Enhancement of the functional stability of solubilized nucleoside transporters by substrates and inhibitors.
Purification of functional nucleoside transporters has been hampered by the instability of detergent-solubilized proteins. The present study was undertaken to determine if the presence of specific transporter ligands in the solubilization medium could enhance the functional stability of the isolated proteins. Ehrlich cell plasma membranes were solubilized with 1% (w/v) octylglucoside (+/- transporter ligands) and reconstituted into liposomal membranes either immediately after solubilization or after storage for 48 hr at 6 degrees. Storage resulted in a parallel loss (approximately 60%) of [3H]nitrobenzylthioinosine (NBMPR) binding and reconstituted [3H]uridine uptake activities. furthermore, upon storage, the relative amount of NBMPR-resistant [3H]uridine uptake by the reconstituted system dropped from 19 +/- 2 to 8 +/- 1% of the total mediated influx. The inclusion of high concentrations (> 10 mM) of adenosine in the solubilization medium completely prevented the storage-induced loss of both [3H]NBMPR binding and [3H]uridine influx activity, and prevented the shift in NBMPR sensitivity. In addition, inclusion of adenosine in the solubilization procedure increased the relative amount of NBMPR-resistant [3H]uridine uptake to 33 +/- 2% of the total influx in proteoliposomes prepared immediately after the proteins were extracted from the plasma membrane (i.e. no storage). A partial protection of [3H]NBMPR binding activity was also obtained using 2'-deoxyadenosine, 2-chloroadenosine, uridine, and non-radiolabelled NBMPR, but not with cytidine, inosine, diazepam, dipyridamole, or dilazep. These results suggest that both NBMPR sensitivity and transporter stability are dependent upon the conformational state of the protein. The protective effects of adenosine analogues and other nucleosides are likely due to their binding to the substrate translocation site, thereby effectively "locking" the transporter in a stable conformation.[1]References
- Enhancement of the functional stability of solubilized nucleoside transporters by substrates and inhibitors. Hammond, J.R. Biochem. Pharmacol. (1997) [Pubmed]
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