Molecular Gastroenterology and Hepatology, Kyoto Prefectural University of Medicine, Kamigyo-ku, Kyoto 602-8566, Japan.
J Clin Biochem Nutr. 2012 Jan;50(1):47-52. doi: 10.3164/jcbn.11-39. Epub 2011 Sep 3.
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.
N(ε)-(己酰基)赖氨酸是赖氨酸与 n-6 脂质氢过氧化物反应形成的一种脂质过氧化标记物，是氧化应激初始阶段的标志物。本研究旨在通过 N-甲基-N'-硝基-N-亚硝基胍鉴定肿瘤转化胃黏膜细胞中的 N(ε)-(己酰基)赖氨酸修饰蛋白，并比较胃黏膜细胞和正常胃细胞中这些蛋白的水平。2-[6-(4'-羟基)苯氧基-3H-呫吨-3-酮-9-基]苯甲酸的荧光强度明显大于正常胃细胞，这是细胞内活性氧水平的指标。通过 SDS-PAGE 或二维电泳和 Western 印迹用抗 N(ε)-(己酰基)赖氨酸多克隆抗体检测 N(ε)-(己酰基)赖氨酸修饰蛋白，与正常胃细胞相比，胃黏膜细胞中 30-40 kDa 的蛋白带明显增加。通过基质辅助激光解吸电离飞行时间质谱分析，用 MASCOT 搜索，在胃黏膜细胞中鉴定出两个 N(ε)-(己酰基)赖氨酸修饰蛋白斑点为原肌球蛋白 1 蛋白。通过 SDS-PAGE 分离或二维电泳分离蛋白的 Western 印迹以及用抗原肌球蛋白 1 抗体进行免疫沉淀，证实了原肌球蛋白 1 中存在 N(ε)-(己酰基)赖氨酸修饰。这些数据表明，N-甲基-N'-硝基-N-亚硝基胍诱导胃上皮细胞发生肿瘤转化时，原肌球蛋白 1 中的 N(ε)-(己酰基)赖氨酸修饰可能与之相关。
