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Journal of Biochemistry Advance Access originally published online on October 26, 2007
Journal of Biochemistry 2007 142(5):633-638; doi:10.1093/jb/mvm173
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© 2007 The Japanese Biochemical Society

Expression of Glutamate Decarboxylase Isoform, GAD65, in Human Mononuclear Leucocytes: A Possible Implication of C-terminal End Deletion by Western Blot and RT–PCR Study

Satoko Matsukawa and Hiroshi Ueno*

Laboratory of Applied Microbiology & Biochemistry, Nara Women's University, Nara 630-8506, Japan

*To whom correspondence should be addressed. Tel: +81-742-20-3493, Fax: +81-742-20-3448, E-mail: hueno{at}cc.nara-wu.ac.jp

Received August 5, 2007; Accepted August 29, 2007


  Abstract

Human peripheral blood leucocyte was examined for the expression of glutamate decarboxylase (GAD). Peripheral blood from healthy individuals was fractionated into polynuclear leucocytes and mononuclear leucocytes. Cell lysate from the mononuclear leucocytes was analysed by SDS–PAGE and Western blot. With antibody raised against unique C-terminal sequence for GAD65, two protein bands at 30 and 80 kDa were detected. However, with anti-GAD65/67 antibody recognizing very end of C-terminal, about 40 residues toward C-terminal, no protein bands were observed. Expression of mRNA coding for the epitope of these two antibodies was examined by using PCR technique. Results showed an evidence that mononuclear leucocytes express GAD65 with its C-terminal segment truncated. Our results have suggested an expression of GAD with the novel molecular weight may be caused by possible mononuclear leucocyte specific splicing errors.

Key Words: GABA, GAD, leucocyte, splicing

Abbreviations: DTT, dithiothreitol; GABA, {gamma}-aminobutyric acid; GAD, glutamate decarboxylase; GAPDH, glyceraldehyde 3'-phosphate dehydrogenase; LiDS, lithium dodecyl sulphate; RNase, ribonuclease; RT-PCR, reverse transcriptase-polymerase chain reaction; TBS, Tris buffered saline; TCA, trichloroacetic acid


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