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Effect of detergents on ligand binding and translocation activities of solubilized/reconstituted nucleoside transporters.

Abstract：

: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.

journal_name

Arch Biochem Biophys

journal_title

Archives of biochemistry and biophysics

authors

Hammond JR,Zarenda M

doi

10.1006/abbi.1996.0347

subject

Has Abstract

pub_date

1996-08-15 00:00:00

pages

313-22

issue

eissn

0003-9861

issn

1096-0384

pii

S0003-9861(96)90347-1

journal_volume

332

pub_type

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Effect of detergents on ligand binding and translocation activities of solubilized/reconstituted nucleoside transporters.