斑马鱼肌肉GPT酶的反应体系及对Nic敏感性研究

Study on the reaction system of GPT from zebrafish(Danio rerio)and its sensitivity to Niclosamide

以斑马鱼肌肉匀浆上清液为酶液,用L_(25)(5~6)正交和极(方)差分析确定GPT最佳反应条件:酶浓度1.6 mg/mL、α-酮戊二酸4 mmol/L、反应体系pH8.0、反应温度45℃及反应时间45 min,其中酶浓度、温度及时间对GPT活性影响均达到极显著水平(P<0.01).在GPT反应最适条件下测得0.1、0.2、0.3、0.4、0.5及0.6 mg/L的Nic急性水浴暴露斑马鱼4 d时,其活性诱导率分别为12.42、23.86、32.03、42.37、50.65及64.16%,Nic暴露浓度与GPT活性诱导呈良好的直线关系(y= 0.6259x+0.7036,R^2=0.9259).初步推定斑马鱼肌肉GPT是Nic致其生化毒性的靶点.

The zebrafish muscle tissue homogenate supernatants were used as enzyme solution. L25 （5^6） orthogonal matrix was adopted to determine the optimal reaction system of the enzyme solution. Through analyzing the data from orthogonal experiment with range analysis and variance analysis, we found that the optimal conditions were enzyme concentration 1.6 mg/mL, a-ketoglutarie acid concentration 4 umol/L, pH value of the reaction system 8.0,reaction temperature 45℃ ,and reaction time 45 min. The results revealed that the measuring of the activity of GPT were remarkably affected by enzyme concentration,reaction temperature and reaction time （p〈0.01）. After acute exposure to different concentrations of Nic （0.1,0.2,0.3,0.4,0.5 and 0.6 mg/L） 4 days,the GPT enzyme activities of the fishes were measured under the optimal conditions. The results showed that the induction percentages of GPT activity of the fishes were 12.42, 23.86,32.03,42.37,50.65 and 64.16%, respectively, compared to the control. There was a significant liner regression between the concentration of Nic and GPT activities which were inducted by Nic（y=-0. 6259x＋0. 7036 ,R^2 =0. 9259）. Preliminary analysis showed that GPT is one target of biochemical toxicity which caused by Nic.