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Characterization of the East Asian variant of aldehyde dehydrogenase-2: bioactivation of nitroglycerin and effects of Alda-1.

Beretta M, Gorren AC, Wenzl MV, Weis R, Russwurm M, Koesling D, Schmidt K, Mayer B - J. Biol. Chem. (2009)

Bottom Line: In the present study we compared the wild type enzyme (ALDH2*1) with the Asian variant (ALDH2*2) regarding GTN bioactivation and the effects of Alda-1.The present results indicate that Alda-1 stimulates established ALDH2 activities by improving NAD binding but does not improve the GTN binding affinity of the Asian variant.In addition, our data revealed an unexpected discrepancy between GTN reductase activity and sGC activation, suggesting that GTN denitration and bioactivation may reflect independent pathways of ALDH2-catalyzed GTN biotransformation.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology and Toxicology, Karl-Franzens-Universität Graz, 8010 Graz, Austria

ABSTRACT
The East Asian variant of mitochondrial aldehyde dehydrogenase (ALDH2) exhibits significantly reduced dehydrogenase, esterase, and nitroglycerin (GTN) denitrating activities. The small molecule Alda-1 was reported to partly restore low acetaldehyde dehydrogenase activity of this variant. In the present study we compared the wild type enzyme (ALDH2*1) with the Asian variant (ALDH2*2) regarding GTN bioactivation and the effects of Alda-1. Alda-1 increased acetaldehyde oxidation by ALDH2*1 and ALDH2*2 approximately 1.5- and 6-fold, respectively, and stimulated the esterase activities of both enzymes to similar extent as the coenzyme NAD. The effect of NAD was biphasic with pronounced inhibition occurring at > or = 5 mM. In the presence of 1 mM NAD, Alda-1 stimulated ALDH2*2-catalyzed ester hydrolysis 73-fold, whereas the NAD-stimulated activity of ALDH2*1 was inhibited because of 20-fold increased inhibitory potency of NAD in the presence of the drug. Although ALDH2*2 exhibited 7-fold lower GTN denitrating activity and GTN affinity than ALDH2*1, the rate of nitric oxide formation was only reduced 2-fold, and soluble guanylate cyclase (sGC) activation was more pronounced than with wild type ALDH2 at saturating GTN. Alda-1 caused slight inhibition of GTN denitration and did not increase GTN-induced sGC activation in the presence of either variant. The present results indicate that Alda-1 stimulates established ALDH2 activities by improving NAD binding but does not improve the GTN binding affinity of the Asian variant. In addition, our data revealed an unexpected discrepancy between GTN reductase activity and sGC activation, suggesting that GTN denitration and bioactivation may reflect independent pathways of ALDH2-catalyzed GTN biotransformation.

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Dependence of sGC activation on GTN (A) and ALDH2 (B) concentration. Purified sGC (50 ng) was incubated at 37 °C for 10 min in the presence of 0.5 mm [α-32P]GTP (∼250 000 cpm), 3 mm MgCl2, 1 mm cGMP, 2 mm DTT and 0.1 mm DTPA. A: Activation of sGC by increasing concentrations of GTN in the presence of ALDH2*1 or ALDH2*2 (25 μg each). B: Activation of sGC by 0.1 mm GTN in the presence of the indicated amounts of ALDH2*1 or ALDH2*2. [32P]cGMP was isolated and quantified as described in “Experimental Procedures”. Data are mean values ± S.E. of three independent experiments.
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Figure 2: Dependence of sGC activation on GTN (A) and ALDH2 (B) concentration. Purified sGC (50 ng) was incubated at 37 °C for 10 min in the presence of 0.5 mm [α-32P]GTP (∼250 000 cpm), 3 mm MgCl2, 1 mm cGMP, 2 mm DTT and 0.1 mm DTPA. A: Activation of sGC by increasing concentrations of GTN in the presence of ALDH2*1 or ALDH2*2 (25 μg each). B: Activation of sGC by 0.1 mm GTN in the presence of the indicated amounts of ALDH2*1 or ALDH2*2. [32P]cGMP was isolated and quantified as described in “Experimental Procedures”. Data are mean values ± S.E. of three independent experiments.

Mentions: Bioactivation of GTN was assayed by coincubation of ALDH2 with purified sGC and determination of GTN-induced cGMP formation. As shown in Fig. 2A, GTN caused biphasic activation of sGC in the presence of both ALDH2 variants with apparent EC50 values of 3.4 ± 0.20 and 15 ± 1.5 μm for ALDH2*1 and ALDH2*2, respectively. Although the ∼5-fold lower apparent GTN affinity of the East Asian variant agrees well with the denitration data shown in Fig. 1A, the even slightly increased maximal effect on cGMP caused by ALDH2*2 (6.0 ± 0.23 versus 4.6 ± 0.12 μmol cGMP × min−1 × mg−1 at 0.1 mm GTN) was unexpected considering the lower GTN reductase activity of the Asian variant. The apparent discrepancy between GTN denitration and sGC activation became even more evident at higher ALDH2 concentrations. Formation of cGMP increased with both enzymes until a limit was reached at ∼100 μg of ALDH2 (Fig. 2B). However, whereas ALDH2*2 caused maximal sGC activation (18 ± 0.94 μmol cGMP × min−1 × mg−1) as determined with the NO donor DEA/NO (20 ± 0.06 μmol × min−1 × mg−1), the maximal effect of ALDH2*1 was 2-fold lower (9.0 ± 0.38 μmol × min−1 × mg−1).


Characterization of the East Asian variant of aldehyde dehydrogenase-2: bioactivation of nitroglycerin and effects of Alda-1.

Beretta M, Gorren AC, Wenzl MV, Weis R, Russwurm M, Koesling D, Schmidt K, Mayer B - J. Biol. Chem. (2009)

Dependence of sGC activation on GTN (A) and ALDH2 (B) concentration. Purified sGC (50 ng) was incubated at 37 °C for 10 min in the presence of 0.5 mm [α-32P]GTP (∼250 000 cpm), 3 mm MgCl2, 1 mm cGMP, 2 mm DTT and 0.1 mm DTPA. A: Activation of sGC by increasing concentrations of GTN in the presence of ALDH2*1 or ALDH2*2 (25 μg each). B: Activation of sGC by 0.1 mm GTN in the presence of the indicated amounts of ALDH2*1 or ALDH2*2. [32P]cGMP was isolated and quantified as described in “Experimental Procedures”. Data are mean values ± S.E. of three independent experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2801295&req=5

Figure 2: Dependence of sGC activation on GTN (A) and ALDH2 (B) concentration. Purified sGC (50 ng) was incubated at 37 °C for 10 min in the presence of 0.5 mm [α-32P]GTP (∼250 000 cpm), 3 mm MgCl2, 1 mm cGMP, 2 mm DTT and 0.1 mm DTPA. A: Activation of sGC by increasing concentrations of GTN in the presence of ALDH2*1 or ALDH2*2 (25 μg each). B: Activation of sGC by 0.1 mm GTN in the presence of the indicated amounts of ALDH2*1 or ALDH2*2. [32P]cGMP was isolated and quantified as described in “Experimental Procedures”. Data are mean values ± S.E. of three independent experiments.
Mentions: Bioactivation of GTN was assayed by coincubation of ALDH2 with purified sGC and determination of GTN-induced cGMP formation. As shown in Fig. 2A, GTN caused biphasic activation of sGC in the presence of both ALDH2 variants with apparent EC50 values of 3.4 ± 0.20 and 15 ± 1.5 μm for ALDH2*1 and ALDH2*2, respectively. Although the ∼5-fold lower apparent GTN affinity of the East Asian variant agrees well with the denitration data shown in Fig. 1A, the even slightly increased maximal effect on cGMP caused by ALDH2*2 (6.0 ± 0.23 versus 4.6 ± 0.12 μmol cGMP × min−1 × mg−1 at 0.1 mm GTN) was unexpected considering the lower GTN reductase activity of the Asian variant. The apparent discrepancy between GTN denitration and sGC activation became even more evident at higher ALDH2 concentrations. Formation of cGMP increased with both enzymes until a limit was reached at ∼100 μg of ALDH2 (Fig. 2B). However, whereas ALDH2*2 caused maximal sGC activation (18 ± 0.94 μmol cGMP × min−1 × mg−1) as determined with the NO donor DEA/NO (20 ± 0.06 μmol × min−1 × mg−1), the maximal effect of ALDH2*1 was 2-fold lower (9.0 ± 0.38 μmol × min−1 × mg−1).

Bottom Line: In the present study we compared the wild type enzyme (ALDH2*1) with the Asian variant (ALDH2*2) regarding GTN bioactivation and the effects of Alda-1.The present results indicate that Alda-1 stimulates established ALDH2 activities by improving NAD binding but does not improve the GTN binding affinity of the Asian variant.In addition, our data revealed an unexpected discrepancy between GTN reductase activity and sGC activation, suggesting that GTN denitration and bioactivation may reflect independent pathways of ALDH2-catalyzed GTN biotransformation.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology and Toxicology, Karl-Franzens-Universität Graz, 8010 Graz, Austria

ABSTRACT
The East Asian variant of mitochondrial aldehyde dehydrogenase (ALDH2) exhibits significantly reduced dehydrogenase, esterase, and nitroglycerin (GTN) denitrating activities. The small molecule Alda-1 was reported to partly restore low acetaldehyde dehydrogenase activity of this variant. In the present study we compared the wild type enzyme (ALDH2*1) with the Asian variant (ALDH2*2) regarding GTN bioactivation and the effects of Alda-1. Alda-1 increased acetaldehyde oxidation by ALDH2*1 and ALDH2*2 approximately 1.5- and 6-fold, respectively, and stimulated the esterase activities of both enzymes to similar extent as the coenzyme NAD. The effect of NAD was biphasic with pronounced inhibition occurring at > or = 5 mM. In the presence of 1 mM NAD, Alda-1 stimulated ALDH2*2-catalyzed ester hydrolysis 73-fold, whereas the NAD-stimulated activity of ALDH2*1 was inhibited because of 20-fold increased inhibitory potency of NAD in the presence of the drug. Although ALDH2*2 exhibited 7-fold lower GTN denitrating activity and GTN affinity than ALDH2*1, the rate of nitric oxide formation was only reduced 2-fold, and soluble guanylate cyclase (sGC) activation was more pronounced than with wild type ALDH2 at saturating GTN. Alda-1 caused slight inhibition of GTN denitration and did not increase GTN-induced sGC activation in the presence of either variant. The present results indicate that Alda-1 stimulates established ALDH2 activities by improving NAD binding but does not improve the GTN binding affinity of the Asian variant. In addition, our data revealed an unexpected discrepancy between GTN reductase activity and sGC activation, suggesting that GTN denitration and bioactivation may reflect independent pathways of ALDH2-catalyzed GTN biotransformation.

Show MeSH
Related in: MedlinePlus