Photoaffinity labeling of the adenosine transport system in adipocyte plasma membranes

Abstract

A membrane component involved in the transport of adenosine in adipocytes has been identified utilizing the techniques of photoaffinity labeling with the adenosine derivative, 8-azidoadenosine. In the absence of light, adenosine and 8-azidoadenosine exhibited similar transport characteristics. In addition, adenosine was shown to be a competitive inhibitor of 8-azidoadenosine uptake, and the photoprobe, a competitive inhibitor of adenosine uptake. Analysis of the nucleotide metabolites indicated that the photoprobe was metabolized in a similar fashion to that observed for adenosine. Several nucleoside transport inhibitors were also equally effective in inhibiting the uptake of both nucleosides. These results suggest that 8-azidoadenosine is transported by the same membrane system as adenosine. Photolysis of 8-azido[2- 3H]adenosine in the presence of adipocytes resulted in the covalent incorporation of the photoprobe into the plasma membrane fraction. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that essentially all of the radioactivity was incorporated into a glycoprotein with a molecular weight of 56,000. This labeling was inhibited by greater than 90% when the photolysis was carried out in the presence of excess adenosine or the transport inhibitors, persantin or theophylline. Fractionation of the labeled plasma membranes by dialysis against water (pH 9.5) indicated that approximately 75% of the radioactivity was associated with a glycoprotein which resisted solubilization by this procedure. These results suggest that the major labeled species is a 56,000 M r intrinsic membrane glycoprotein which may function as a component of a transmembrane assembly involved in the transport of adenosine.