Oriented reconstitution of red cell membrane proteins and assessment of their transmembrane disposition by immunoquenching of fluorescence.
The two major membrane glycoproteins of human red cells, glycophorin and band 3, the anion exchange protein, were isolated from cells exofacially labeled with fluorescein and reconstituted into vesicles with defined transmembrane disposition. Uniform orientation of polypeptides was accomplished by two procedures: Vesicles with single protein units were obtained by a one-step dilution of a protein/detergent suspension with a vast excess of phospholipid. Vesicles with uniform orientation of protein were selected by affinity chromatography on derivatized Sepharoses (organomercurial, wheat germ agglutinin, aminoethyl or diethylaminoethyl). Vesicles with multiple protein units with uniform orientation were generated by vectorial immobilization of detergent solubilized proteins on the above affinity matrices and in situ formation of proteoliposomes by detergent substitution for phospholipid. The proteoliposomes were released from the column by addition of excess free ligand. The orientation of band 3 and glycophorin in the reconstituted vesicles was first assessed by immunofluorescence quenching, using anti-fluorescein antibodies, to quantitatively quench fluorescein residues exposed on the outer surface of vesicles. Further assessment was achieved by chromatographing the vesicles through various affinity and ionic matrices. Vesicle populations of higher than 90% homogeneity in protein orientation (right-side-out or inside-out) were obtained with both procedures. The above methods provide a convenient experimental tool for the oriented reconstitution of proteins and the evaluation of their transmembrane disposition.
D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; D012856:4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid / Q000031:analogs & derivatives / Q000378:metabolism; D001457:Anion Exchange Protein 1, Erythrocyte / Q000032:analysis; D004910:Erythrocyte Membrane / Q000032:analysis*; D019793:Fluorescein; D005452:Fluoresceins; D006021:Glycophorins / Q000032:analysis; D006801:Humans; D007158:Immunologic Techniques; D008565:Membrane Proteins / Q000032:analysis*; D008722:Methods; D008854:Microscopy, Electron; D008958:Models, Molecular; D012996:Solutions; D013050:Spectrometry, Fluorescence
D001457:Anion Exchange Protein 1, Erythrocyte; D005452:Fluoresceins; D006021:Glycophorins; D008565:Membrane Proteins; D012996:Solutions; D012856:4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; D019793:Fluorescein