Thy-1 solubilization from mouse T cells by phosphatidylinositol-specific phospholipase C: biochemical and antigenic characterization.
Membrane anchorage of mouse and rat Thy-1 antigens results from the post-translational attachment of a non-proteic tail terminated by a phosphatidylinositol group. In order to determine the biochemical and antigenic properties of the material released by phosphatidylinositol-specific phospholipase C (PI-PLC), we studied, by one-(1-D) and two-dimensional (2-D) gel electrophoresis and by immunoprecipitation, the supernatant of surface-labelled mouse T cells treated with purified Staphylococcus aureus PI-PLC. The major protein released by this enzymatic treatment showed an apparent molecular weight (MW) and an isoelectric focusing (IEF) pattern identical to those of detergent-solubilized, immunoprecipitated Thy-1. In addition, a sandwich radioimmunoassay (RIA) utilizing two Thy-1-specific monoclonal antibodies (mAb) was used to quantitate the amounts of PI-PLC-released and spontaneously shed Thy-1. Considerable differences in susceptibility to enzymatic cleavage and in spontaneous shedding were observed for a variety of mouse T-cell populations, including thymocytes and hybridoma, helper and cytotoxic cloned T cells, even though time-course experiments demonstrated that excess enzyme was used. It might be useful to consider these differences in the cell biology of Thy-1 and the occurrence of other PI-linked proteins of the lymphocyte surface in terms of their implications in the transduction of activation signals.
D000818:Animals; D000911:Antibodies, Monoclonal; D000954:Antigens, Surface / Q000276:immunology / Q000302:isolation & purification*; D004591:Electrophoresis, Polyacrylamide Gel; D005455:Fluorescent Antibody Technique; D051379:Mice; D008806:Mice, Inbred AKR; D008807:Mice, Inbred BALB C; D008810:Mice, Inbred C57BL; D009132:Muscles / Q000276:immunology*; D043265:Phosphatidylinositol Diacylglycerol-Lyase; D054801:Phosphoinositide Phospholipase C; D010727:Phosphoric Diester Hydrolases; D011863:Radioimmunoassay; D051381:Rats; D012995:Solubility; D013601:T-Lymphocytes / Q000276:immunology*; D018800:Thy-1 Antigens
D000911:Antibodies, Monoclonal; D000954:Antigens, Surface; D018800:Thy-1 Antigens; D010727:Phosphoric Diester Hydrolases; D054801:Phosphoinositide Phospholipase C; D043265:Phosphatidylinositol Diacylglycerol-Lyase