Solubilization, reconstitution, and attempted affinity chromatography of the sugar transporter of the erythrocyte membrane

Abstract

Reconstitution of the sugar transport system of human erythrocytes into artificial liposomes was achieved by freezing, thawing, and sonicating preformed phospholipid vesicles in the presence of intact ghosts, protein-depleted ghosts, or detergent-treated ghosts. D-glucose equilibrium exchange activities and affinity constants in the range of the reported erythrocyte values were reached in the best experiments. Whereas the extraction of peripheral membrane proteins did not depress the transport function crucially after reconstituting these protein-depleted ghosts, the selective solubilization of integral membrane proteins by a variety of nonionic detergents resulted in an uncontrollable, continuously increasing inactivation of the carrier. However, Emulphogene BC-720 extracts could be prepared in which the glucose transporter retained activity for days at 4 degrees C. These extracts were applied to affinity chromatography matrices of phloretin-Agarose, prepared by coupling phloretinyl-3'-benzylamine (PBA) to CH-Sepharose 4B and to Affigel 202. Although the solubilized sugar transporter appeared to be selectively adsorbed to both PBA matrices, it could not be eluted by specific counter ligands or gentle eluants in a biologically active form. However, chaotropic agents could be used to elute intrinsic proteins, including bands 3 and 4.5, from the Affigel affinity medium.

Purification of proteins of the Na/Cl cotransporter from membranes of Ehrlich ascites cells using a bumetanide-sepharose affinity column

“…For example, Pimplikar and Reithmeier (1986) successfully used affinity columns with stilbene derivatives as ligands to isolate the anion exchanger from red cells. Weber et al (1985) have reported some success in isolating the glucose transporter by affinity chromatography. Finally Cherksey et al (1987) have used a furosemide affinity gel to isolate proteins from Ehrlich ascites cells which are part of a K channel.…”