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Suh, Pann-Ghill
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Monoclonal antibodies to three phospholipase C isozymes from bovine brain

Author(s)
Suh, Pann-GhillRyu, Sung HoChoi, Won ChulLee, Kee-YoungRhee, Sue Goo
Issued Date
1988-10
URI
https://scholarworks.unist.ac.kr/handle/201301/16508
Fulltext
http://www.jbc.org/content/263/28/14497.long
Citation
JOURNAL OF BIOLOGICAL CHEMISTRY, v.263, no.28, pp.14497 - 14504
Abstract
Murine hybridoma cell lines secreting antibodies against the three bovine isozymes of phosphoinositide-specific phospholipase C (PLC) were established: 6, 23, and 12 lines were obtained for PLC-I (150 kDa), PLC-II (145 kDa), and PLC-III (85 kDa), respectively. The antibodies were purified from ascites fluid, and their properties were studied in detail. All the antibodies cross-reacted with their corresponding PLC enzymes, but not with the other two isozymes, suggesting that the three enzymes contain very different antigenic determinants. The six antibodies elicited by bovine PLC-I also cross-reacted with human and rat enzyme, whereas three each from anti-PLC-II antibodies and anti-PLC-III antibodies did not react with the enzymes from different species. Each antibody exerts different effects on the phosphatidylinositol-hydrolyzing activity of PLC. The most inhibitory antibody for either isozyme PLC-I or PLC-II exhibits 80% inhibition, whereas no more than 20% inhibition was observed for the anti-PLC-III antibodies. Purified PLC-I frequently contains catalytically active 140- and 100-kDa forms and an inactive 41-kDa protein in addition to the intact 150-kDa form, probably due to its high sensitivity to an unidentified endogenous protease. The five anti-PLC-I antibodies which bind to the denaturated 150-kDa polypeptide also recognized the 140-kDa form, whereas only three cross-reacted with the 100-kDa form, and the remaining two bound to the 41-kDa protein. Competitive binding studies with intact PLC enzymes and Western blot experiments with proteolytic digests revealed that the 6 anti-PLC-I, 23 anti-PLC-II and 12 anti-PLC-III antibodies bind at least five, six, and seven different epitopes on PLC-I, PLC-II and PLC-III, respectively. The fact that these monoclonal antibodies bind to different epitopes on the same enzyme allowed one to develop a highly specific and sensitive tandem radioimmunoassay for quantitating PLC-I, PLC-II and PLC-III. The principle of the assay is that binding of an 125I-labeled antibody to the antigen immobilized by another antibody at a distinctive binding site is proportional to the amount of antigen present. By using this method, PLC-I, PLC-II and PLC-III could be measured quantitatively in the presence of other proteins, detergents, lipids, polyanions, and metal ions, all of which greatly affect the activity of PLC enzymes
Publisher
AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC
ISSN
0021-9258

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