Blackburn M L, Podgorski I, Bull A W
Department of Chemistry, Oakland University, Rochester, MI 48309-4477, USA.
Biochim Biophys Acta. 1999 Sep 22;1440(2-3):225-34. doi: 10.1016/s1388-1981(99)00123-7.
The linoleic acid metabolite, 13-oxooctadecadienoic acid (13-OXO), is reactive with cellular thiols. In the present report, incubations of HT-29 or CaCo-2 homogenates with 13-OXO and GSH indicate that HT-29 cell homogenates produce a 13-OXO-GSH conjugate. The conjugate formed was likely of enzymatic origin as chiral-phase HPLC showed the major product consisted of only one of two possible diastereomers. The glutathione transferase activity (GST), using chlorodinitrobenzene, was found to be 126 nmol/mg/min in HT-29 cells and 21 nmol/mg/min in CaCo-2 cells. These levels of activity are consistent with the relative ability of the two cell lines to conjugate GSH to 13-OXO. Incubation of intact HT-29 cells with either 13-OXO, or the metabolic precursor 13-hydroxyoctadecadienoic acid (13-HODE), showed detectable 13-OXO-GSH conjugate in the media, but none in the cells. The stereochemistry of the extracellular conjugate suggested an enzymatic origin. In additional experiments, the labeling of cellular protein by 13-HODE was much more specific than the labeling of protein by 13-OXO suggesting that in situ generation of 13-OXO from 13-HODE confers selectivity on the reactions between cellular thiols and 13-OXO. These results demonstrate that in HT-29 cells, 13-HODE is converted to 13-OXO which then either reacts with cellular protein or is conjugated to GSH by GST. The 13-OXO-GSH conjugate is then exported from the cell.
亚油酸代谢产物13 - 氧代十八碳二烯酸（13 - OXO）可与细胞内的硫醇发生反应。在本报告中，HT - 29或CaCo - 2匀浆与13 - OXO和谷胱甘肽（GSH）一起孵育表明，HT - 29细胞匀浆产生了一种13 - OXO - GSH共轭物。所形成的共轭物可能起源于酶促反应，因为手性相高效液相色谱显示主要产物仅由两种可能的非对映异构体中的一种组成。使用氯二硝基苯测定的谷胱甘肽转移酶（GST）活性在HT - 29细胞中为126 nmol/mg/min，在CaCo - 2细胞中为21 nmol/mg/min。这些活性水平与两种细胞系将GSH与13 - OXO共轭的相对能力一致。用13 - OXO或代谢前体13 - 羟基十八碳二烯酸（13 - HODE）孵育完整的HT - 29细胞，在培养基中可检测到13 - OXO - GSH共轭物，但在细胞中未检测到。细胞外共轭物的立体化学表明其起源于酶促反应。在额外的实验中，13 - HODE对细胞蛋白的标记比13 - OXO对蛋白的标记更具特异性，这表明13 - HODE原位生成13 - OXO赋予了细胞硫醇与13 - OXO之间反应的选择性。这些结果表明，在HT - 29细胞中，13 - HODE转化为13 - OXO，然后13 - OXO要么与细胞蛋白反应，要么通过GST与GSH共轭。然后，13 - OXO - GSH共轭物从细胞中输出。
