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Evaluation by dot-immunoassay of the differential distribution of cell surface and intracellular proteins in glycosylphosphatidylinositol-rich plasma membrane domains
|Published in||Analytical Biochemistry. 1996, vol. 235, no. 1, p. 49-56|
|Abstract||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 p56lck partially 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.|
|Keywords||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|