Exam 2: Biochemistry

Researchers measured the activity of two enzymes that catalyze the same reaction, with results shown below.  Which of the enzymes must have multiple active sites?

Which event could alter a protein's primary structure?

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 tested tripeptide variants most likely have which of the following characteristics in common?Primary structureIsoelectric pointHydrophobicity

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. -Given that pyruvate synthesis is generally unidirectional, which of the following statements regarding the thermodynamics of Reaction 1 is most accurate?

During prolonged fasting, which of the following liver enzymes has upregulated activity?

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. -If the pathological effects observed in patients with the L156P mutation are caused by decreased Complex V activity, the effects could be best counteracted by a drug that increases which of the following?

Passage Destabilase, an enzyme found in the saliva of leeches, exhibits at least two activities: isopeptidase activity and glycosidase activity.  Isopeptidase activity is believed to prevent blood clotting by hydrolyzing the isopeptide bond between the ε-amino group of lysine and the γ-carboxyl group of glutamate in fibrin monomers.  The enzyme's glycosidase activity digests sugars found in the peptidoglycans of bacterial cell walls and may play a role in innate immunity.Two isoforms of destabilase were tagged with a polyhistidine tail and recombinantly expressed in E. coli cells.  Both isoforms were then purified by affinity chromatography and eluted by imidazole, which competes with histidine-tagged proteins for column binding.  Each isoform eluted efficiently with 300 mM imidazole.  Five microliters of each purified enzyme were then provided with saturating levels of substrate and assessed for isopeptidase and glycosidase activity at various pH levels, which allowed the optimal pH of enzymatic activities for each isoform to be identified (Figure 1).  Further studies showed that at optimal pH, both isoforms had approximately the same k_cat values, but isoform 2 had a lower K_m than isoform 1 for both enzymatic activities. Figure 1  Isopeptidase activity (A) and glycosidase activity (B) of 5 μL of destabilase isoforms 1 and 2 at various pH valuesIsoform 2 was further characterized by mutating specific residues and measuring enzymatic activities at optimal pH.  The results are shown in Figure 2. Figure 2  Isopeptidase and glycosidase activity of isoform 2 of destabilase with various mutations -Which conclusion is LEAST supported by the data in Figure 2?

Passage The recent advent of multidrug-resistant Mycobacterium tuberculosis (Mtb) has led to the search for novel agents to combat tuberculosis.  Mtb secretes over 70 serine proteases that may be used as viable drug targets.  Hydrolase important for pathogenesis 1 (Hip1) is a serine protease found in Mtb with proteolytic activity that is required for dampening the host inflammatory response.  Given the role of Hip1 in Mtb virulence, analyzing Hip1 substrate specificity is a critical step in designing selective inhibitors for effective tuberculosis treatment.Fluorogenic substrates are nonfluorescent compounds that release a fluorescent reporter group when acted upon by an enzyme.  Such substrates are useful biomolecular imaging tools that allow monitoring of enzymatic activity in vitro and in vivo.  A large library of peptides was screened against Hip1, and enzymatic cleavage was analyzed using mass spectrometry to facilitate the design of fluorogenic substrates (Figure 1). Figure 1  Schematic design of Hip1 fluorogenic substrates.  P1, P2, P3, and P4 in Group 1 represent the positions of variable amino acids in the peptide.  Group 2 depicts the fluorescent compound released on Hip1 cleavage.Researchers reported Michaelis-Menten kinetic parameters for substrates from two peptide families: the WKLL substrates and the CSL substrates.Table 1  Kinetic Parameters of Fluorogenic Substrates for Mtb Hip1 Adapted from Lentz CS, Ordonez AA, Kasperkiewicz P, et al. Design of Selective Substrates and Activity-Based Probes for Hydrolase Important for Pathogenesis 1 (HIP1) from Mycobacterium tuberculosis. ACS Infect Dis. 2016;2(11):807-815. -Which of the following substrates yields the most optimal catalytic turnover and catalytic efficiency, respectively?

Passage The bacterium Clostridium difficile secretes protein toxins TcdA and TcdB, two homologues associated with C. difficile-related colitis.  Tcd proteins enter host intestinal cells via endocytosis induced by interactions between the Tcd C-terminal domain and cell surface receptors.  Endocytosis is followed by translocation of the N-terminal glucosyltransferase domain (GTD) across the endosomal membrane.  The GTD remains anchored to endosomes by the central region of the protein, a transmembrane domain believed to form membrane pores and facilitate translocation.  Inositol hexakisphosphate (IP6) binds Tcd proteins, an interaction that is stabilized by salt bridges in the binding pocket (Figure 1). Figure 1  Salt bridges formed between IP6 and the amino acids in the binding pocketThis interaction induces a conformational change that causes a molecular flap to move away from the catalytic site.  The catalytic site then mediates intramolecular cleavage in a reaction known as autoprocessing.  This reaction results in the release of GTD into the cytosol, where it becomes active.To better understand the relationship between IP6 binding and catalytic activity, autoprocessing assays were performed.  Researchers mutated histidine to alanine at positions 759 and 757 in the flap regions of TcdA and TcdB, respectively.  They then compared the autoprocessing activities of H759A and H757A mutants to those of wild-type TcdA and TcdB at varying concentrations of IP6 (Figure 2). Figure 2  Percent cleavage of wild-type and mutant Tcd by autoprocessing Adapted from Chumbler NM, Rutherford SA, Zhang Z, et al. Crystal structure of Clostridium difficile toxin A. Nat Microbiol. 2016;1:15002. -Which conclusion about the effect of the H759A and H757A mutations is best supported by Figure 2?

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. -Which of the following experiments could be used to determine whether the lactate secreted by astrocytes can serve as a fuel source in neurons in the hippocampus?

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 side chain of the C-terminal amino acid residue in GAH is derived from which structure?

During anaerobic exercise, the Cori cycle connects which two metabolic pathways?

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 Figure 1, which of the following statements is true regarding R510Q pyruvate kinase as compared to WT pyruvate kinase?

Passage Aldehyde dehydrogenase 2 (ALDH2) is essential for the oxidation of toxic aldehydes, and uses NAD⁺ as the oxidizing agent.  Mitochondrial ALDH2 exists as a 220-kD homotetrameric protein containing a catalytic, nucleotide-binding, and coenzyme-binding domain.  The most prevalent ALDH2 mutation (ALDH2*2) involves an E to K (E478K) substitution in the catalytic domain that leads to decreased enzymatic activity.Scientists hypothesize that Asian populations have higher rates of ALDH2*2 incidence.  The properties of ALDH2 variants in Caucasian and Japanese populations were analyzed in the following experiments.Experiment 1Mitochondrial extracts from both populations were combined in a single mixture and separated by native gel electrophoresis.  Proteins 210-230 kD in size were removed from the agarose gel and further isolated using affinity columns with antibodies designed to separate wild-type ALDH2 from ALDH2*2 by binding the catalytic domain of the wild-type but not the mutant protein.  Concentrated eluates E1 and E2 were produced by washing the affinity columns with buffers that remove unbound proteins and dislodge bound proteins from antibodies, respectively (Figure 1). Figure 1  Purification of ALDH2 proteinsALDH2 activity in each eluate was quantified by adding acetaldehyde and NAD⁺, and then monitoring the rate of NADH production.  The results are shown in Figure 2. Figure 2  Enzymatic activity of E1 and E2 eluatesExperiment 2Researchers predict that mutations at conserved amino acid sites containing S or T may prevent ALDH2 from being phosphorylated by protein kinase C epsilon (εPKC).  The activity of wild-type ALDH2 (WT) and ALDH2 mutants with substitutions at positions T185 and S279 in the presence or absence of εPKC are summarized in Table 1.Table 1  Effect of εPKC on ALDH2 Variants Adapted from Chen CH, Ferreira JC, Gross ER, Mochly-rosen D. Targeting aldehyde dehydrogenase 2: new therapeutic opportunities. Physiol Rev. 2014;94(1):1-34. -Increasing the concentration of sodium chloride (NaCl) in the E2 eluate could affect the rate of NADH production in what way?

Passage Upon entering muscle cells, glucose is immediately phosphorylated by hexokinase to produce glucose 6-phosphate (G6P).  This phosphorylation event prevents glucose from exiting the cell.  G6P may then enter one of three metabolic pathways, depending on the needs of the cell (Figure 1). Figure 1  Three potential fates of G6P, determined by the needs of the cellUnder low ATP conditions, G6P enters glycolysis, where it is converted to two pyruvate molecules.  The energy released in this process produces a net total of two ATP molecules per glucose molecule consumed.  The resulting pyruvate molecules can then enter the mitochondria, where they participate in additional metabolic reactions that produce more ATP.When the cell experiences oxidative stress, G6P enters the pentose phosphate pathway (PPP) and produces NADPH, a reducing agent.  During this process, G6P loses one carbon atom in the form of CO₂ and is converted to ribose 5-phosphate (R5P), which is required for nucleotide synthesis.  In addition to low NADPH conditions, G6P may also enter the PPP during replication, when nucleotides are needed for DNA synthesis.  If nucleotides are not needed but energy is, R5P can be converted back to glycolytic intermediates and reenter glycolysis.  Each glucose molecule that is converted to R5P prior to glycolysis yields an average of 1.67 ATP molecules.When all the cell's metabolic requirements are met, additional G6P can enter glycogenesis and produce glycogen, which stores glucose for later use; this process consumes ATP.  When signal molecules bind G protein-coupled receptors on muscle cells, glycogenolysis is activated to release individual monomers from glycogen, allowing them to enter the appropriate pathway. -Under low ATP conditions, which of the following enzymes would be LEAST beneficial to the cell?

Passage Destabilase, an enzyme found in the saliva of leeches, exhibits at least two activities: isopeptidase activity and glycosidase activity.  Isopeptidase activity is believed to prevent blood clotting by hydrolyzing the isopeptide bond between the ε-amino group of lysine and the γ-carboxyl group of glutamate in fibrin monomers.  The enzyme's glycosidase activity digests sugars found in the peptidoglycans of bacterial cell walls and may play a role in innate immunity.Two isoforms of destabilase were tagged with a polyhistidine tail and recombinantly expressed in E. coli cells.  Both isoforms were then purified by affinity chromatography and eluted by imidazole, which competes with histidine-tagged proteins for column binding.  Each isoform eluted efficiently with 300 mM imidazole.  Five microliters of each purified enzyme were then provided with saturating levels of substrate and assessed for isopeptidase and glycosidase activity at various pH levels, which allowed the optimal pH of enzymatic activities for each isoform to be identified (Figure 1).  Further studies showed that at optimal pH, both isoforms had approximately the same k_cat values, but isoform 2 had a lower K_m than isoform 1 for both enzymatic activities. Figure 1  Isopeptidase activity (A) and glycosidase activity (B) of 5 μL of destabilase isoforms 1 and 2 at various pH valuesIsoform 2 was further characterized by mutating specific residues and measuring enzymatic activities at optimal pH.  The results are shown in Figure 2. Figure 2  Isopeptidase and glycosidase activity of isoform 2 of destabilase with various mutations - The Lineweaver-Burk plot shown above depicts the glycosidase activity of isoform 1.  If both isoforms were tested at equal enzyme concentrations, which of the following would describe the appearance of the plot for isoform 2 relative to isoform 1?

Passage Glycolysis and gluconeogenesis are tightly regulated, opposing metabolic pathways that help control blood glucose levels.  Glycolysis converts glucose to two pyruvate molecules, whereas gluconeogenesis consumes 6 ATP equivalents to convert two pyruvate molecules back to glucose.  When glycolysis is upregulated, gluconeogenesis is downregulated, and vice versa.As shown in Figure 1, glycolysis and gluconeogenesis in the liver are largely regulated by the allosteric action of the small molecule fructose-2,6-bisphosphate (F2,6BP) on the enzymes phosphofructokinase-1 (PFK-1) and fructose-1,6-bisphosphatase (F1,6BPase).  PFK-1 is a kinase that uses ATP to phosphorylate fructose-6-phosphate (F6P) in an irreversible step of glycolysis, forming fructose-1,6-bisphosphate (F1,6BP) and ADP.  During gluconeogenesis, F1,6BPase removes the phosphate group by hydrolysis. Figure 1  Activities of (A) PFK-1 and (B) F1,6BPase in the presence (solid lines) and absence (dashed lines) of F2,6BPA bifunctional enzyme that contains a phosphofructokinase-2 (PFK-2) domain and a fructose-2,6-bisphosphatase (F2,6BPase) domain controls F2,6BP levels in the liver.  The PFK-2 domain converts F6P to F2,6BP, and the F2,6BPase domain converts F2,6BP back to F6P.  When blood glucose levels are low, the enzyme becomes phosphorylated.  This phosphorylation event simultaneously activates the F2,6BPase domain and inactivates the PFK-2 domain.  Under high blood glucose conditions, the enzyme becomes dephosphorylated, activating the PFK-2 domain and inactivating the F2,6BPase domain. -Which metabolic process most likely provides the energy necessary for sustained gluconeogenesis?

Which amino acid substitution would result in the greatest change in the net charge of a polypeptide at pH 7?

Which series depicts the order in which the precursors of steroid hormones are synthesized?

Passage Dystroglycan (Dg) is a transmembrane protein that mediates interactions between the cytoskeleton and the extracellular matrix (ECM).  It binds the ECM protein laminin in an interaction that is required for intercellular communication and muscle membrane structural stability.  The Dg-laminin interaction is facilitated by a long carbohydrate chain called an O-mannosyl glycan, which is covalently linked to Dg (Figure 1). Figure 1  Schematic of interaction between Dg and laminin, mediated by the Dg-linked O-mannosyl glycanThe O-mannosyl glycan is added to Dg by a series of enzymes called glycosyltransferases.  The first enzyme in this series, protein O-mannosyltransferase (POMT), attaches D-mannose to the hydroxyl groups of serine and threonine residues in an α-linkage through the anomeric carbon.  The next sugar in the chain then forms a β-1,4 linkage to the mannose residue, followed by several more steps to produce the full glycan.  Genetic inactivation of any of the enzymes in the pathway results in an incomplete glycan and failure to bind laminin, and is the underlying cause of several severe forms of muscular dystrophy known as dystroglycanopathies.Researchers investigating dystroglycanopathies recently discovered the functions of two enzymes in the pathway:  protein O-mannose kinase (POMK) and fukutin.  POMK phosphorylates Dg-linked mannose at carbon 6, and fukutin adds the sugar ribitol 5-phosphate to the glycan chain.  To assess the roles of POMK and fukutin relative to each other, scientists conducted experiments in which lysates from muscle cells deficient in either POMK or fukutin were incubated with ³²P-labeled ATP or ³H-labeled activated ribitol 5-phosphate.  Combined lysates containing both fukutin and POMK were also tested.  Dg was then purified and detected by autoradiography and western blot using an antibody against Dg, as shown in Figure 2. Figure 2  Autoradiograms and western blots of Dg purified from lysates incubated with ³²P-labeled ATP or ³H-labeled ribitol 5-phosphate Adapted from Kanagawa M, Kobayashi K, Tajiri M, et al. Identification of a Post-translational Modification with Ribitol-Phosphate and Its Defect in Muscular Dystrophy. Cell Rep. 2016;14(9):2209-23. -Alpha mannosidase cleaves the link between D-mannose and the hydroxyl group of serine or threonine.  If researchers treated the radiolabeled Dg from each autoradiogram with alpha mannosidase, where would the radiolabels be found?