A refined method for the photoaffinity labelling of the nitrobenzylthioinosine-sensitive nucleoside transport protein: application to cell membranes of calf lung tissue.
A refined method for the photoaffinity labelling of the NBI-sensitive nucleoside transport protein is described. It involves the use of low concentrations of the photolabile probe [3H]nitrobenzylthioinosine ([3H]NBI), whereas the usual inclusion of dithiothreitol in the protocol is omitted. The method was successfully applied to cell membranes of calf lung tissue, which was shown to be a rich source of this physiologically important protein with all the characteristics (both in membrane bound and solubilized form) known from similar proteins on other cell types. Specific covalent incorporation of radioactivity appeared to be pH independent. SDS-polyacrylamide gel electrophoresis revealed a specifically labelled protein with an apparent molecular weight of 55 kDa.
IJzerman
A P
AP
Division of Medicinal Chemistry, State University of Leiden, The Netherlands.
Menkveld
G J
GJ
Thedinga
K H
KH
D000241:Adenosine / Q000494:pharmacology; D000345:Affinity Labels; D000818:Animals; D002352:Carrier Proteins / Q000032:analysis* / Q000378:metabolism; D002417:Cattle; D002462:Cell Membrane / Q000032:analysis; D004229:Dithiothreitol / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D006863:Hydrogen-Ion Concentration; D007288:Inosine / Q000031:analogs & derivatives* / Q000494:pharmacology; D008168:Lung / Q000032:analysis*; D008565:Membrane Proteins / Q000032:analysis* / Q000378:metabolism; D033703:Nucleoside Transport Proteins; D010777:Photochemistry; D013868:Thioinosine / Q000031:analogs & derivatives* / Q000378:metabolism
D000345:Affinity Labels; D002352:Carrier Proteins; D008565:Membrane Proteins; D033703:Nucleoside Transport Proteins; D013868:Thioinosine; D007288:Inosine; C001789:4-nitrobenzylthioinosine; D000241:Adenosine; D004229:Dithiothreitol