title: Glyceraldehyde-3-phosphate dehydrogenase as a quinone reductase in the suppression of 1,2-naphthoquinone protein adduct formation.
abstract: 1,2-Naphthoquinone (1,2-NQ) is electrophilic, and forms covalent bonds with protein thiols, but its two-electron reduction product 1,2-dihydroxynaphthalene (1,2-NQH(2)) is not, so enzymes catalyzing the reduction with reduced pyridine nucleotides as cofactors could protect cells from electrophile-based chemical insults. To assess this possibility, we examined proteins isolated from the 9000g supernatant from mouse liver for 1,2-NQ reductase activity using an HPLC assay procedure for the hydroquinone of 1,2-NQ and Cibacron Blue 3GA column chromatography and Western blot analysis with specific antibody to determine 1,2-NQ-bound proteins. Among the proteins with high affinities for pyridine nucleotides that also inhibited 1,2-NQ-protein adduct formation in the presence of NADH, a 37-kDa protein was found and identified as glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Using recombinant human GAPDH, we found that this glycolytic enzyme indeed catalyzes the two-electron reduction of 1,2-NQ accompanied by extensive NADH consumption under 20% oxygen conditions. When either 1,2-NQH(2) or 1,2-NQ was incubated with GAPDH in the presence of NADH, minimal covalent bonding to the enzyme occurred compared to that in its absence. These results indicate that GAPDH can inhibit 1,2-NQ-based electrophilic protein modification by conversion to the nonelectrophilic 1,2-NQH(2) via an NADH-dependent process.
authors.affiliation: Doctoral Program in Biomedical Sciences, Graduate School of Comprehensive Human Sciences, University of Tsukuba, Tsukuba, Ibaraki, Japan.
authors.forename: Takashi
authors.initials: T
authors.lastname: Miura
authors.forename: Yasuhiro
authors.initials: Y
authors.lastname: Shinkai
authors.forename: Reiko
authors.initials: R
authors.lastname: Hirose
authors.forename: Noriko
authors.initials: N
authors.lastname: Iwamoto
authors.forename: Arthur K
authors.initials: AK
authors.lastname: Cho
authors.forename: Yoshito
authors.initials: Y
authors.lastname: Kumagai
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D055162:Biocatalysis; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000302:isolation & purification / Q000378:metabolism*; D006801:Humans; D008297:Male; D051379:Mice; D008813:Mice, Inbred ICR; D008969:Molecular Sequence Data; D009243:NAD / Q000378:metabolism; D016660:NAD(P)H Dehydrogenase (Quinone) / Q000302:isolation & purification / Q000378:metabolism*; D009285:Naphthoquinones / Q000737:chemistry / Q000378:metabolism*
chemical_list: D009285:Naphthoquinones; D009243:NAD; C040756:1,2-naphthoquinone; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases; D016660:NAD(P)H Dehydrogenase (Quinone)