Evaluation by Dot-Immunoassay of the Differential Distribution of Cell Surface and Intracellular Proteins in Glycosylphosphatidylinositol-Rich Plasma Membrane Domains

The dot-immunoassay has been adapted for rapid detection and partial characterization of glycosylphosphatidylinositol (GPI)-linked, transmembrane, and intracellular proteins in Triton X-100 (TX-100) extracts of lymphoma cells and intestinal tissue. The GPI-anchored proteins tend to concentrate into...

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Bibliographic Details
Published in:Analytical biochemistry 1996-03, Vol.235 (1), p.49-56
Main Authors: Ilangumaran, Subburaj, Arni, Stephan, Chicheportiche, Yves, Briol, Anne, Hoessli, Daniel C.
Format: Article
Language:English
Subjects:
Animals
Cell Membrane - metabolism
Evaluation Studies as Topic
Glycosylphosphatidylinositols - metabolism
Immunoassay
Membrane Proteins - metabolism
Mice
Mice, Inbred BALB C
Octoxynol
Phosphatidylinositol Diacylglycerol-Lyase
Phosphoinositide Phospholipase C
Phosphoric Diester Hydrolases - metabolism
Sensitivity and Specificity
Tumor Cells, Cultured
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Summary:The dot-immunoassay has been adapted for rapid detection and partial characterization of glycosylphosphatidylinositol (GPI)-linked, transmembrane, and intracellular proteins in Triton X-100 (TX-100) extracts of lymphoma cells and intestinal tissue. The GPI-anchored proteins tend to concentrate into specialized plasma membrane domains enriched in glycosphingolipids. The dot-immunoassay has been successfully used to demonstrate the differential distribution of GPI-linked and transmembrane surface glycoproteins of T lymphocytes in sucrose density gradient fractions of TX-100 lysate. The type II transmembrane protein CD26 and the intracellular tyrosine kinase p56lckpartially cofractionated with GPI-linked glycoproteins, and the extent to which they partition into GPI-rich plasma membrane domains could be evaluated. Preferential association of the acidic glycosphingolipid GM1 with these domains could be demonstrated by cholera toxin binding directly to the dot-blotted sucrose density gradient fractions. Treatment of whole cell TX-100 lysates or sucrose gradient fractions dotted onto nitrocellulose filter strips with bacterial phosphatidylinositol-specific phospholipase C (PI-PLC) proved to be an efficient method to assay for the presence of a GPI-anchor in Thy-1 and Ly6 surface glycoproteins. We have used three criteria, namely flotation to light density fractions in sucrose gradients, colocalization with GM1, and sensitivity to PI-PLC cleavage, to assess the presence of a GPI modification in a putative GPI-linked protein in intestinal tissue extract. It is envisaged that the techniques described in this report would find a wider application to rapidly assess the contents of GPI-rich plasma membrane domains in different cells and tissues.
ISSN:0003-2697
1096-0309
DOI:10.1006/abio.1996.0090