Study On The Interaction Of Fish GABA Receptor With Drug By Fluorescent Labeling

Fluorescent labeling is a kind of inexpensive, high sensitivity and environment- friendly non-radioactive labeling methods. In this study, the interactions between γ- aminobutyric acid(GABA) receptor in hypophthalmichthys nobilis brain with benzodiazepines and pesticides were studied by fluorescence- labeling method, to further clarify the difference of GABA receptor in the brain of fish and mammalian, which plays a very important role in investigating the new environment- friendly pesticides of high selectivity.In this experiment, six kinds of detergent were used to dissolve GABA receptor protein. Comparing the efficiency of dissolution by 1% Nonidet P-40, 1% Triton X-100, 4% CHAPS, 0.5% Lubrol-PX, 2% OCTG and 2% sodium deoxycholate solubilization, we found that protein amount eluted by 1%Nonidet P-40 was 728μg/m L, which proved to be more than others.Meantime, the separation efficiency of the two affinity chromatography about Ro7-1986 / 1 and fipronil was studied. Compared with affinity ligand fipronil, Ro7-1986/1 affinity ligand more suitable for separate the GABA receptor, the recovery was 32%.Two kinds of GABA receptor ligands, Ro7-1986/1 and fipronil, were exploited for preparing fluorescein conjugates including FITC-Ro7-1986/1 and FITC- fipronil. Fourier transform infrared spectroscopy(FTIR) and H- nuclear magnetic resonance(1H-NMR) were used to characterize the structure of the synthesized products.The interactions between GABA receptor in hypophthalmichthys nobilis brain and both ligands were studied by fluorescence- labeling method. 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 Kd and [RT] between fipronil and receptor were 346±6 nmol/L and 40.6±3.5 pmol/mg protein, respectively.In addition, the effects of GABA on the binding of Ro7-1986/1 and GABA receptor were also investigated. 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.