Bighead Isolation And Purification Of GABA Receptors

γ-amino butyric acid (GABA) is the major inhibitory neurotransmitter in thecentral nervous system of vertebrates and invertebrates, whose transmission effect ismediated by the GABA receptor. The receptor is the target of various organochlorineinsecticides and fipronilavermectin. GABA receptor is also closely related to manyneurological diseases. It is reported that fipronil insecticide is safe and nontoxic tomammals, but highly toxic to fish, bees due to the difference of GABA receptorbetween them. The difference of fish and mammalian GABA receptor can beobserved by separating and purifying GABA receptor of fish, and this work plays avery important role in the screening of highly selective new environment-friendlypesticides. We achieved following results:Firstly, we chose bighead as the object of this study. Six kinds of detergent wereused to dissolve GABA receptor protein, and we found that protein amount eluted by1%(V/V) Nonidet P-40was729.01μg/mL, which proved to be more than others.Secondly, we prepared affinity chromatography media by couplingRo7-1986/1to the epoxy-modified agarose (Sepharose6B). We measured that theepoxy superficial density of agarose was66.8μmol/g gum by thiosulfate titration,the density of coupled ligand Ro7-1986/1was63.12μmol/g gum.Thirdly, we can get the bighead brain GABA receptor by the use of theprepared affinity column, combining with ultrafiltration method, polyethylene glycoldialysis method, and DEAE-Sephacel ion-exchange chromatography. The GABAreceptor was two clear bands, whose molecular weight was46KDa and42KDa,which might be the α and β subunits of the GABA receptor of bighead respectively.Bighead GABA receptor was similar with the GABA receptor subunit of mammalswhich was about50KDa. We used Bradford assay to measure protein concentrationto be4.6μg/mL.Lastly, we obtained the fluorescent ligand (FITC-GABA) through modifyingGABA with fluorescein isothiocyanate (FITC).We measured dissociation constant (Kd)and maximum binding capacity (Bmax) between FITC-GABA and bighead GABA receptor crude extract to be88.89nM and1.81pM/mg protein, both of which werebigger than mammals. The result means that the amount of GABA binding by fishGABA receptor was more than mammals, but the combination was of low stability.And we also measured dissociation constant (Kd) and maximum binding capacity(Bmax) between FITC-GABA and the purified GABA receptor to be13.84nM and824.5pM/mg protein. The maximum binding capacity of the purified receptor is455times more than crude extract, which means the combination of a series ofpurification method for the separation is effective.