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.
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.
Matsukawa
Satoko
S
Laboratory of Applied Microbiology & Biochemistry, Nara Women's University, Nara 630-8506, Japan.
Ueno
Hiroshi
H
D000595:Amino Acid Sequence; D000906:Antibodies / Q000276:immunology; D015153:Blotting, Western / Q000379:methods; D000939:Epitopes / Q000276:immunology; D015971:Gene Expression Regulation, Enzymologic / Q000235:genetics*; D005968:Glutamate Decarboxylase / Q000097:blood / Q000737:chemistry / Q000235:genetics*; D006801:Humans; D007527:Isoenzymes / Q000097:blood / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D007963:Leukocytes, Mononuclear / Q000378:metabolism* / Q000473:pathology; D008969:Molecular Sequence Data; D008970:Molecular Weight; D020033:Protein Isoforms / Q000097:blood / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods
D000906:Antibodies; D000939:Epitopes; D007527:Isoenzymes; D020033:Protein Isoforms; D012333:RNA, Messenger; D005968:Glutamate Decarboxylase