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. [125I]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.
Luescher
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Department of Pathology, Washington University School of Medicine, St Louis, MO 63110.
Unanue
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D000345:Affinity Labels; D000911:Antibodies, Monoclonal / Q000276:immunology; D001386:Azides; D002462:Cell Membrane / Q000737:chemistry; D002846:Chromatography, Affinity / Q000379:methods*; D003840:Deoxycholic Acid / Q000494:pharmacology; D003902:Detergents; D004591:Electrophoresis, Polyacrylamide Gel; D005227:Fatty Acids; D005944:Glucosamine / Q000031:analogs & derivatives; D005960:Glucosides / Q000494:pharmacology; D000949:Histocompatibility Antigens Class II / Q000302:isolation & purification*; D006863:Hydrogen-Ion Concentration; D009113:Muramidase; D010446:Peptide Fragments; D013012:Sorbitol / Q000031:analogs & derivatives; D014407:Tumor Cells, Cultured
D000345:Affinity Labels; D000911:Antibodies, Monoclonal; D001386:Azides; D003902:Detergents; D005227:Fatty Acids; D005960:Glucosides; D000949:Histocompatibility Antigens Class II; D010446:Peptide Fragments; D003840:Deoxycholic Acid; C018619:octyl-beta-D-glucoside; D013012:Sorbitol; C042222:nonanoyl-N-methylglucamide; C063161:MEGA 8; D009113:Muramidase; D005944:Glucosamine