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

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Kd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Kd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.

Two kinds of y-aminobutyric acid（GABA） receptor ligands, Ro7-1986/1 and Fipronil, were ex- plored 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 （Kd ） 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 Ka 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.