Exam 2: Biochemistry

Passage A newborn girl is diagnosed with a rare genetic disorder that causes premature lysis of red blood cells.  Through genome sequencing, physicians identified a specific mutation (R510Q) in both copies of her pyruvate kinase gene, which encodes the enzyme that catalyzes the final step of glycolysis.  Pyruvate kinase produces pyruvate and ATP from phosphoenolpyruvate (PEP) and ADP.PEP + ADP ↔ Pyruvate + ATPReaction 1Because red blood cells lack mitochondria, pyruvate in erythrocytes does not enter the citric acid cycle and the electron transport chain.  Therefore, red blood cells rely almost exclusively on glycolysis to produce sufficient ATP for cellular functions.  In patients with pyruvate kinase deficiency, it is thought that a lack of ATP compromises the integrity of the red blood cell membrane, leading to premature lysis in the spleen.  Although these observations are well established, the exact mechanism by which low ATP alters the cell membrane is unclear.Scientists interested in analyzing this particular patient's gene variant expressed and purified recombinant R510Q pyruvate kinase.  They performed several biochemical tests to characterize the R510Q mutant with respect to wild-type (WT) pyruvate kinase.  In the first experiment, WT and R510Q pyruvate kinases were each incubated with a constant amount of ADP and varying amounts of PEP.  The activity of the enzyme was measured at each concentration of PEP and plotted.  They then repeated the experiment in the presence of ATP and found that ATP is a noncompetitive inhibitor of the WT enzyme, but a mixed inhibitor of the R510Q enzyme.  The results are shown in Figure 1.  All experiments were done at the same enzyme concentration. Figure 1  Pyruvate kinase activity with and without ATP as a function of [PEP].  Open markers are shown at ½V_max for each plot. Adapted from Wang C, Chiarelli LR, Bianchi P, et al. Human erythrocyte pyruvate kinase: characterization of the recombinant enzyme and a mutant form (R510Q) causing nonspherocytic hemolytic anemia. Blood. 2001;98(10):3113-20. -Based on information in the passage, pyruvate in red blood cells can be further metabolized only by:

Passage The influenza A virus is an enveloped RNA virus with potential for zoonosis (transmission from animals to humans).  It obtains its plasma membrane from its host cell, but the composition of the viral envelope and vertebrate plasma membrane are distinct.  The envelope of H6-G225D consists mostly of phosphatidylethanolamine as well as some negatively charged glycerophospholipids.  The outer membrane contains hemagglutinin (HA) proteins that bind to the carbohydrate known as sialic acid (SA) on host cell receptor proteins, facilitating viral entry.  It also contains neuraminidase enzymes that break the bond between SA and galactose in the host membrane glycans, allowing new virions to be released.Minute changes in the genes that code for HA proteins can alter the virulence, host, and attachment mechanism.  In general, influenza A infects avian species by attaching to tracheal tissue, which displays SA groups bound to receptors through an α-2,3-glycosidic linkage to galactose (Figure 1).  Avian viruses generally cannot attack human cells, in which SA is linked to galactose through an α-2,6-glycosidic linkage instead of an α-2,3 linkage. Figure 1  Structure of the influenza A receptor in avian cell membranesIn a recent study, however, a G225D substitution in HA, caused by a GGC to GAU codon mutation, enabled avian virions to infect human cells.  The stability of G225D HA proteins was compared to that of H6-P186L, which is created by a single CCC to CUC mutation and exclusively infects avian cells, and H1N1, which targets only humans.  The viral RNA of H6-P186L, H6-G225D, and H1N1 was cloned using cDNA and expressed in embryonic kidney cells.  The melting curve of each purified HA protein was determined using differential scanning calorimetry, with peaks representing melting temperatures (Figure 2). Figure 2  Hemagglutinin melting curves for H6N1 and H1N1 influenza A strains Adapted from De vries RP, Tzarum N, Peng W, et al.  A single mutation in Taiwanese H6N1 influenza hemagglutinin switches binding to human-type receptors.  EMBO Mol Med. 2017;9(9):1314-1325. -Which statement best describes the structure of the avian receptor of influenza A shown in Figure 1?

Passage Many cephalopods can change color to blend in with their surroundings by altering the conformations of particular proteins within their skin cells.  The reflectin proteins play a major role in this camouflage mechanism.  Reflectins are a group of intrinsically disordered proteins that do not adopt clearly defined secondary or tertiary structures.  Under certain conditions reflectins aggregate, allowing cells to reflect various wavelengths of light.Skin cells from the squid Doryteuthis opalescens were cultured and three reflectins-DoRA, DoRB, and DoRC-were purified and analyzed to determine amino acid composition.  They were found to contain an abundance of arginine and serine but relatively few cysteine, leucine, isoleucine, valine, phenylalanine, or tryptophan residues.  Figure 1 shows the relative hydrophobicity of each reflectin variant as a function of amino acid position. Figure 1  Hydrophobicity plots of three reflectins isolated from D. opalescens.  (Note: Negative numbers indicate hydrophilic regions.)In D. opalescens skin cells (pH ≈ 7.8), reflectins aggregate in response to acetylcholine (ACh), which causes a signal cascade that changes the phosphorylation state of the proteins.  Cells were incubated with or without ACh, and DoRA and DoRB were purified and assessed for phosphorylation by mass spectrometry.  DoRA became phosphorylated in the presence of ACh whereas DoRB became dephosphorylated.  After examining the isoelectric points of unmodified DoRA and DoRB, as shown in Figure 2, scientists proposed that reflectins aggregate upon charge neutralization. Figure 2  Isoelectric focusing of DoRA and DoRB Adapted from Demartini DG, Izumi M, Weaver AT, Pandolfi E, Morse DE.  Structures, Organization, and Function of Reflectin Proteins in Dynamically Tunable Reflective Cells.  J Biol Chem. 2015;290(24):15238-49. -Which of the reflectin variants is most likely to be integrated into a membrane?

Phospholipases release arachidonic acid from cell membranes as part of the inflammatory response.  Given this information, which type of molecule is derived from arachidonic acid and is involved in this response?

Passage The sirtuins are a class of enzymes that regulate various metabolic processes by removing post-translational modifications from enzymes.  SIRT4 is an autosomally encoded sirtuin protein that colocalizes with the pyruvate dehydrogenase complex.  It deacylates lysine residues on certain metabolic proteins by removing methylglutaryl and hydroxymethylglutaryl units.  Methylglutaryl-CoA and hydroxymethylglutaryl-CoA are intermediates in leucine catabolism, and scientists hypothesized that SIRT4 regulates this pathway.  To test their hypothesis, livers from wild-type (WT) and SIRT4 knockout (KO) mice were treated with α-keto acid derivatives of several amino acids, including the derivative of leucine, α-ketoisocaproate (αKIC).  Figures 1 and 2 show the rate of oxidation of α-keto acids and oxygen consumption in both WT and KO livers, respectively. Figure 1  Box plot showing the rate of oxidation of α-ketoisocaproate (αKIC), α-ketoglutarate (αKG), and pyruvate in SIRT4 knockout livers (Note: Asterisk indicates p < 0.05 relative to WT level) Figure 2  Oxygen consumption in WT and KO livers in the presence and absence of succinate and reduced cytochrome C (Cyt-C)Because leucine is known to regulate insulin secretion, scientists injected WT and KO mice with leucine and measured plasma insulin levels over time.  They then injected mice with insulin and monitored blood glucose levels (Figure 3). Figure 3  (A) Plasma insulin levels after injection with leucine and (B) blood glucose levels after injection with insulin in both WT and KO mice Adapted from Anderson KA, Huynh FK, Fisher-Wellman K, et al. SIRT4 Is a Lysine Deacylase that Controls Leucine Metabolism and Insulin Secretion. Cell Metab. 2017;25(4):838-855.e15. -In the absence of SIRT4, the electrochemical potential across the inner mitochondrial membrane:

During protein folding, which of the following interactions help stabilize secondary, tertiary, or quaternary structure?Hydrogen bondsIonic bondsvan der Waals forcesAmide bonds

Passage The ABO blood groups are determined by the polysaccharide antigens linked to sphingolipids on red blood cell membranes.  The polysaccharide known as the H-antigen (O-type blood) consists of a polysaccharide (R-group) glycosidically linked to D-galactose, which is connected to L-fucose in an α-1,2 linkage.  Type A and type B blood express the A- and B-antigens, respectively (Figure 1).  These antigens are modified forms of the H-antigen, in which D-galactose (B-antigen) or a derivative of D-galactose called GalNAc (A-antigen) is added in an α-1,3 linkage to the D-galactose moiety of the H-antigen. Figure 1  Structures of the H-, A-, and B-antigens.  The R-group represents additional carbohydrates.The addition of GalNAc or galactose to the H-antigen is catalyzed by an enzyme encoded by the ABO gene.  One allele of the gene encodes GalNAcyl transferase and produces the A-antigen, while another allele encodes galactosyl-transferase and yields the B-antigen (Figure 1).  The two alleles differ at seven nucleotide positions, summarized in Table 1.Table 1  Nucleotide Positions Where the Alleles Encoding GalNAcyl Transferase and Galactosyl Transferase Differ Individuals with both alleles express both antigens and have type AB blood.  Those with type O blood have a third allele that encodes an inactive form of the enzyme.  A study of blood type abundance was conducted, and the number of participants with each type is shown in Table 2.  Table 3 shows several fatty-acyl chains found in the sphingolipids to which the antigens were attached.Table 2  Number of Participants That Express Each Blood Type in a Study of 10,000 Individuals Table 3  Types of Sphingolipids to Which Polysaccharide Antigens Were Bound, as Determined by Mass Spectrometry (Note:  For fatty acid chains, the numbers on the left and right of the colon indicate number of carbons and number of C=C double bonds, respectively) -An individual with type B blood would most likely have which nucleotide at position 793 of the ABO gene?

Passage The sirtuins are a class of enzymes that regulate various metabolic processes by removing post-translational modifications from enzymes.  SIRT4 is an autosomally encoded sirtuin protein that colocalizes with the pyruvate dehydrogenase complex.  It deacylates lysine residues on certain metabolic proteins by removing methylglutaryl and hydroxymethylglutaryl units.  Methylglutaryl-CoA and hydroxymethylglutaryl-CoA are intermediates in leucine catabolism, and scientists hypothesized that SIRT4 regulates this pathway.  To test their hypothesis, livers from wild-type (WT) and SIRT4 knockout (KO) mice were treated with α-keto acid derivatives of several amino acids, including the derivative of leucine, α-ketoisocaproate (αKIC).  Figures 1 and 2 show the rate of oxidation of α-keto acids and oxygen consumption in both WT and KO livers, respectively. Figure 1  Box plot showing the rate of oxidation of α-ketoisocaproate (αKIC), α-ketoglutarate (αKG), and pyruvate in SIRT4 knockout livers (Note: Asterisk indicates p < 0.05 relative to WT level) Figure 2  Oxygen consumption in WT and KO livers in the presence and absence of succinate and reduced cytochrome C (Cyt-C)Because leucine is known to regulate insulin secretion, scientists injected WT and KO mice with leucine and measured plasma insulin levels over time.  They then injected mice with insulin and monitored blood glucose levels (Figure 3). Figure 3  (A) Plasma insulin levels after injection with leucine and (B) blood glucose levels after injection with insulin in both WT and KO mice Adapted from Anderson KA, Huynh FK, Fisher-Wellman K, et al. SIRT4 Is a Lysine Deacylase that Controls Leucine Metabolism and Insulin Secretion. Cell Metab. 2017;25(4):838-855.e15. -Based on the passage, the SIRT4 protein most likely contains which of the following?

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. -The data in Table 1 suggest that the domain with the highest affinity for its ligand is:

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. -What is the initial rate of rubisco folding in the presence of GroEL/ES, given a rate constant k_f = 2 × 10⁻¹ s⁻¹?

Which statement correctly describes the cleavage of protein disulfide bonds by the enzyme thioredoxin?

Passage Many cephalopods can change color to blend in with their surroundings by altering the conformations of particular proteins within their skin cells.  The reflectin proteins play a major role in this camouflage mechanism.  Reflectins are a group of intrinsically disordered proteins that do not adopt clearly defined secondary or tertiary structures.  Under certain conditions reflectins aggregate, allowing cells to reflect various wavelengths of light.Skin cells from the squid Doryteuthis opalescens were cultured and three reflectins-DoRA, DoRB, and DoRC-were purified and analyzed to determine amino acid composition.  They were found to contain an abundance of arginine and serine but relatively few cysteine, leucine, isoleucine, valine, phenylalanine, or tryptophan residues.  Figure 1 shows the relative hydrophobicity of each reflectin variant as a function of amino acid position. Figure 1  Hydrophobicity plots of three reflectins isolated from D. opalescens.  (Note: Negative numbers indicate hydrophilic regions.)In D. opalescens skin cells (pH ≈ 7.8), reflectins aggregate in response to acetylcholine (ACh), which causes a signal cascade that changes the phosphorylation state of the proteins.  Cells were incubated with or without ACh, and DoRA and DoRB were purified and assessed for phosphorylation by mass spectrometry.  DoRA became phosphorylated in the presence of ACh whereas DoRB became dephosphorylated.  After examining the isoelectric points of unmodified DoRA and DoRB, as shown in Figure 2, scientists proposed that reflectins aggregate upon charge neutralization. Figure 2  Isoelectric focusing of DoRA and DoRB Adapted from Demartini DG, Izumi M, Weaver AT, Pandolfi E, Morse DE.  Structures, Organization, and Function of Reflectin Proteins in Dynamically Tunable Reflective Cells.  J Biol Chem. 2015;290(24):15238-49. -What effect does the addition of ACh have on the net charge of DoRA and DoRB in D. opalascens skin cells?

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. -OCTN2 facilitates transport of L-carnitine by which mechanism?

Passage Physical activity produces an increased energy requirement in the muscles as well as in the brain.  During periods of intense exercise, glycogenolysis is activated in muscles to provide them with the energy needed for contractions, and research indicates that astrocytes also consume glycogen at increased levels during exercise.  It is hypothesized that during periods of physical exertion, astrocytes convert glycogen to lactate, which is then used as a fuel source in neurons.Monocarboxylate transporters (MCTs) are responsible for the movement of lactate across membranes, and MCT2 is expressed at increased levels in neuronal cells during exercise.  Scientists proposed that lactate is secreted from astrocytes and taken up by neurons through MCT2.  Lactate is then converted to pyruvate and enters the mitochondria, where it is further metabolized.  To test their predictions, scientists examined the effect of exercise on glycogen metabolism in rodents.Experiment 1Rats were either allowed to rest or exercised to exhaustion.  The levels of several metabolites were then quantified in both the plantaris muscle and the hippocampus. Figure 1  Levels of several metabolites in plantaris muscle (A) and hippocampus (B) from rats at rest or after exercising to exhaustionExperiment 2Rats were injected with 1,4-dideoxy-1,4-imino-D-arabinitol (DAB) or saline, and glycogen and lactate levels were measured in the hippocampus of rats at rest or after exercise to exhaustion. Figure 2  Levels of glycogen (A) and lactate (B) in rats injected with DAB or saline, after rest or upon exhaustion Adapted from Matsui T, Omuro H, Liu Y-F, et al. Astrocytic glycogen-derived lactate fuels the brain during exhaustive exercise to maintain endurance capacity. Proceedings of the National Academy of Sciences. 2017;114(24):6358-6363. -Assuming the proposed mechanism in the passage is correct, what is the fate of the carbon atoms in lactate after they pass through MCT2 channels?

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. -Which of the following graphs accurately predicts the aspartate decarboxylation activity of PanD and ADC between pH 3 and pH 9?

Which of the following molecules could decrease the activity of one or more Krebs cycle enzymes?

Researchers isolate a protein that runs as a single band on a nonreducing SDS-PAGE gel but as two distinct bands on a reducing gel.  The protein is most likely a:

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. -Residues in the binding pockets of domains 3, 5, and 9 interact with carbohydrates through hydrogen bonds.  In Experiment 2, tyrosine residues were mutated to remove hydrogen bonding while minimizing changes in the shape of the active site.  Tyrosine residues were most likely mutated to:

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 -What is the lowest tested concentration of GAH that produces a significant reduction in extracellular LDH activity when 5 mM H₂O₂ is used?

Proline differs from the other standard amino acids in that only the former: