Hypertonic Salt Extraction of HL-A Antigens: Assessment of Protease Activity 1

Abstract

Human histocompatibility antigens (HL-A) are genetically segregating cell surface markers which differ from person to person. The gene products of the HL-A locus thus provide a set of chemical markers of biological individuality which can be used in studying antigenic recognition of foreign graft and immune responsiveness to organ and tissue transplantation. Because of their potential usefulness, many efforts have been made during the last decade to solubilize these antigens from their hydrophobic site on the cell membrane in order to determine their chemical and molecular structure (1). Of the solubilization methods that have been tried, hypertonic salt (3 M KCl) extraction of cultured human lymphoid cells has proved to be the most simple, reproducible, and effective (2). This method is now widely used for the solubilization of H-2 (3) and a variety of tumor-specific antigens (4).

The objective of this study was to find out how this method solubilizes HL-A antigens from their hydrophobic sites on the cell surface. Specifically, we wanted to determine whether the method's effectiveness is attributable to the cleavage of protein fragments by intracellular proteases or to the dissociation of hydrophobic bonds brought about by the chaotropic effect of KCl.

Materials and Methods. Cellular extracts. The Wl-L₂ cultured cell line was propagated in our laboratory and we obtained cell lines RPMI 1788 and RPMI 4098 from Associated Biomedic Systems, Buffalo, NY. Cultured cells were washed three times with saline and then extracted with 3 M KCl at 4°C (2).