.  Amino acids were classified into separate categories according to their metabolic intermediates.Table 1  Classification of Amino Acids in Blood Plasma Figure 1  Serum concentrations of amino acids in patients with ME and healthy controls (HC)Next, researchers hypothesized that the inhibition of pyruvate dehydrogenase (PDH) and impairment of fatty acid oxidation were responsible for the fluctuations in amino acid levels.  PDH function and lipid metabolism were studied in peripheral blood mononuclear cells (PBMCs) by measuring the gene expression of proteins within regulatory pathways.  Researchers monitored the mRNA levels of the inhibitor pyruvate dehydrogenase kinase 1 (PDK1) and of sirtuin 4 (SIRT4), which removes lipoic acid groups from the PDH complex.  They also examined lipid metabolism by measuring the mRNA levels of peroxisome proliferator-activated receptor-α (PPARα), a transcription factor that upregulates the uptake of fatty acids.  The mRNA level of each protein in PBMCs of patients with ME was evaluated against healthy controls, as shown in Figure 2. Figure 2  Gene expression of PDK1, SIRT4, and PPARα in PBMCs Adapted from Fluge Ø, Mella O, Bruland O, et al.. JCI Insight. 2016;1(21):e89376. -If lower phospholipid levels in ME patients are due to inhibition of fatty acid synthesis, researchers would most likely observe which of the following?

Passage Whole grains have been shown to reduce the risk of cardiovascular disorders.  A team of scientists discovered that the compound 5-aminovaleric acid betaine (5-AVAB) was present at high levels in cardiomyocytes of mice given a diet rich in whole grains and decided to investigate its role in metabolism.  Preliminary evidence indicated that 5-AVAB did not alter the citric acid cycle in these cardiomyocytes, but that it did alter fatty acid metabolism.The plasma membrane transport protein organic cation/carnitine transporter 2 (OCTN2) transports extracellular L-carnitine into the cell against its concentration gradient using the energy provided by the simultaneous transport of Na⁺ into the cell.  Cellular L-carnitine is required for the transport of fatty acids from the cytosol into the mitochondria.  Based on the structures of 5-AVAB and L-carnitine (Figure 1), scientists hypothesized that 5-AVAB may interfere with OCTN2-mediated transport of L-carnitine. Figure 1  Structures of L-carnitine and 5-AVABTo test their hypothesis, scientists incubated mouse cardiomyocytes with various concentrations of 5-AVAB for 24 hours.  Cells were then provided with the fatty acid palmitate for energy and were deprived of all other energy sources.  The cardiomyocytes were also given 50 μM L-carnitine (endogenous extracellular concentration) or 2 mM L-carnitine, and the rate of oxygen consumption was monitored (Figure 2). Figure 2  Oxygen consumption rate of cardiomyocytes at various concentrations of 5-AVAB and L-carnitineIn a follow-up experiment, the scientists used siRNA to silence expression of OCTN2 and found that intracellular L-carnitine levels were similarly depleted in siRNA-treated cells as in cells treated with 100 μM 5-AVAB. Adapted from Kärkkäinen O, Tuomainen T, Koistinen V, et al. Whole grain intake associated molecule 5-aminovaleric acid betaine decreases ß-oxidation of fatty acids in mouse cardiomyocytes. Sci Rep. 2018;8(1):13036. -When palmitate is the only energy source, silencing of OCTN2 expression would impair mitochondrial synthesis of which metabolite?

Passage Oxidative stress occurs in cells when reactive oxygen species (ROS) such as H₂O₂, superoxide (O₂⁻), and hydroxyl radicals (^•OH) interact destructively with biological molecules.  Several studies have indicated that histidine and certain peptides that contain histidine can decrease oxidative stress.A common marker of oxidative stress in cells is leakage of the enzyme lactate dehydrogenase (LDH) into the extracellular space.  Researchers incubated cultured rat cells (PC12 cells) with H₂O₂ in the presence of different tripeptides, each containing one alanine, one glycine, and one histidine residue.  Every possible sequence was tested, but the researchers found that the sequence GAH was the most effective in limiting extracellular LDH activity.  After obtaining these preliminary results, the following experiments were conducted.Experiment 1PC12 cells were incubated with or without 5 mM H₂O₂ and with varying concentrations of GAH for 4 hours.  Cells were centrifuged, and the supernatant (ie, extracellular material) was extracted and assayed for LDH activity (Figure 1). Figure 1  Extracellular LDH activity from PC12 cells exposed to H₂O₂ and varying concentrations of GAH.  p-values are relative to 5 mM H₂O₂ and 0 mg/mL GAH.Experiment 2LDH was purified and assayed for activity in the presence of 5 mM H₂O₂ and varying concentrations of GAH (Figure 2). Figure 2  Activity of purified LDH with varying concentrations of GAHExperiment 3PC12 cells were incubated with or without 5 mM H₂O₂ in the presence and absence of 1 mg/mL GAH.  Cells were then stained with the fluorescent marker TUNEL, which binds strongly to apoptotic cells.  The percentage of apoptotic cells in each condition is shown in Figure 3. Figure 3  Percentage of apoptotic cells in the presence or absence of H₂O₂ and GAH -The results of Experiment 3 best support which of the following statements?

Passage NDPK is an enzyme in budding yeast that uses ATP to phosphorylate nucleotide diphosphates (NDPs and dNDPs), yielding the triphosphate form of each (NTPs and dNTPs).  dNTPs are necessary for DNA synthesis, yet yeast cells that lack NDPK grow and divide at the same rate as wild-type yeast.  Therefore, triphosphate generation is most likely carried out by one or more other enzymes.The enzyme TMPK converts deoxythymidine monophosphate (dTMP) to the diphosphate form (dTDP) by transferring the γ-phosphate of ATP to dTMP.  Yeast cells that lack TMPK activity cannot synthesize DNA and become arrested during replication.  It was proposed that TMPK may partially compensate for NDPK loss by phosphorylating dTDP as well as dTMP.To better understand the role of TMPK in nucleotide phosphorylation, wild-type TMPK and the inactive E75K variant were purified by affinity chromatography, and the following experiments were performed.Experiment 1After purification, a sample containing 3 μg of protein was added to a reaction mixture containing radiolabeled nucleotide monophosphates (NMPs or dNMPs) and unlabeled ATP.  The reaction proceeded for 30 minutes, and products were separated by thin-layer chromatography (TLC) on a PEI-cellulose plate.  PEI-cellulose separates nucleotides by charge (Figure 1), and migration distance decreases with increasing negative charge.  Spots were visualized by autoradiography, and it was determined that 0.30 nmol of dTMP and 0.15 nmol of uridine monophosphate (UMP) became phosphorylated, respectively. Figure 1  TLC analysis of nucleotide monophosphates after exposure to TMPK and ATP.  (Note: Different nucleotides with the same phosphorylation level migrate different distances.)Experiment 2The experiment was repeated using radiolabeled dTDP as the starting material.  The reaction proceeded in buffer only, in the presence of E75K TMPK, or in the presence of wild-type TMPK.  Products were separated by TLC and visualized (Figure 2). Figure 2  TLC analysis of dTDP phosphorylation with buffer only (Lane 1), E75K TMPK (Lane 2), or wild-type TMPK (Lane 3) Adapted from Chien CY, Chen BR, Chou CK, Sclafani RA, Su JY. The yeast Cdc8 exhibits both deoxythymidine monophosphate and diphosphate kinase activities. FEBS Lett. 2009;583(13):2281-6. -NDPK and TMPK are members of a class of enzymes called:

Passage Interactions between positively charged histones and negatively charged nucleic acids are altered by acetylation and methylation of histone subunits.  These different post-translational modifications are recognized by histone-binding proteins such as BROMO domain Adjacent to Zinc finger 2B (BAZ2B).  BAZ2B has a net negative charge but contains a lysine-rich regulatory region between its two histone-binding domains.NMR data revealed potential interactions between BAZ2B and histone subunit H3 (Figure 1).  Binding was confirmed by isothermal titration calorimetry (ITC), which measures enthalpy of binding when a protein solution is titrated with ligand injections of equal volume and concentration.  Each injection results in a negative peak as heat is released.  Figure 2 shows ITC results when wild-type BAZ2B (WT) or BAZ2B with the regulatory region removed (10M+) were titrated with unmodified histone 3 (H3) or H3 that was acetylated at lysine-14 (H3K14ac).  In each case, the Hill coefficient was approximately 1.To test binding in a biological setting (in vivo), WT and 10M+ BAZ2B were extracted from cell lysates on affinity columns containing H3 or H3K14ac covalently attached to beads.  BAZ2B was then eluted and binding was quantified by Western blot (Figure 3). Figure 1  Electrostatic interactions between H3 and BAZ2B amino acid residues Figure 2  ITC traces (A) and K_d values (B) of WT BAZ2B and 10M+ BAZ2B titrated with either unmodified H3 or H3Ka4ac Figure 3  Western blot of BAZ2B recovered from unmodified or acetylated H3 columns Adapted from Kostrhon S, Kontaxis G, Kaufmann T, et al. A histone-mimicking interdomain linker in a multidomain protein modulates multivalent histone binding. J Biol Chem. 2017;292(43):17643-17657. -Which of the proteins mentioned in the passage has the highest isoelectric point?

Passage Porphyrin biosynthesis is a multistep process that is required for production of porphyrin derivatives such as heme, which is a crucial component of hemoglobin, myoglobin, and the cytochrome cofactors of the electron transport chain.  In the first step of this pathway, the enzyme porphobilinogen synthase catalyzes the production of porphobilinogen (PBGN) from two δ-aminolevulinate molecules, as shown in Reaction 1. Reaction 1In the next step, the monomeric enzyme hydroxymethylbilane synthase (HMBS) combines four PBGN molecules to form hydroxymethylbilane (Reaction 2).  Several additional steps are required to convert hydroxymethylbilane to heme. Reaction 2Defects in HMBS activity can lead to accumulation of δ-aminolevulinate and porphobilinogen in the liver and blood plasma.  The accumulation of these molecules can cause acute intermittent porphyria, a disorder characterized by episodes of severe abdominal pain.  Researchers expressed, purified, and assessed several variants of HMBS for enzymatic activity (Table 1) and thermal stability (Figure 1).Table 1  Michaelis-Menten equations generated from several HMBS variants, with V₀ and V_max measured in nmol/hr and [PBGN] and K_m measured in μM.  (Note: All reactions were carried out with 1 μM HMBS.) Figure 1  Thermal stability of several HMBS variants -Which statement most accurately describes the binding of PBGN to HMBS?  (Note: Assume binding is significantly faster than catalysis.)

Aspirin inhibits the enzyme cyclooxygenase.  Researchers discovered a new drug that inhibits cyclooxygenase more effectively.  If they report that the difference between aspirin and the new drug is statistically significant, it can be assumed that they:

Passage Tumor hypoxia is a marker of resistance to chemotherapy and radiation.  The low oxygen saturation in solid tumors activates hypoxia-inducible factors (HIFs), which stimulate the expression of genes required for angiogenesis, erythropoiesis, and glucose utilization.  Under normal conditions, HIF-DNA binding is interrupted by HIF-prolyl hydroxylases (HIF-PHDs), enzymes that hydroxylate aliphatic residues such as leucine or proline on HIFs.  These hydroxylated residues serve as markers for the ubiquitin-proteasome system.  Researchers have proposed that changes in oxygen-dependent pathways such as the citric acid cycle can regulate HIF-PHD activity.Experiment 1A kinematic study was conducted to test the effect of citric acid cycle intermediates on HIF-PHDs.  Fumarate and succinate were examined with purified HIF-PHD against increasing concentrations of 2-oxoglutarate (2-OG), an HIF analog. Figure 1  Activity of HIF-PHD exposed to succinate and fumarate in vitro based on 2-OG catalysisExperiment 2Researchers measured HIF-1a, a HIF subunit, using western blot analysis in wild-type (FH⁺) and fumarate-deficient (FH⁻) cells that were treated with fumarate under normoxic and hypoxic conditions (Figure 2). Figure 2  HIF-1a in FH⁺ and FH⁻ cells grown in vitro under normoxic (N) and hypoxic (H) conditions Adapted from Koivunen P, Hirsilä M, Remes AM, Hassinen IE, Kivirikko KI, Myllyharju J. Inhibition of hypoxia-inducible factor (HIF) hydroxylases by citric acid cycle intermediates: possible links between cell metabolism and stabilization of HIF. J Biol Chem. 2007;282(7):4524-32. -Based on the passage, culturing wild-type and FH⁻ cells in atmospheric oxygen conditions will lead to:

Passage Although the structure of the cell membrane has been investigated repeatedly, few studies examine the contribution of its lipid components.  Lipid composition influences the fluidity and permeability of cell membranes in the human body.  Chronic diseases such as atherosclerosis and psoriasis arise from alterations to the lipid composition.Atherosclerosis is caused by the accumulation of cholesterol in the endothelial cells lining the arterial wall.  Cholesterol carried by low-density lipoprotein (LDL) is deposited into cell membranes, where it forms cholesterol crystalline domains (CCDs).  In damaged cells, these CCDs increase membrane permeability and contribute to the formation of cholesterol plaques that can potentially block the artery itself.Omega-3 fatty acids (O3FAs) derived from marine animals may be used to slow the development of atherosclerosis.  Formulations of O3FA contain varying concentrations of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) (Figure 1).  Present research suggests that formulations of O3FA should contain EPA exclusively because it prevents cholesterol from organizing into CCDs.  Nonetheless, atherosclerosis is generally treated by administering drugs referred to as statins, which decrease the free cholesterol in blood plasma by inhibiting the production of a cholesterol precursor.  Statin therapy combined with O3FAs may be used to reduce atherosclerosis in patients with persistent hypertriglyceridemia (high triglyceride plasma levels). Figure 1  Organization of membranes in endothelial and epidermal cellsWater loss from the uppermost layer of the epidermis, the stratum corneum (SC), is prevented by specialized lipids called ceramides (cer).  Psoriasis is a skin disease in which the permeability of the SC is compromised due to a significant decrease in the level of ceramides.  The scarcity of ceramides impedes the formation of a lipid matrix called the long periodicity phase (LPP) in the cell membrane.  Increasing the concentration of ceramides such as esterified omega-hydroxyacyl-sphingosine (cer-EOS) has been shown to reduce the permeability of SC cell membranes and may be used to treat psoriasis scabs (Figure 1). Adapted from Mason RP, Jacob RF, Shrivastava S, Sherratt SC, Chattopadhyay A. Eicosapentaenoic acid reduces membrane fluidity, inhibits cholesterol domain formation, and normalizes bilayer width in atherosclerotic-like model membranes. Biochim Biophys Acta. 2016;1858(12):3131-3140. -Based on the passage, individuals with atherosclerosis or psoriasis would benefit from therapies that result in which of the following changes, respectively?

Passage Oxidative stress occurs in cells when reactive oxygen species (ROS) such as H₂O₂, superoxide (O₂⁻), and hydroxyl radicals (^•OH) interact destructively with biological molecules.  Several studies have indicated that histidine and certain peptides that contain histidine can decrease oxidative stress.A common marker of oxidative stress in cells is leakage of the enzyme lactate dehydrogenase (LDH) into the extracellular space.  Researchers incubated cultured rat cells (PC12 cells) with H₂O₂ in the presence of different tripeptides, each containing one alanine, one glycine, and one histidine residue.  Every possible sequence was tested, but the researchers found that the sequence GAH was the most effective in limiting extracellular LDH activity.  After obtaining these preliminary results, the following experiments were conducted.Experiment 1PC12 cells were incubated with or without 5 mM H₂O₂ and with varying concentrations of GAH for 4 hours.  Cells were centrifuged, and the supernatant (ie, extracellular material) was extracted and assayed for LDH activity (Figure 1). Figure 1  Extracellular LDH activity from PC12 cells exposed to H₂O₂ and varying concentrations of GAH.  p-values are relative to 5 mM H₂O₂ and 0 mg/mL GAH.Experiment 2LDH was purified and assayed for activity in the presence of 5 mM H₂O₂ and varying concentrations of GAH (Figure 2). Figure 2  Activity of purified LDH with varying concentrations of GAHExperiment 3PC12 cells were incubated with or without 5 mM H₂O₂ in the presence and absence of 1 mg/mL GAH.  Cells were then stained with the fluorescent marker TUNEL, which binds strongly to apoptotic cells.  The percentage of apoptotic cells in each condition is shown in Figure 3. Figure 3  Percentage of apoptotic cells in the presence or absence of H₂O₂ and GAH -Which of the following expressions gives the number of peptides that were tested for their ability to reduce extracellular LDH activity?

Passage Synthesis of coenzyme A (CoA) requires vitamin B₅ as a substrate.  In bacteria, vitamin B₅ is synthesized by condensation of β-alanine and pantoic acid.  β-alanine is produced by decarboxylation of L-aspartate, mediated by the enzyme aspartate α-decarboxylase (ADC).  Active ADC is formed when its inactive precursor, PanD, binds to a regulatory complex composed of acetyl coenzyme A (AcCoA) and a protein known as PanZ.  Formation of the PanD-PanZ-AcCoA complex causes the activation loop of PanD to fold into a β-pleated sheet, resulting in PanD autocleavage.  Researchers hypothesize that this conformational change allows the Thr57 residue to cleave the N-terminal end of PanD to form ADC.The limited information on the role of PanZ in the synthesis of CoA has stimulated an investigation of its effect on ADC activity.  Escherichia coli cells were provided with varying concentrations of PanZ, and the rate of β-alanine production was measured in three different conditions (Figure 1).  Reaction vessels were maintained at pH 6 to ensure optimal decarboxylation activity. Figure 1  Catalytic activity of ADC in E. coli with increasing concentrations of L-aspartate Adapted from Monteiro DC, Patel V, Bartlett CP, et al. The structure of the PanD/PanZ protein complex reveals negative feedback regulation of pantothenate biosynthesis by coenzyme A. Chem Biol. 2015;22(4):492-503. -Based on the results shown in Figure 1, what is the ratio of k_cat in Condition 1 to k_cat in Condition 3 for ADC?

Passage ATP synthase, also known as Complex V, produces the majority of the ATP in eukaryotic cells.  It is a transmembrane protein encoded by both nuclear and mitochondrial genes in humans, and is found in the inner mitochondrial membrane.  Complex V consists of over 20 subunits, including three sets of heterodimers arranged in a circle in the mitochondrial matrix.  Each dimer contains an α subunit and a β subunit, and each β subunit exists in one of three conformations: one that binds ATP, one that binds ADP and inorganic phosphate, and one that binds neither.As protons flow from the intermembrane space through Complex V and into the matrix, they cause the transmembrane γ subunit to rotate and interact with each β subunit in turn.  Four protons provide enough energy for rotation from one β subunit to the next, and each rotation causes a β subunit to release a new ATP molecule as it changes from the ATP-binding to the nonbinding conformation (Figure 1).  Thus 12 protons produce 3 ATP molecules each time the γ subunit makes a complete revolution.  Every NADH molecule that enters the electron transport chain (ETC) pumps 10 protons into the intermembrane space for use by Complex V whereas only 6 protons are pumped per FADH₂ molecule. Figure 1  Depictions of ATP synthase viewed from the side (left) and the bottom (right)Researchers visualized the rotation of the γ subunit by attaching it to a fluorescently labeled actin filament.  The observed rotation over time is shown in Figure 2. Figure 2  Counterclockwise rotation of the γ subunit of ATP synthase (bottom view) over time in seconds as visualized by a fluorescent labelAn L156P mutation in subunit A of Complex V is linked to neuropathy, ataxia, and retinal degeneration.  Although the exact molecular bases of these pathological states are unknown, the following hypotheses are currently under study:Hypothesis 1L156P Complex V functions normally but is less abundant than wild-type Complex V.Hypothesis 2The mutation partially decouples proton flow from ATP synthesis.Hypothesis 3The mutation leads to increased oxidative stress from the ETC, resulting in apoptosis. Adapted from Kucharczyk R, Zick M, Bietenhader M, et al. Mitochondrial ATP synthase disorders: molecular mechanisms and the quest for curative therapeutic approaches. Biochim Biophys Acta. 2009;1793(1):186-99. -When pyruvate enters the mitochondria, it is converted to acetyl-CoA and further oxidized in a series of reactions that produces four NADH molecules and one FADH₂ molecule.  If all of the NADH and FADH₂ produced by 2 moles of pyruvate digestion in the mitochondria enter the ETC, how many moles of ATP can wild-type Complex V produce?

Passage Ribulose-1,5-bisphosphate carboxylase/oxygenase (rubisco) is one of the most abundant enzymes on Earth.  In carbon-fixing organisms, it consumes CO₂ to carboxylate the sugar ribulose-1,5-bisphosphate (Ru1,5BP), and forms two molecules of 3-phosphoglycerate (3PG) as a product.  The functional form of rubisco from the bacterium Rhodospirillum rubrum is a noncovalent homodimer composed of two 51 kDa monomers.  Each subunit requires a magnesium ion as a prosthetic group in the active site.Rubisco is often used as a model for protein folding studies.  On their own, rubisco monomers typically cannot fold to completion and instead are trapped in a form known as a "kinetic intermediate."  These kinetically trapped monomers cannot dimerize correctly and are prone to aggregation.Scientists interested in studying protein folding dynamics labeled the rubisco kinetic intermediate with a set of fluorophores known as a FRET (fluorescence resonance energy transfer) pair, with one fluorophore at the N-terminus and the other at the C-terminus.  A FRET signal occurs when the emission spectrum of one fluorophore (the donor) overlaps with the excitation spectrum of the other (the acceptor).  When the two fluorophores are sufficiently close to each other, the donor can transfer energy from the light it absorbs directly to the acceptor, causing the acceptor to emit light when the donor absorbs light.The GroEL/ES complex is a chaperone protein that binds and releases misfolded proteins, hydrolyzing ATP each time the protein is released.  A misfolded protein may undergo multiple rounds of binding and release before adopting its correct conformation, or it may never fold correctly and instead be targeted for destruction (Figure 1). Figure 1  Representation of GroEL/ES-mediated protein foldingResearchers diluted the labeled kinetic intermediate into a refolding buffer in the presence or absence of 200 nM GroEL/ES and then monitored the FRET signal over time.  The results are shown in Figure 2. Figure 2  FRET signal of labeled rubisco with and without GroEL Lin Z, Rye HS. Expansion and compression of a protein folding intermediate by GroEL. Mol Cell. 2004;16(1):23-34. -Which of the following conclusions can be drawn based on the data from Figure 2?

Passage Many lysosomal proteins are transported to the lysosome by the cation-independent mannose 6-phosphate receptor (CI-MPR).  CI-MPR is a transmembrane protein found in the Golgi apparatus and vesicles.  It contains 15 globular domains folded similarly on the luminal side of the membrane, each of which has up to 3 glycosylation sites and 6−12 cysteine residues that aid in folding.The ability of CI-MPR to recognize multiple different ligands was studied by fluorescent microarray analysis.  Microarray analysis was performed by cross-linking candidate ligands such as plasminogen, insulin-like growth factor 2 (IGF-II), and mannose 6-phosphate (M6P), to glass slides.  The slides were then incubated with full-length CI-MPR or recombinantly expressed CI-MPR domains, followed by incubation with an anti−CI-MPR antibody.  A fluorescently labeled antibody that binds the first antibody was then added, and the relative fluorescence levels of each candidate ligand were compared.  Higher fluorescence levels correspond to greater binding.Experiment 1Microarray analysis was carried out on slides containing varying levels of candidate ligands, and binding affinities of each CI-MPR domain for its primary ligand were estimated.  Table 1 shows the results.Experiment 2Domains 3, 5, and 9 bind carbohydrates.  Amino acids in these domains were mutated to determine which are required for binding.  Based on this experiment, scientists concluded that Arg, Glu, and Tyr are required for carbohydrate binding by domains 3, 5, and 9 of CI-MPR.Table 1 Adapted from Bohnsack RN, Song X, Olson LJ, et al. Cation-independent mannose 6-phosphate receptor: a composite of distinct phosphomannosyl binding sites. J Biol Chem. 2009;284(50):35215-26. -Based on information in the passage and the data in Table 1, the 15 domains of CI-MPR are most likely similar to each other in which of the following ways?

The graph below shows the effect of an inhibitor on enzyme activity when the enzyme and inhibitor were preincubated prior to addition of substrate, and when no preincubation occurred.  Based on the graph, which of the following statements is true?

Passage Tumor hypoxia is a marker of resistance to chemotherapy and radiation.  The low oxygen saturation in solid tumors activates hypoxia-inducible factors (HIFs), which stimulate the expression of genes required for angiogenesis, erythropoiesis, and glucose utilization.  Under normal conditions, HIF-DNA binding is interrupted by HIF-prolyl hydroxylases (HIF-PHDs), enzymes that hydroxylate aliphatic residues such as leucine or proline on HIFs.  These hydroxylated residues serve as markers for the ubiquitin-proteasome system.  Researchers have proposed that changes in oxygen-dependent pathways such as the citric acid cycle can regulate HIF-PHD activity.Experiment 1A kinematic study was conducted to test the effect of citric acid cycle intermediates on HIF-PHDs.  Fumarate and succinate were examined with purified HIF-PHD against increasing concentrations of 2-oxoglutarate (2-OG), an HIF analog. Figure 1  Activity of HIF-PHD exposed to succinate and fumarate in vitro based on 2-OG catalysisExperiment 2Researchers measured HIF-1a, a HIF subunit, using western blot analysis in wild-type (FH⁺) and fumarate-deficient (FH⁻) cells that were treated with fumarate under normoxic and hypoxic conditions (Figure 2). Figure 2  HIF-1a in FH⁺ and FH⁻ cells grown in vitro under normoxic (N) and hypoxic (H) conditions Adapted from Koivunen P, Hirsilä M, Remes AM, Hassinen IE, Kivirikko KI, Myllyharju J. Inhibition of hypoxia-inducible factor (HIF) hydroxylases by citric acid cycle intermediates: possible links between cell metabolism and stabilization of HIF. J Biol Chem. 2007;282(7):4524-32. -During hypoxia, skeletal muscles have been shown to accumulate succinate and ADP molecules.  Which of the following explains this result?

Atpenins are succinate-ubiquinone reductase inhibitors with antifungal properties.  Atpenins illustrate the fact that:

Passage NDPK is an enzyme in budding yeast that uses ATP to phosphorylate nucleotide diphosphates (NDPs and dNDPs), yielding the triphosphate form of each (NTPs and dNTPs).  dNTPs are necessary for DNA synthesis, yet yeast cells that lack NDPK grow and divide at the same rate as wild-type yeast.  Therefore, triphosphate generation is most likely carried out by one or more other enzymes.The enzyme TMPK converts deoxythymidine monophosphate (dTMP) to the diphosphate form (dTDP) by transferring the γ-phosphate of ATP to dTMP.  Yeast cells that lack TMPK activity cannot synthesize DNA and become arrested during replication.  It was proposed that TMPK may partially compensate for NDPK loss by phosphorylating dTDP as well as dTMP.To better understand the role of TMPK in nucleotide phosphorylation, wild-type TMPK and the inactive E75K variant were purified by affinity chromatography, and the following experiments were performed.Experiment 1After purification, a sample containing 3 μg of protein was added to a reaction mixture containing radiolabeled nucleotide monophosphates (NMPs or dNMPs) and unlabeled ATP.  The reaction proceeded for 30 minutes, and products were separated by thin-layer chromatography (TLC) on a PEI-cellulose plate.  PEI-cellulose separates nucleotides by charge (Figure 1), and migration distance decreases with increasing negative charge.  Spots were visualized by autoradiography, and it was determined that 0.30 nmol of dTMP and 0.15 nmol of uridine monophosphate (UMP) became phosphorylated, respectively. Figure 1  TLC analysis of nucleotide monophosphates after exposure to TMPK and ATP.  (Note: Different nucleotides with the same phosphorylation level migrate different distances.)Experiment 2The experiment was repeated using radiolabeled dTDP as the starting material.  The reaction proceeded in buffer only, in the presence of E75K TMPK, or in the presence of wild-type TMPK.  Products were separated by TLC and visualized (Figure 2). Figure 2  TLC analysis of dTDP phosphorylation with buffer only (Lane 1), E75K TMPK (Lane 2), or wild-type TMPK (Lane 3) Adapted from Chien CY, Chen BR, Chou CK, Sclafani RA, Su JY. The yeast Cdc8 exhibits both deoxythymidine monophosphate and diphosphate kinase activities. FEBS Lett. 2009;583(13):2281-6. -Given that 1 unit of activity equals 1 nmol of substrate converted per minute, what was the specific activity of purified TMPK toward dTMP in Experiment 1?  (Note: Assume the rate of phosphorylation is constant throughout the experiment.)

Passage The Ankyrin repeat and SOCS box containing protein 4 (Asb-4) is highly expressed in the cytosol of hypothalamus cells during the well-fed state but is downregulated during fasting.  In addition, the protein insulin receptor substrate 4 (IRS4), known to be involved in appetite regulation, is expressed in the cytosol of the same cells as Asb-4.  Accordingly, it was hypothesized that Asb-4 suppresses appetite through interactions with IRS4.Small peptides such as myc (EQKLISEEDL) and flag (DYKDDDDK) are often attached to recombinant proteins to facilitate their purification and detection.  HEK293 cells were transfected with plasmids encoding Asb-4 and IRS4 tagged with myc or flag at their N-termini (myc-Asb4 and flag-IRS4, respectively).  Cell lysates were incubated with beads linked to α-flag, an antibody that binds flag, and proteins that did not bind were washed away.  The bound proteins were then visualized by Western blot using α-myc antibodies.  The α-myc antibody detected the presence of myc, indicating that myc-Asb-4 binds to flag-IRS4.To better understand interactions between Asb-4 and IRS4, cells were transfected with 1 μg of flag-IRS4 plasmid and varying amounts of either myc-Asb-4 plasmid or a plasmid with the sb domain deleted (myc-Asb-4Δsb).  Flag-IRS4 levels from each set of cells were then quantified, as shown in Figure 1. Figure 1  Flag-IRS4 levels in cells with varying amounts of myc-Asb-4 or myc-Asb-4Δsb plasmid addedIn a follow-up experiment, lysates from transfected cells were again incubated with the α-flag beads, and a Western blot was performed on the precipitated proteins using α-flag, α-myc, and an antibody against ubiquitin (α-ub).  The results are shown in Figure 2. Figure 2  Ubiquitin, flag-IRS4, and myc-Asb-4 levels in cells transfected with flag-IRS4 and either an empty vector (control), myc-Asb-4 plasmid, or myc-Asb-4Δsb plasmid Adapted from Li JY, Chai B, Zhang W, et al. Ankyrin repeat and SOCS box containing protein 4 (Asb-4) colocalizes with insulin receptor substrate 4 (IRS4) in the hypothalamic neurons and mediates IRS4 degradation. BMC Neurosci. 2011;12:95. -To conclude that IRS4 binds Asb-4, what assumption must be made about the recombinant proteins?