Analysis in deoxycholate of three antigenic specificities associated with the rat Thy‐1 molecule

Abstract

Three antigens similar in tissue distribution can be identified on rat thymocytes; the Thy-1.1 antigen, a rat specific xenoantigen, and a rat-mouse cross-reacting xenoantigen. To determine if these three antigens were on the same molecule their behavior in detergent-solubilized extracts from thymocytes was studied. Membrane fragments containing Thy-1.1 activity were prepared by a rapid method involving the use of Tween-40 detergent, and were solubilized in deoxycholate. The 150 000 × g supernatant from this extract contained aproximately 50 % of the original Thy-1.1 and xenoantigen activity. The supernatant was chromatographed on Sephadex G-200, and subjected to zone sedimentation on sucrose gradients in H₂O and ²H₂O to determine the hydrodynamic properties of the antigens. The three antigens migrated in identical fashion in all cases, and behaved as a molecule of 28 000 daltons molecular weight. When the antigenically active fraction, recovered after chromatography on Sephadex G-200, was passed through an immunoabsorbent consisting of rabbit antibody to one of the xenoantigens, all three antigens were equally depleted compared with passage through a control column. The results of these experiments suggested that Thy-1.1 antigen and the two xenoantigens were closely associated and most probably all on the Thy-1 molecule.