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Solubilization, reconstitution, and attempted affinity chromatography of the sugar transporter of the erythrocyte membrane
Author(s) -
Weber Jakob,
Warden Dean Allan,
Semenza Giorgio,
Diedrich Donald F.
Publication year - 1985
Publication title -
journal of cellular biochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.028
H-Index - 165
eISSN - 1097-4644
pISSN - 0730-2312
DOI - 10.1002/jcb.240270203
Subject(s) - chemistry , chaotropic agent , affinity chromatography , membrane , chromatography , elution , biochemistry , phloretin , vesicle , enzyme
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‐depicted 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°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.