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.
-Replacing native gel electrophoresis with nonreducing sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (PAGE) during the purification step results in eluates with no enzymatic activity. The best explanation of this observation would be:

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. -Replacing native gel electrophoresis with nonreducing sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (PAGE) during the purification step results in eluates with no enzymatic activity. The best explanation of this observation would be: A) ALDH2 must retain its quaternary structure to catalyze reactions. B) the SDS-PAGE breaks disulfide bonds between ALDH2 subunits. C) ALDH2 has the mobility of a 55-kD protein in native PAGE. D) the primary structure of the ALDH2 active site has been modified.

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.
-Replacing native gel electrophoresis with nonreducing sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (PAGE) during the purification step results in eluates with no enzymatic activity. The best explanation of this observation would be:

Quizplus · Accepted Answer

11ecba2d_0df8_65c6_a3bf_19a77d3eaaab_MD0008_00 ALDH2 proteins were initially isolated by native polyacrylamide gel electrophoresis (PAGE), which is used to separate proteins by charge and molecular weight without affecting their three-dimensinal structure. In this procedure, an electric field is applied to a gel and proteins migrate toward one of the electrodes based on their mass and charge. Under these conditions, proteins maintain their biological function because they retain their tertiary and quaternary structure during native PAGE purification. In contrast, SDS-PAGE separates proteins only on the basis of molecular weight. To accomplish this, proteins are coated with sodium dodecyl sulfate (SDS), a detergent that confers a negative charge. Under these conditions, each protein in the gel has a similar charge and migrates toward the anode. Although this technique is superior for estimating the molecular weight of a protein, detergents such as SDS disrupt noncovalent interactions and cause proteins to unfold, or become denatured. As a result, proteins exist only in their primary structure (linear sequence of amino acids) and cannot perform their biological function. Replacing native PAGE with SDS-PAGE results in the denaturation of active ALDH2 tetramers. The lack of enzymatic activity in eluates obtained from SDS-PAGE purification indicates that ALDH2 proteins lost their quaternary structure, preventing them from functioning. (Choice B) Reducing agents can separate multisubunit proteins into individual subunits by cleaving disulfide bonds, but nonreducing SDS-PAGE does not contain reducing agents. (Choice C) ALDH2 would migrate as a 220-kD tetramer (four subunits) in native PAGE. (Choice D) SDS does not alter the primary structure (amino acid sequence) of proteins. Educational objective: Native polyacrylamide gel electrophoresis (PAGE) separates proteins by charge and mass without denaturing proteins whereas SDS-PAGE separates proteins only on the basis of mass. Sodium dodecyl sulfate (SDS) is a detergent that coats proteins with a negative charge and denatures proteins by disrupting noncovalent interactions. Proteins separated by SDS-PAGE are unfolded into their primary structure, and are unable to perform their biological functions.

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