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Suh, Pann-Ghill
BioSignal Network Lab (BSN)
Research Interests
  • Signal transduction, cancer, metabolism, phospholipase C

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Monoclonal antibodies to three phospholipase C isozymes from bovine brain

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Title
Monoclonal antibodies to three phospholipase C isozymes from bovine brain
Author
Suh, Pann-GhillRyu, Sung HoChoi, Won ChulLee, Kee-YoungRhee, Sue Goo
Issue Date
1988-10
Publisher
AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC
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
URI
https://scholarworks.unist.ac.kr/handle/201301/16508
URL
http://www.jbc.org/content/263/28/14497.long
ISSN
0021-9258
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