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Cross-linking of Ly 6-linked alloantigens: association between ThB and Ly 5
B A Houlden1, I F McKenzie, P M Hogarth
1Department of Pathology, University of Melbourne, Parkville, Victoria, Australia.
The bifunctional cross-linking reagent dithiobis(succinimidyl propionate) (DSP) was used to cross-link 125I surface-labelled glycoproteins from viable thymocytes. The cells were solubilized, and the cross-linked material immunoprecipitated and analysed by SDS-PAGE. When DSP cross-linked thymocyte material was immunoprecipitated with either anti-ThB or anti-Ly 5 monoclonal antibodies, and then cleaved, molecules with masses identical to Ly 5 (Mr 180 kD) and ThB (Mr 16-18 kD) were obtained. However, if the cross-linker was not cleaved, the intact product had a molecular mass of greater than 200 kD. The identity of these co-precipitated, cross-linked moieties was formally proved by limited proteolysis peptide map analysis. The data indicated that the ThB and Ly 5 antigens were associated on the thymocyte cell surface but no such association could be found on peripheral lymphocytes. The ThB-Ly 5 interaction may indicate an association relevant to the differentiation of thymocytes.
The bifunctional cross-linking reagent dithiobis(succinimidyl propionate) (DSP) was used to cross-link 125I surface-labelled glycoproteins from viable thymocytes. The cells were solubilized, and the cross-linked material immunoprecipitated and analysed by SDS-PAGE. When DSP cross-linked thymocyte material was immunoprecipitated with either anti-ThB or anti-Ly 5 monoclonal antibodies, and then cleaved, molecules with masses identical to Ly 5 (Mr 180 kD) and ThB (Mr 16-18 kD) were obtained. However, if the cross-linker was not cleaved, the intact product had a molecular mass of greater than 200 kD. The identity of these co-precipitated, cross-linked moieties was formally proved by limited proteolysis peptide map analysis. The data indicated that the ThB and Ly 5 antigens were associated on the thymocyte cell surface but no such association could be found on peripheral lymphocytes. The ThB-Ly 5 interaction may indicate an association relevant to the differentiation of thymocytes.