Detection of Nε-(hexanoyl)lysine in the tropomyosin 1 protein in N-methyl-N'-nitro-N-nitrosoguanidine-induced rat gastric cancer cells

Nε-(Hexanoyl)lysine, formed by the reaction of lysine with n-6 lipid hydroperoxide, is a lipid peroxidation marker during the initial stage of oxidative stress. The aim of the present study is to indentify Nε-(hexanoyl)lysine-modified proteins in neoplastic transformed gastric mucosal cells by N-methyl-N’-nitro-N-nitrosoguanidine, and to compare the levels of these proteins between gastric mucosal cells and normal gastric cells. Much greater fluorescence of 2-[6-(4'-hydroxy)phenoxyl-3H-xanthen-3-on-9-yl]benzoic acid, an index of the intracellular levels of reactive oxygen species, was observed for gastric mucosal cells compared to normal gastric cells. Nε-(Hexanoyl)lysine-modified proteins were detected by SDS-PAGE or two-dimensional electrophoresis and Western blotting using anti-Nε-(hexanoyl)lysine polyclonal antibody, and a protein band of between 30–40 kDa was clearly increased in gastric mucosal cells compared to normal gastric cells. Two Nε-(hexanoyl)lysine-modified protein spots in gastric mucosal cells were identified as the tropomyosin 1 protein by mass spectrometry using a MASCOT search. The existence of Nε-(hexanoyl)lysine modification in tropomyosin 1 was confirmed by Western blotting of SDS-PAGE-separated or two-dimensional electrophoresis-separated proteins as well as by the immunoprecipitation with anti-tropomyosin 1 antibody. These data indicate that Nε-(hexanoyl)lysine modification of tropomyosin 1 may be related to neoplastic transformation by N-methyl-N'-nitro-N-nitrosoguanidine in gastric epithelial cells.