Functional reconstitution of pharmacologically distinct subtypes of nucleoside transporters in liposomal membranes.
We examined the functional and pharmacological characteristics of liposome-reconstituted nucleoside transporter proteins obtained by detergent (octylglucoside) extraction from Ehrlich ascites tumor cell plasma membranes. Optimal reconstitution was achieved using a lipid composition of phosphatidylcholine, cholesterol, phosphatidylethanolamine and phosphatidylserine in a molar ratio of 33:33:26:8 and with a lipid-to-protein ratio of 30. This preparation had Km and Vmax values of 280 microM and 570 pmol/mg/sec, respectively, for the transporter-mediated uptake of [3H]uridine and bound 8.3 pmol of [3H]nitrobenzyl-thioinosine per milligram of protein. In general, the reconstituted system had kinetic and pharmacological characteristics comparable to those of the native membrane-located system, including an 80:20 ratio of nitrobenzylthioinosine-sensitive to -resistant [3H]uridine influx. The uridine translocation capacity of the optimally reconstituted system was 56 molecules per transporter per second compared with 104 molecules per transporter per second in intact cells, indicating that more than half of the inserted proteins were capable of mediating the influx of [3H]uridine (assuming that each functioning transporter was operating at optimal efficiency). Differences between the native and reconstituted transporters included the appearance of a [3H]uridine influx component (inhibited by adenosine) that was resistant to inhibition by R75231 (18%) and dipyridamole (10%). Dilazep was also significantly less effective in inhibiting the nitrobenzylthioinosine-sensitive transporter in the reconstituted preparations (IC50 = 41 nM) relative to that seen in intact Ehrlich cells (IC50 = 1.4 nM). These results suggest that inhibitor sensitivity may be a factor of the lipid microenvironment of the transporter or may involve other cellular components that could dissociate from the complex on detergent solubilization.(ABSTRACT TRUNCATED AT 250 WORDS)
Department of Pharmacology and Toxicology, University of Western Ontario, London, Canada.
J R
JR
Hammond
D000818:Animals; D002352:Carrier Proteins / Q000187:drug effects / Q000302:isolation & purification* / Q000502:physiology*; D005960:Glucosides / Q000494:pharmacology; D007700:Kinetics; D008055:Lipids / Q000032:analysis; D008081:Liposomes / Q000737:chemistry*; D008297:Male; D008565:Membrane Proteins / Q000187:drug effects / Q000302:isolation & purification* / Q000502:physiology*; D051379:Mice; D033703:Nucleoside Transport Proteins; D013868:Thioinosine / Q000031:analogs & derivatives / Q000378:metabolism; D014529:Uridine / Q000378:metabolism
D002352:Carrier Proteins; D005960:Glucosides; D008055:Lipids; D008081:Liposomes; D008565:Membrane Proteins; D033703:Nucleoside Transport Proteins; C018619:octyl-beta-D-glucoside; D013868:Thioinosine; C001789:4-nitrobenzylthioinosine; D014529:Uridine