Reconstitution of purified detergent-soluble HLA-A and HLA-B antigens into phospholipid vesicles.

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RESUMO

Purified detergent-soluble human histocmpatibility antigens (HLA-A and HLA-B) were reconstituted into phospholipid vesicles by mixing the protein and lipid together in the presence of either octylglucoside (octyl-beta-D-glucopyranoside) or deoxycholate and removing the detergent by dialysis. The resulting preparation consisted of lipid vesicles containing all or most of the added protein. The protein in the vesicles was antigenically active, as demonstrated by specific binding to anti-beta2-microglobulin IgG-Sepharose beads and by specific inhibition of alloantibody and complement-mediated cytotoxicity. Protein incorporated into vesicles at a protein/phospholipid ratio of 1:10 showed an asymmetric distribution of the HLA-A and HLA-B molecules, with virtually all of the antigens oriented facing the external medium. Cleavage experiments with proteases showed that the molecule was attached to the vesicle membrane via the COOH terminus, consistent with its proposed structure in intact cellular plasma membranes. Electron micrographs of the vesicles showed 50-60 A knobs on the outer surface similar to structures observed for other membrane proteins. HLA-A and HLA-B could also be incoporated into vesicles together with Semliki Forest virus membrane proteins. The resulting preparations should be useful in defining the molecular interactions involving HLA-A and HLA-B antigens in the immune response.

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