摘要
目的 探讨龙葵碱诱导HepG2细胞凋亡的芳香胺N-乙酰化转移酶(NAT1的影响。方法 采用高效液相色谱法(HPLC,以对氨基苯甲酸(PABA为底物,以PABA被NAT1乙酰化为乙酰对氨基苯甲酸(Ac-PABA的量反应NAT1酶的活性。观察不同浓度、不同时间龙葵碱对完整HepG2细胞NAT1酶活性的影响;龙葵碱对HepG2细胞细胞质中NAT1酶活性的影响;通过改变底物PABA浓度,采用双倒数作图法,以底物PABA浓度的倒数(1/S对NAT1反应速率的倒数(1/V作直线,得出回归方程,计算Km和Vmax。结果 在NAT1酶活性测定中,龙葵碱能显著降低HepG2完整细胞NAT1的活性;龙葵碱能够降低HepG2细胞质内NAT1的活性;随着作用时间的增加Ac-PABA生成的量逐渐增加,但在相同作用时间段龙葵碱能显著降低Ac-PABA生成的量。动力学研究表明,以PABA为底物,对于HepG2完整细胞,阴性对照组的Km和Vmax分别为(1.04×10-3±8.36×10-5mmol·L -1、(1.64×10-4±9.57×10-6 nmol·106 cells-1,龙葵碱组的Km和Vmax分别为(1.06×10-3±6.97×10-5 mmol·L-1和(1.48×10-4±4.28×10-6 nmol·106 cells-1·h-1。对于HepG2细胞质,阴性对照组的Km和Vmax分别为(3.32×10-1±2.35×10-4mmol·L -1、(2.60×10-3±6.79×10-6 nmol·h-1·mg pro-1,龙葵碱组的Km和Vmax分别为(3.35×10-1±1.66×10-4 mmol·L-1和(2.22×10-3±8.12×10-6 nmol·h-1·mg(Pro-1,经统计学处理表明,对于HepG2完整细胞和细胞质,阴性对照组和龙葵碱组的Km没有差异,而Vmax差异显著。结论 龙葵碱是HepG2细胞NAT1酶的非竞争性抑制剂。龙葵碱通过作用于NAT1与PABA结合位点以外的其他位点抑制NAT1酶的活性而诱导HepG2细胞凋亡。
Abstract
OBJECTIVE To explore the relationship between N-acetyltransferase 1 activity and apoptosis inducing by solanine in HepG2 cell. METHODS The speed of PABA acetylation reaction mediated by NAT1 was teken as the indicator of the activity of NAT1 in intact HepG2 cells and their cytoplasm. HPLC was employed to determine the concentration of AC-PABA, the effects of different concentrations of solanine and time on the intact HepG2 cell or cytoplasm were investigated. Taking 1/S(the recip of PABA concentration and 1/V(the recip of reaction rate of NATase as coordinates, regression equation was obtanined using double reciprocal plot, and Km and Vmax were caculated. RESULTS Solanine could inhibit the activity of NAT1 in intace HepG2 cell and the cytoplasm. For intact HepG2 cells, Km and Vmax of control group were(1.04×10-3±8.36×10-5 mmol·L -1、(1.64×10-4±9.57×10-6 nmol·106 cells-1, respectively, Km and Vmax of the solanine group were(1.06×10-3±6.97×10-5 mmol·L-1 and (1.48×10-4±4.28×10-6 nmol·106 cells-1·h-1 respectively. For the cytoplasm of HepG2 cells, Km and Vmax of control group were(3.32×10-1±2.35×10-4 mmol·L -1 and(2.60×10-3±6.79×10-6 nmol·h-1·mg-1 Protein,Km and Vmax of solanine group were(3.35×10-1±1.66×10-4 mmol·L-1 and(2.22×10-3±8.12×10-6 nmol·h-1·mg-1 protein, respectively. For intact HepG2 cells and their cytoplasm, there was no difference between the Km of control group and that of solanine group, but there was remarkable difference between Vmax of control group and that of solanine group(P<0.01 for intact cell and P<0.05 for cytoplasm. CONCLUSION Solanine is a noncompetitive inhibitor of NAT1 in HepG2. Solanine induces HepG2 cell apoptosis by acting on some target point other than NAT1 and PABA binding sites.
关键词
龙葵碱 /
HepG2人肝癌细胞 /
N-乙酰化转移酶 /
米氏常数 /
最大反应速率
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Key words
solanine /
HepG2 cell /
N-acetyltransferase 1 /
michaelis constant /
maximum reaction rate
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高世勇 苏怡君 季宇彬 .
龙葵碱对HepG2细胞NAT1酶活性及动力学影响的研究[J]. 中国药学杂志, 2011, 46(8): 589-594
GO Shi-yong;SU Yi-jun;JI Yu-in;.
Study on the Effect of Solanine on N-acetyltransferase 1 Activity in HepG2 Cell[J]. Chinese Pharmaceutical Journal, 2011, 46(8): 589-594
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脚注
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