荧光标记法研究鱼类GABA受体与Ro7-1986/1和Fipronil的相互作用

doi:10.7503/cjcu20140942

高等学校化学学报 ›› 2015, Vol. 36 ›› Issue (3): 456.doi: 10.7503/cjcu20140942

荧光标记法研究鱼类GABA受体与Ro7-1986/1和Fipronil的相互作用

杨姗¹, 张博², 张元¹, 任天瑞¹()

1. 上海师范大学生命科学与环境科学学院, 资源化学教育部重点实验室, 上海 200234
2. 贵州大学精细化工研究开发中心, 绿色农药与农业生物工程国家重点实验室培育基地/教育部重点实验室, 贵阳 550025

收稿日期:2014-10-24 出版日期:2015-03-10 发布日期:2015-01-30
作者简介:联系人简介: 任天瑞, 男, 博士, 教授, 博士生导师, 主要从事农药靶标研究. E-mail: trren@shnu.edu.cn
基金资助:
国家自然科学基金(批准号: 21172147)和国家“八六三”计划项目(批准号: 2011AA100503)资助

Interaction of GABA Receptor in the Fish Brain with Ro7-1986/1 and Fipronil by Fluorescent-labeled Ligand^†

YANG Shan¹, ZHANG Bo², ZHANG Yuan¹, REN Tianrui^1,^*()

1. Key Laboratory of Resource Chemical of Ministry of Education, College of Life and Environment Science,Shanghai Normal University, Shanghai 200234, China
2. State Key Laboratory Breeding Base of Green Pesticide and Agricultural Bioengineering,Key Laboratory of Green Pesticide and Bioengineering of Ministry of Education,Center for Research and Development of Fine Chemicals, Guizhou University, Guiyang 550025, China

Received:2014-10-24 Online:2015-03-10 Published:2015-01-30
Contact: REN Tianrui E-mail:trren@shnu.edu.cn
Supported by:
† Supported by the National Natural Science Foundation of China(No.21172147) and the National High Technology Research and Development Program of China(No.2011AA100503)

摘要/Abstract

摘要：

以苯二氮卓类化合物Ro7-1986/1和氟虫腈(Fipronil)分别与异硫氰酸荧光素(FITC)反应合成了2种γ-氨基丁酸(GABA)受体的配体荧光复合物, 即FITC-Ro7-1986/1(简称FITC-Ro7)和FITC-Fipronil; 利用傅里叶变换红外光谱(FTIR)和核磁共振氢谱(¹H NMR)等手段对其结构进行了表征. 以2种荧光配体为探针, 采用荧光标记法研究了Ro7-1986/1和Fipronil与鳙鱼(Hypophthalmichthys nobilis)脑内GABA受体的相互作用, 得到了亲和常数K_d和最大结合量[RT]. 同时考察了GABA对Ro7-1986/1与受体相互作用的影响. 研究结果表明, Ro7-1986/1和Fipronil与受体的亲和常数K_d分别为(67±5) nmol/L和(346±6) nmol/L; 最大结合量[RT]分别为(13.8±1.8) pmol/mg protein和(40.6±3.5) pmol/mg protein; GABA促进了Ro7-1986/1与受体的结合, 进一步的研究结果表明, 鱼类与哺乳动物脑中GABA受体结构相似; 相对于哺乳动物, 苯吡唑类杀虫剂Fipronil对鱼类GABA的亲和力较高.

关键词: Ro7-1986/1, 氟虫腈, 荧光标记法, γ-氨基丁酸(GABA)受体

Abstract:

Two kinds of γ-aminobutyric acid(GABA) receptor ligands, Ro7-1986/1 and Fipronil, were explored for preparing fluorescein conjugates including FITC-Ro7-1986/1 and FITC-Fipronil. Fourier transform infrared spectroscopy(FTIR) and ¹H NMR were used to characterize the structures of the synthesized products. The interactions between GABA receptor in hypophthalmichthys nobilis brain and both ligands were studied by fluorescence-labeling method. In addition, the effects of GABA on the binding of Ro7-1986/1 and GABA receptors were also investigated. The results showed that the equilibrium dissociation constants(K_d) and the maximum number of binding sites [RT] between Ro7-1986/1 and receptor were (67±5) nmol/L and (13.8±1.8) pmol/mg protein, respectively. The values of K_d and [RT] between Fipronil and receptor were (346±6) nmol/L and (40.6±3.5) pmol/mg protein, respectively. Meanwhile, GABA was able to significantly elevate the binding of between ligand and receptor, which indicated the pharmacological properties of GABA receptor in fish brain are remarkably similar to those in mammalian brain. Moreover, Fipronil exhibited more excellent affinities to fish by comparison to mammals.

Key words: Ro7-1986/1, Fipronil, Fluorescence-labeled method, γ-Aminobutyric acid receptor

中图分类号:

O657

TrendMD:

杨姗, 张博, 张元, 任天瑞. 荧光标记法研究鱼类GABA受体与Ro7-1986/1和Fipronil的相互作用. 高等学校化学学报, 2015, 36(3): 456.

YANG Shan, ZHANG Bo, ZHANG Yuan, REN Tianrui. Interaction of GABA Receptor in the Fish Brain with Ro7-1986/1 and Fipronil by Fluorescent-labeled Ligand^†. Chem. J. Chinese Universities, 2015, 36(3): 456.

图/表 7

Fig.1 Structures of FITC-Ro7(A) and FITC-Fipronil(B)

Fig.2 FTIR spectra of FITC(a), Ro7-1986/1(b), FITC-Ro7(c), Fipronil(d) and FITC-Fipronil(e)

Fig.3 Excitation(a) and emission(b) spectra of FITC(A), FITC-Ro7(B) and FITC-Fipronil(C)

Fig.4 Fluorescence intensity vs. FITC-Ro7(A) and FITC-Fipronil(B) concentration a. In 50% methanol; b. in tissue suspension; c. in buffer.

Fig.5 Saturability of FITC-Ro7(A), FITC-Fipronil(B) and Scatchard analysis of FITC-Ro7(C),FITC-Fipronil(D) binding to cortical membranes by different methods Supernatant(S1), tissue suspension(P1), supernatant after dissociation(S2) in the binding assay of FITC-Ro7. Supernatant(S1'), tissue suspension(P1'), supernatant after dissociation(S2') in the binding assay of FITC- Fipronil.

Table 1 Equilibrium binding properties of FITC-Ro7 and FITC-Fipronil binding to GABA receptor

Comp.	K_d/(nmol·L^-1)	[RT]/(pmol·mg^-1 protein)	Comp.	K_d/(nmol·L^-1)	[RT]/(pmol·mg^-1 protein)
FITC-Ro7(S₁ )	109±9	21.3±2.5	FITC-Fipronil(S₁')	502±8	56.2±5.0
FITC-Ro7(P₁)	86±7	16.0±2.2	FITC-Fipronil(P₁')	377±7	39.3±3.7
FITC-Ro7(S₂)	67±5	13.8±1.8	FITC-Fipronil(S₂')	346±6	40.6±3.5

Fig.6 GABA enhancement of FITC-Ro7 binding to hypophthalmichthys nobilis brain membranes Each point represents the ratio of binding in the presence and absence of GABA. The data shown are representative of three independent experiments, each point in triplicate.

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荧光标记法研究鱼类GABA受体与Ro7-1986/1和Fipronil的相互作用

Interaction of GABA Receptor in the Fish Brain with Ro7-1986/1 and Fipronil by Fluorescent-labeled Ligand^†

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荧光标记法研究鱼类GABA受体与Ro7-1986/1和Fipronil的相互作用

Interaction of GABA Receptor in the Fish Brain with Ro7-1986/1 and Fipronil by Fluorescent-labeled Ligand†

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Interaction of GABA Receptor in the Fish Brain with Ro7-1986/1 and Fipronil by Fluorescent-labeled Ligand^†