Photoaffinity-labeled hapten-binding T-cell receptor on a suppressor T-cell hybridoma.
A T-cell hydridoma, 7C3-13-Ag6, which produces a (4-hydroxy-3-nitrophenyl)acetyl (NP)-specific suppressor T-cell factor associated with an I-J determinant, was utilized to study the hapten-binding receptor of T-cells. This hybridoma had been shown to express NP-binding receptor molecules on the cell surface with heteroclitic fine specificity for a cross-reactive hapten, (4-hydroxy-5-iodo-3-nitrophenyl) acetyl (NIP). The stoichiometric analysis of the hapten binding by 7C3-13-Ag6 cells was performed by the measurement of direct binding of highly radioactive haptens to the cell surface. The affinity constant (Ka) of the receptor for N125IP-epsilon-aminocaproic acid (N125IP-cap), as calculated from a Hill plot, was 5.75 X 10(7) M-1 [Hill coefficient (a) = 0.86; expression of receptor sites per cell = approximately 1 X 10(3) on average]. The receptor molecule was specifically affinity labeled with photoreactive nitroaryl azide derivatives of N125IP (510-570 Ci/mmole). The specificity of photoaffinity labeling was demonstrated both by competitive inhibition of labeling with NIP- or NP-cap and by differential photoaffinity labeling based on the reversibility of hapten-receptor interaction. The gel electrophoretic analysis of the photoaffinity-labeled molecule indicated that the hapten-binding receptor of 7C3-13-Ag6 has a mol. wt of 28,000 +/- 3000 and an isoelectric point of 5.6-5.7. No immunoglobulin determinants were detected on the molecule. A comparative immunoprecipitation analysis of the membrane lysate of 7C3-13-Ag6 with monoclonal anti-I-J reagents identified a separate I-J molecule of 25,000 +/- 1000 mol. wt that is distinct from the photoaffinity-labeled hapten-binding molecule.
D000345:Affinity Labels; D001665:Binding Sites; D005453:Fluorescence; D006241:Haptens / Q000276:immunology*; D006825:Hybridomas / Q000276:immunology*; D008722:Methods; D009592:Nitrohydroxyiodophenylacetate / Q000276:immunology; D009596:Nitrophenols / Q000276:immunology; D010648:Phenylacetates; D010782:Photolysis; D011971:Receptors, Immunologic; D013601:T-Lymphocytes / Q000276:immunology*; D050378:T-Lymphocytes, Regulatory / Q000276:immunology*
D000345:Affinity Labels; D006241:Haptens; D009596:Nitrophenols; D010648:Phenylacetates; D011971:Receptors, Immunologic; C026242:4-hydroxy-5-nitrophenyl acetic acid; D009592:Nitrohydroxyiodophenylacetate