金属硫蛋白对羟自由基损伤的大鼠肝细胞核核苷三磷酸酶的保护作用

目的:研究金属硫蛋白(MTs)是否对羟自由基(hydroxylradical,·OH^-)损伤的大鼠肝细胞核核苷三磷酸酶(NTPase)具有保护作用。方法:以羟自由基发生系统Fe³⁺/H₂O₂单独或与MTs共同孵育大鼠离体肝细胞核,检测分别用ATP和GTP作底物时大鼠肝细胞核NTPase活性。结果:不同浓度Fe³⁺/H₂O₂(μmol·L^-1/μmol·L^-1:0.1/0.5、0.5/2.5、1/5、5/25)孵育肝细胞核,浓度依赖地增强核NTPase活性,与对照组差异显著(P＜0.01)。用不同浓度的MT(10^-9-10^-4mol·L^-1)与Fe²⁺/H₂O₂(1μmol·L^-1/5μmol·L^-1)共孵育,浓度依赖地拮抗Fe³⁺/H₂O₂诱导的效应(P＜0.01)。用MT单独孵育肝细胞核对NTPase的活性没有影响(P＞0.05)。结论:Fe³⁺/H₂O₂系统产生的·OH对核NTPase活性具有强烈的抑制效应,MT浓度依赖地拮抗·OH导致的NTPase活性降低。

Metallothionein protects rat hepatic nuclear nucleoside triphosphatase from hydroxyl radical-induced suppression

AIM:Metallothioneins (MTs) are cysteine-rich metal-binding proteins that exert cytoprotection during metal exposure and oxidative stress. The present study was designed to investigate whether MT can directly protect NTPase on nuclear envelope from damage induced by hydroxyl radical.METHODS:Isolated hepatic nuclei from rat liver were exposed to Fe²⁺/H₂O₂ with or without MT, and the NTPase activity on nuclei was assayed using ATP and GTP as substrate, respectively.RESULTS:Incubation of rat hepatic nuclei with the Fe²⁺/H₂O₂ (in μmol·L^-1/μmol·L^-1 : 0.1/0.5, 0.5/2.5, 1/5, 5/25) resulted in a concentration-dependent decrease in nuclear NTPase activities (P＜0.01). Incubation of hepatic nuclei with different concentrations of MT (10^-9-10^-4mol·L^-1)and Fe²⁺/H₂O₂ (1 μmol·L^-1/5 μmol·L^-1) for 10 min, nuclear NTPase activities were increased in a MT concentration-dependent fashion as compared with that of incubation with Fe²⁺/H₂O₂(1 μmol·L^-1/5 μmol·L^-1) alone. When MT was at 10^-4 mol·L^-1, TNPase activities reversed to (102±10) nmol·mg^-1 protein·min^-1(for ATP as substrated) and (131±12) μmol·g^-1 protein·min^-1(for GTP as substrate), which had no significant defferences from that of the controls (112±8 and 142±10 μmol·g^-1 protein·min^-1, respectively) (P＞0.05). In addition, incubation of hepatic nuclei with only MT had no effect on nuclear NTPase activity. CONCLUSION:These data demonstrate that hydroxyl radical generated from Fe²⁺/H₂O₂ might attack nuclear NTPase. MT antagonistically reduces toxicity of Fe²⁺/H₂O₂ system to the NTPase.