色谱

色谱

• 研究论文 • 上一篇    下一篇

敬钊毒素-V剪切体Y1-JZTX-V的化学合成与氧化复性及其钠通道活性鉴定

全妙华1,2,曾雄智1,皮建辉1,2,邓梅春1,梁宋平1   

  1. 1.The Key Laboratory of Protein Chemistry and Developmental Biology of Ministry of Education,
    Hunan Normal University, Changsha 410081, China; 2.Department of Bioengineering,
    Huaihua University, Huaihua 418008, China
  • 收稿日期:2007-01-16 修回日期:2007-03-17 出版日期:2007-07-30 发布日期:1985-06-25

Synthesis and Oxidative Refolding of an N-Terminal Truncate of Jingzhaotoxin-V and Characterization of Its Activities of Sodium Channels

QUAN Miaohua1,2, ZENG Xiongzhi1, PI Jianhui1,2, DENG Meichun1, LIANG Songping1   

  1. 1.The Key Laboratory of Protein Chemistry and Developmental Biology of Ministry of Education,
    Hunan Normal University, Changsha 410081, China; 2.Department of Bioengineering,
    Huaihua University, Huaihua 418008, China
  • Received:2007-01-16 Revised:2007-03-17 Online:2007-07-30 Published:1985-06-25

PDF

566

摘要： 应用芴甲氧羰基(Fmoc)固相方法化学合成了敬钊毒素-V（JZTX-V）分子N-端酪氨酸残基剪切体(Y1-JZTX-V),并且通过反相高效液相色谱和质谱对不同条件下的氧化复性结果进行监测,从而得到该剪切体的最佳氧化复性条件:0.1 mol/L Tris-HCl缓冲液、pH 7.50、1 mmol/L还原型谷胱甘肽(GSH)、0.1 mmol/L氧化型谷胱甘肽(GSSG)、样品浓度为0.05 mg/L、复性温度为4 ℃。膜片钳电生理实验结果显示敬钊毒素-V剪切体Y1-JZTX-V对大鼠背根神经节(DRG)细胞上表达的河豚毒素不敏感型(TTX-R)与河豚毒素敏感型(TTX-S)钠电流均有抑制作用,其半数抑制浓度(IC50)分别为(160±2.5) nmol/L和(39.6±3.2) nmol/L。与天然的敬钊毒素-V相比,该剪切体对大鼠DRG细胞上的TTX-S钠电流的抑制作用基本一致,但对TTX-R钠电流的抑制作用却大大降低,表明敬钊毒素-V分子N-端的酪氨酸残基是一个与TTX-R钠通道结合活性相关的氨基酸残基。

关键词: 复性, 化学合成, 敬钊毒素-V剪切体, 钠离子通道

Abstract:

An N-terminal tyrosine residue truncate of Jingzhaotoxin-V (Y1-JZTX-V) was synthesized by solid-phase chemical methods using Fmoc-protected amino acids. Reversed-phase high performance liquid chromatography (RP-HPLC) and matrix assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF/TOF MS) were used to monitor the oxidative refolding of Y1-JZTX-V to find the optimal renaturation conditions of the synthetic linear peptide. When Y1-JZTX-V (0.05 mg/L) was dissolved in 0.1 mol/L Tris-HCl buffer containing 1 mmol/L GSH and 0.1 mmol/L GSSG at pH 7.50 and 4 ℃, the best renaturation yield of the truncate toxin was obtained. Under the whole-cell patch-clamp mode, Y1-JZTX-V could inhibit tetrodotoxin-resistant (TTX-R) and tetrodotoxin-sensitive (TTX-S) sodium currents in adult rat dorsal root ganglion neurons with IC50 values of 160 nmol/L and 39.6 nmol/L, respectively. The inhibition potentiality of Y1-JZTX-V on TTX-S sodium currents was almost the same as the natural JZTX-V, while that on TTX-R sodium currents was obviously weakened. The IC50 value of Y1-JZTX-V on TTX-R sodium currents was 5.8 times as many as that of natural JZTX-V. Present findings indicated that the first tyrosine residue (Y1) in the N-terminal of JZTX-V was involved in the binding activities of JZTX-V to TTX-R sodium channels.

Key words: N-terminal tyrosine residue truncate of Jingzhaotoxin-V (Y1-JZTX-V), renaturation, sodium channels , chemical synthesis