title: Detection of N-(hexanoyl)lysine in the tropomyosin 1 protein in N-methyl-N'-nitro-N-nitrosoguanidine-induced rat gastric cancer cells.
abstract: N(ε)-(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 N(ε)-(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. N(ε)-(Hexanoyl)lysine-modified proteins were detected by SDS-PAGE or two-dimensional electrophoresis and Western blotting using anti-N(ε)-(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 N(ε)-(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 N(ε)-(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 N(ε)-(hexanoyl)lysine modification of tropomyosin 1 may be related to neoplastic transformation by N-methyl-N'-nitro-N-nitrosoguanidine in gastric epithelial cells.
authors.affiliation: Molecular Gastroenterology and Hepatology, Kyoto Prefectural University of Medicine, Kamigyo-ku, Kyoto 602-8566, Japan.
authors.forename: Hitomi
authors.initials: H
authors.lastname: Okada
authors.forename: Yuji
authors.initials: Y
authors.lastname: Naito
authors.forename: Tomohisa
authors.initials: T
authors.lastname: Takagi
authors.forename: Megumi
authors.initials: M
authors.lastname: Takaoka
authors.forename: Tomoko
authors.initials: T
authors.lastname: Oya-Ito
authors.forename: Kohei
authors.initials: K
authors.lastname: Fukumoto
authors.forename: Kazuhiko
authors.initials: K
authors.lastname: Uchiyama
authors.forename: Osamu
authors.initials: O
authors.lastname: Handa
authors.forename: Satoshi
authors.initials: S
authors.lastname: Kokura
authors.forename: Yumiko
authors.initials: Y
authors.lastname: Nagano
authors.forename: Hirofumi
authors.initials: H
authors.lastname: Matsui
authors.forename: Yoji
authors.initials: Y
authors.lastname: Kato
authors.forename: Toshihiko
authors.initials: T
authors.lastname: Osawa
authors.forename: Toshikazu
authors.initials: T
authors.lastname: Yoshikawa