Sielicka A, Pelikant-Malecka I, Kaniewska E, Olkowicz M, Pankrac Z, Swiatkowska-Freund M, Preis K, Gorska M, Slominska E M, Smolenski R T
a Department of Biochemistry , Medical University of Gdansk , Gdansk , Poland.
Nucleosides Nucleotides Nucleic Acids. 2014;33(4-6):342-6. doi: 10.1080/15257770.2014.898069.
Mechanisms of free radical injury involve chemical modification of proteins, lipid derivatives and nucleic acids and consequent loss of its function. However, specific targets and exact sequence of events has not been fully clarified. We determined whether extracellular enzymes that are involved in adenosine formation such as ecto-5'nucleotidase (e5N) and removal such as extracellular form of adenosine deaminase (eADA) could be affected by peroxynitrite. We used intact cell assay system that involves exposure of cultured HMEC-1 cells to substrates followed by HPLC analysis of conversion of substrates into products. We found that e5N and ADA activities decreased by 20-40% after incubation for 20 or 60 minutes with 30 μM peroxynitrite. Decrease of cellular ATP and NAD was also observed. We conclude that besides other cytotoxic effects modification of extracellular enzymes of nucleotide metabolism could be important target for free radical injury.
自由基损伤的机制涉及蛋白质、脂质衍生物和核酸的化学修饰以及随之而来的功能丧失。然而，具体的靶点和确切的事件顺序尚未完全阐明。我们确定参与腺苷形成的细胞外酶，如ecto-5'核苷酸酶（e5N）和参与腺苷清除的酶，如细胞外形式的腺苷脱氨酶（eADA）是否会受到过氧亚硝酸盐的影响。我们使用完整细胞检测系统，即将培养的HMEC-1细胞暴露于底物，然后通过HPLC分析底物向产物的转化。我们发现，用30μM过氧亚硝酸盐孵育20或60分钟后，e5N和ADA活性降低了20-40%。还观察到细胞ATP和NAD的减少。我们得出结论，除了其他细胞毒性作用外，核苷酸代谢细胞外酶的修饰可能是自由基损伤的重要靶点。
