PURIFICATION AND PHOTOAFFINITY-LABELING OF THE I-AK HISTOCOMPATIBILITY MOLECULE

Photoaffinity labeling was used to evaluate optimal conditions for purification of I-A(k) histocompatibility molecules in functionally active form. We assessed the biological activity of I-A(k) primarily by its binding of the hen egg-white lysozyme (HEL) peptide from residues 46-61. [I-125]iodo,4-azidosalicyloly(HEL)46-61 (IASA-46-61)-labeled I-A(k) on B cell hybridoma membranes and their detergent solubilisates, at the alpha-chain. Following extensive detergent dialysis, the intensity of this labeling remained unchanged in the case of MEGA 8 and MEGA 9 detergents, but decreased in the case of deoxycholate and n-octylglucoside. Conditions for affinity purifications were assessed on one hand by determining the dissociation conditions of I-A(k) from various monoclonal antibodies and by determining the denaturation of I-A(k) under these conditions. Effective dissociation in the absence of detectable denaturation was observed for 10.3.6.2 and 40.LH monoclonal antibody at pH 3.5 and to a lesser extent at low concentrations of ammonium thiocyanate and guanidine thiocyanate at neutral pH. I-A(k) purified from cell membranes using MEGA 8 and MEGA 9 detergent mixtures and acid elution from 10.3.6.2 Sepharose was efficiently labeled by IASA-46-61. Thus, I-A(k) was active in antigen presentation to a T cell hybridoma when reconstituted in planar membranes. In contrast to I-A(k) on cell membranes, purified I-A(k) in detergent showed extensive labeling of the beta-chain. The overall labeling intensity and the extent of beta-chain labeling substantially changed upon addition of certain lysophosphatides.