Effect of detergents on ligand binding and translocation activities of solubilized/reconstituted nucleoside transporters.
Purification of equilibrative nucleoside transporters has been hampered by the functional instability of the detergent-solubilized proteins. A variety of nonionic detergents were compared with octylglucoside (the most commonly used detergent in this regard) for their abilities to solubilize functionally stable nucleoside transporter proteins from Ehrlich cell plasma membranes. Transporter stability was assessed through the binding of the specific probe [3H]nitrobenzylthioinosine to freshly solubilized and stored (48 h/6 degrees C) preparations. The most promising detergents were decylmaltoside and cyclohexylbutylmaltoside, both of which, like octylglucoside, solubilized over 70% of the transporters from the membrane. Decylmaltoside- and cyclohexylbutylmaltoside-solubilized transport proteins retained 61 and 83%, respectively, of their [3H]-nitrobenzylthioinosine binding activity upon storage, compared to about 30% using octylglucoside. Decylmaltoside was also superior to octylglucoside in its capacity to solubilize the transporter in a state that retained its high affinity for the transport inhibitors dilazep (Ki = 11 nM, vs 75 nM in octylglucoside) and dipyridamole (Ki = 260 nM, vs 12 microM in octylglucoside). Reconstitution studies indicated that both the decylmaltoside- and cyclohexylbutylmaltoside-solubilized transporters were capable of mediating the uptake of [3H]uridine. Decylmaltoside was superior to cyclohexylbutylmaltoside, however, in both the enhanced transport activity of the resulting proteoliposomes (Vi = 21 pmol/mg/s vs 13 pmol/mg/s, respectively) and the lower nonmediated uptake observed in the decylmaltoside-derived vesicles (27% of total uptake at 4 min incubation). Nevertheless, cyclohexylbutylmaltoside may be useful in initial solubilization procedures due to its ability to selectively solubilize the nucleoside transporter from the plasma membrane. The rational use of these detergents will enable a more extensive purification of functional nucleoside transporters.
Hammond
J R
JR
Department of Pharmacology and Toxicology, University of Western Ontario, London, Canada. jhammo@julian.uwo ca
Zarenda
M
M
D000818:Animals; D002286:Carcinoma, Ehrlich Tumor / Q000378:metabolism; D002352:Carrier Proteins / Q000037:antagonists & inhibitors / Q000302:isolation & purification* / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D003902:Detergents; D005960:Glucosides; D066298:In Vitro Techniques; D007700:Kinetics; D008024:Ligands; D008297:Male; D008565:Membrane Proteins / Q000037:antagonists & inhibitors / Q000302:isolation & purification* / Q000378:metabolism*; D051379:Mice; D015335:Molecular Probes; D033703:Nucleoside Transport Proteins; D009705:Nucleosides / Q000378:metabolism*; D012995:Solubility; D013868:Thioinosine / Q000031:analogs & derivatives / Q000378:metabolism
D002352:Carrier Proteins; D003902:Detergents; D005960:Glucosides; D008024:Ligands; D008565:Membrane Proteins; D015335:Molecular Probes; D033703:Nucleoside Transport Proteins; D009705:Nucleosides; C018619:octyl-beta-D-glucoside; D013868:Thioinosine; C001789:4-nitrobenzylthioinosine