Purification of papain-solubilized histocompatibility antigens from a cultured human lymphoblastoid line, RPMI 4265.
HL-A antigens having specificities HL-A2, HL-A7, HL-A12 have been solubilized by papain treatment of membrane preparations from the cultured human lymphoblastoid cell line RPMI 4265 and purified about 80-fold by chromatography on carboxymethylcellulose, Sephadex G-150, and diethylaminoethylcellulose columns. Separation of HL-A2 from a mixture of HL-A7 and HL-A12 was achieved on the final DEAE-cellulose column. The yield was about 1 mg of protein of each antigen preparation per 100 g of packed, frozen cells. On sodium dodecyl sulfate gel electrophoresis both preparations showed two polypeptide bands. The smaller subunit of 12,000 daltons is common to all HL-A preparations and has been shown to be identical with beta2-microglobulin. The larger subunit is a glycopeptide and in the HL-A7, 12 preparation was resolved into a duplex of 34,000 and 37,000 daltons. The HL-A2 preparation showed a single band at 34,000 daltons. On isoelectric focusing under nondenaturing conditions, the preparation showed multiple bands, all of which contained both subunits and retained antigenic activity. On isoelectric focusing in the presence of 6 M urea a single band for beta2-microglobulin and multiple bands for the larger subunit were seen. This charge heterogeneity of the larger subunit has been shown to be due to variable amounts of sialic acid. When HL-A antigen preparations were subjected to Sephadex G-100 chromatography in the presence of 3 M KCl, no separation of the two subunits was observed.
D002460:Cell Line; D002462:Cell Membrane / Q000032:analysis; D002501:Centrifugation, Zonal; D002848:Chromatography, DEAE-Cellulose; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D006649:Histocompatibility Antigens / Q000302:isolation & purification*; D006801:Humans; D007525:Isoelectric Focusing; D007700:Kinetics; D008214:Lymphocytes / Q000032:analysis*; D010206:Papain; D013997:Time Factors
D006649:Histocompatibility Antigens; D010206:Papain