Molecular cloning and amino acid sequence of brain L-glutamate decarboxylase

Abstract

We used specific polyclonal antibodies against L-glutamate decarboxylase (GAD) to screen a mouse brain cDNA library that was constructed in the expression vector lambda gt11. We obtained 1.5 x 10(6) recombinant DNA clones in the mouse brain cDNA library. One of the clones was positively identified as a GAD clone on the basis of the following results: (i) the clone and its secondary and tertiary clones all reacted strongly with anti-GAD antibodies; (ii) the fusion protein obtained from lambda GAD-Y1089 showed good GAD enzyme activity as determined by both CO2 and gamma-aminobutyric acid methods. The GAD clone thus obtained contains GAD cDNA of approximately 2.6 kilobases that has one internal EcoRI site. After GAD cDNA was cut at the EcoRI site, two DNA fragments of about 1.6 and 1.0 kilobases were obtained at the 5' and 3' ends, respectively. The cDNA insert was found to be composed of 2632 base pairs, the translation initiation site was assigned to the methionine codon ATG, and the termination site was found to be TGA (positions 2216-2218). Furthermore, the coding region in 2169 base pairs was found to consist of 723 amino acids. The protein has a molecular weight of 83,207 and contains 83 strongly basic, 108 strongly acidic, 226 hydrophobic, and 221 polar amino acids with an isoelectric point of 5.355. The relationship of this GAD cDNA to other forms of GAD is discussed.