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.
-The active site of wild-type ALDH2 most likely interacts with which of the following amino acid residues on the antibodies shown in Figure 1?

Question

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) .

Passage Aldehyde dehydrogenase 2 (ALDH2) is essential for the oxidation of toxic aldehydes, and uses NAD<sup>+</sup> 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) . <strong>Figure 1</strong> Purification of ALDH2 proteinsALDH2 activity in each eluate was quantified by adding acetaldehyde and NAD<sup>+</sup>, and then monitoring the rate of NADH production. The results are shown in Figure 2. <strong>Figure 2</strong> 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.<strong>Table 1</strong> 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. -The active site of wild-type ALDH2 most likely interacts with which of the following amino acid residues on the antibodies shown in Figure 1? A) Aspartate B) Phenylalanine C) Isoleucine D) Arginine

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.
-The active site of wild-type ALDH2 most likely interacts with which of the following amino acid residues on the antibodies shown in Figure 1?

Quizplus · Accepted Answer

11ecba2d_0df9_9e47_a3bf_49fc4b83a987_MD0008_00 Affinity chromatography is a separation technique that isolates proteins based on highly specific noncovalent binding interactions. Affinity columns can be created by attaching antibodies (proteins with high affinity for their targets) to beads known as the stationary phase, that serve as the solid support. When a mixture of proteins passes through the column, only the target protein is bound. Contaminant proteins and other molecules with low affinity for the antibody remain unbound and are washed out of the column. The affinity column shown in Figure 1 was specifically designed to separate wild-type ALDH2 from ALDH2*2 by selectively binding the catalytic domain of wild-type ALDH2. The first buffer wash removed the unbound ALDH2*2 and collected it in E1 whereas the second wash dislodged wild-type ALDH2 from antibodies. The catalytic domain of wild-type ALDH2 contains a negatively charged glutamate residue (E), which could interact with positively charged amino acids on the antibodies, such as arginine. Therefore, an arginine residue in the antibodies most likely facilitates binding to the active site of wild-type ALDH2. ALDH2*2, on the other hand, has positively charged lysine (K) in place of glutamate and will be repelled by positively charged residues on the antibody. (Choice A) The negatively charged carboxyl group in the R group of aspartate would repel glutamate in the catalytic domain of wild-type ALDH2. (Choices B and C) Phenylalanine and isoleucine are hydrophobic amino acids that do not participate in electrostatic interactions such as ionic bonds. Educational objective: Affinity chromatography separates proteins based on their specific noncovalent-binding interactions with antibodies or other ligands attached to a column. Negatively charged amino acids such as aspartate and glutamate interact with positively charged amino acids such as arginine, histidine, and lysine to form ionic bonds.

Passage Aldehyde Dehydrogenase 2 (ALDH2) Is Essential for the Oxidation of of Toxic