Preliminary characterization of Thy-1.1 and Ag-B antigens from rat tissues solubilized in detergents.
1. A radioactive binding assay for Thy-1.1 alloantigen which functions in the presence of detergents was established by using glutaraldehyde-fixed thymocytes as target cells. Thy-1.1 activity in detergent extracts was then assayed by measuring inhibition of the binding assay. 2. Solubilization of Thy-1.1 from whole thymocytes, and their membranes by a large number of non-ionic detergents and deoxycholate was studied. In the same extracts Ag-B(4) histocompatibility antigenic activities were measured. With the exception of Nonidet P-40, the detergents did not affect the antigenicity of Thy-1.1, but only Lubrol-PX and deoxycholate gave effective solubilization as measured by activity remaining in the supernatant after centrifugation at 200000g for 40min. With Ag-B(4) antigen, Triton X-100, Triton X-67 and Nonidet P-40 gave effective solubilization as well as Lubrol-PX and deoxycholate. Solubilization of Thy-1.1 activity from leukaemia cells and a brain homogenate was also studied, but none of the non-ionic detergents gave satisfactory results with these tissues. 3. Extracts from thymocyte membranes were further examined by gel filtration and sucrose gradient centrifugation. The Thy-1.1 activity behaved as a single component in deoxycholate with a density similar to that of a globular protein, but in Lubrol-PX the antigen was contained in a low-density complex. In Lubrol-PX extracts Ag-B(4) was also found in aggregates not observed in deoxycholate. 4. The s(20,w) values for Thy-1.1 and Ag-B(4) antigens in deoxycholate were 2.4 and 4.4, and v values were 0.70 and 0.75 respectively. The Stokes radius observed for Thy-1.1 was 3.1nm and for Ag-B(4) 5.3nm. By using these values the molecular weights for the antigen-detergent complexes were calculated to be 28000 for Thy-1.1 and 100000 for Ag-B(4).
D000818:Animals; D000936:Antigen-Antibody Complex; D001647:Bile Acids and Salts / Q000494:pharmacology; D001921:Brain / Q000276:immunology; D002462:Cell Membrane / Q000032:analysis / Q000276:immunology; D003902:Detergents / Q000494:pharmacology; D005260:Female; D005976:Glutaral; D006680:HLA Antigens / Q000032:analysis; D007519:Isoantigens / Q000032:analysis*; D007938:Leukemia / Q000276:immunology; D008297:Male; D008722:Methods; D051379:Mice; D008970:Molecular Weight; D011485:Protein Binding; D011869:Radioligand Assay; D051381:Rats; D012995:Solubility; D013601:T-Lymphocytes / Q000276:immunology*; D014020:Tissue Extracts
D000936:Antigen-Antibody Complex; D001647:Bile Acids and Salts; D003902:Detergents; D006680:HLA Antigens; D007519:Isoantigens; D014020:Tissue Extracts; D005976:Glutaral