Solubilization and molecular characterization of membrane-bound antigens shared by thymocytes and brain

Abstract

In order to characterize chemically the serologically well-defined antigens shared by thymocytes and nervous tissue of mice and rats, different solubilization procedures have been tested. Treatment of thymocyte membranes with urea-Nonidet-P40 (NP40) proved to be the most valuable method, since nearly 100% of the antigen was solubilized essentially without loss of the antigenicity. By gel filtration in urea-NP40 the molecular weight was estimated to be 35 000 and the Stokes radius in sodium deoxycholate 31.8 A. In gel filtration studies a homogenous peak was obtained, whereas isoelectric focusing yielded two peaks (isoelectric points pH 4.5-5.0 and pH 5.4). The three determinants of this antigenic system were inseparable in gel filtration and isoelectric focusing, supporting the view put forward on the basis of serological studies that they are part of the same molecule. Extraction of murine thymocyte membranes with organic solvents yielded a considerable loss of activity of the thymocyte-brain antigen. There was some residual activity in the protein fraction, but none in the organic phase. Correspondingly, the highly purified ganglioside G Gtet 2b proved unable to absorb any rabbit anti-CBA brain serum. The reduced antigenic activity of the delipidated protein fraction could be restored by addition of lipids, as well as the nonionic detergent NP40. These observations suggest that interaction of protein and lipid is necessary for the antigenicity of the thymocyte-brain antigen.