Deoxycholic Acid-coupled Poly(~-lysyl) Agarose AN AMPHIPATHIC MATRIX WITH BINDING AFFINITY FOR INTEGRAL MEMBRANE

This paper describes techniques for covalently binding deoxycholic acid to poly(L-lysine)-coupled agarose beads (Bio-gel A-5m) to create an insoluble amphipathic or detergent-like matrix with specific affinity for membrane molecules. The deoxycholate/A-5m beads were shown to bind phosphatidylcholine and they were shown to bind, as models for the binding of integral membrane proteins and glycoproteins, deoxycholate-solubilized HLA antigens and the plasma membrane marker enzyme 5’-nucleotidase from human lymphoblastoid cell lines with retention of alloantibodybinding and enzymatic activities, respectively. Various hydrophilic proteins failed to bind detectably. Bovine serum albumin, which possesses four binding sites for deoxycholate, was observed to bind. Binding of membrane proteins to the deoxycholate/A-5m beads was reversed by the addition of deoxycholate, but the interaction was stable in the absence of detergents. HLA antigens were also released from the deoxycholate/A5m beads by proteolysis with papain, in a manner analogous to proteolytic solubilization from membranes, and with an identical yield of inhibitory activity to that of deoxycholate-released material. In studies using deoxycholate-solubilized membrane glycoprotein fractions, purified by affinity chromatography on columns of Lens culinaris hemagglutinin, binding of total protein to the beads was shown to be independent of added phospholipid although the 5’-nucleotidase activity of the deoxycholate/A-5m-associated glycoproteins was markedly enhanced by the simultaneous binding of phosphatidylcholine.