title: Degradation of glyceraldehyde-3-phosphate dehydrogenase induced by acetylleucine chloromethyl ketone in U937 cells.
abstract: We examined whether any changes were induced in cellular proteins by an inhibitor of acylpeptide hydrolase (ACPH) (EC 3.4.19.1), acetylleucine chloromethyl ketone (ALCK), which was shown in our previous report to induce apoptosis of human U937 cells. Extract prepared from U937 cells in 0.05% Triton X-100-PBS was incubated with ALCK at 37 degrees, and then analyzed using SDS-PAGE. A 36kDa protein in the cell extract was decreased markedly during the incubation period. This protein was purified and identified as glyceraldehyde-3-phosphate dehydrogenase (GAPDH, EC 1.2.1.12) by its specific enzyme activity, N-terminal amino acid sequence, and Western blotting. Incubation of purified GAPDH with ALCK resulted in a decrease of GAPDH activity, but not in a decrease in the amount of GAPDH. The ALCK-induced GAPDH decrease in the cell extract was abrogated by co-incubation with a serine protease inhibitor, diisopropyl fluorophosphate, suggesting that GAPDH was first inactivated by ALCK, and subsequently degraded by a serine protease(s). GAPDH degradation was also observed in U937 cell cultures in the presence of ALCK. The significance of GAPDH inhibition in the apoptotic process is discussed.
authors.affiliation: Department of Hygienic Chemistry, Showa Pharmaceutical University, 3-3165 Higashitamagawagakuen, Machida, Tokyo 194-8543, Japan.
authors.forename: Mitsune
authors.initials: M
authors.lastname: Yamaguchi
authors.forename: Yukihiro
authors.initials: Y
authors.lastname: Tsuchiya
authors.forename: Toshiyuki
authors.initials: T
authors.lastname: Chikuma
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Hojo
mesh_terms: D000590:Amino Acid Chloromethyl Ketones / Q000494:pharmacology*; D004591:Electrophoresis, Polyacrylamide Gel; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000378:metabolism*; D006801:Humans; D008970:Molecular Weight; D011480:Protease Inhibitors / Q000494:pharmacology*; D020298:U937 Cells / Q000187:drug effects* / Q000201:enzymology
chemical_list: D000590:Amino Acid Chloromethyl Ketones; D011480:Protease Inhibitors; C121811:acetylleucine chloromethyl ketone; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases