Degradation of glyceraldehyde-3-phosphate dehydrogenase triggered by 4-hydroxy-2-nonenal and 4-hydroxy-2-hexenal
Section snippets
Chemicals
HNE and HHE were purchased from Cayman Chemical (Ann Arbor, MI). Human erythrocyte GAPDH (e-GAPDH) and glyceraldehyde-3-phosphate (GAP) were from Sigma–Aldrich (St. Louis, MO). β-NAD was from Oriental Yeast (Tokyo, Japan). An anti-human GAPDH monoclonal antibody was from Biogenesis (6G5, England, UK). An anti-human GAPDH polyclonal antibody was from Santa Cruz Biotechnology (FL-335, Delaware Avenue, CA). The fluorogenic peptides of l-alanyl-l-alanyl-l-phenylalanine-4-methyl-coumaryl-7-amide
Inhibition and degradation of GAPDH by incubation with lipid peroxidation products
First, we tested the effects of various aldehydes produced by lipid peroxidation on GAPDH activity. Either HNE or HHE strongly inhibited the GAPDH activity of the U937 cell extract and e-GAPDH activity in the IC50 range of 21.0–50.0 μM. However, the other four aldehydes tested, crotonaldehyde, 2-hexenal, 2-nonenal, and glyoxal, did not (IC50 > 1 mM) (Table 1). We then tested the effects of these aldehydes on triggering the degradation of GAPDH in the U937 cell extract (Fig. 1). The density of the
Discussion
Aldehydes produced by lipid peroxidation reactions are mainly classified into three families [3]. HHE and HNE belong to the family of 4-hydroxy-2-alkenals; 2-hexenal, 2-nonenal, and crotonaldehyde to the family of 2-alkenals; and glyoxal to the family of ketoaldehydes. In the present study, HHE and HNE inhibited GAPDH activity with low IC50 values and triggered GAPDH degradation in the U937 cell extract. On the other hand, 2-hexenal, 2-nonenal, crotonaldehyde, and glyoxal did not decrease GAPDH
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