Heterogeneous forms of adenotin-1 of different subcellular localization

Abstract

The localization of the low-affinity adenosine binding protein adenotin-1 with respect to distribution in rat organs and subcellular compartments was investigated. Adenotin-1 was characterized by 5′- N-ethylcarboxamido[2,8- 3H]adenosine ([ 3NECA) binding and Western blotting. Cytosolic as well as membrane fractions of all tissues contained adenotin-1. Highest levels of membrane-bound adenotin-1 were found in the liver (liver > kidney ≈ spleen ≈ lung > forebrain ≈ cerebellum > fat ≈ heart ≈ striated muscle), whereas highest levels of cytosolic adenotin-1 were detected in spleen, liver, lung and fat. Subcellular fractions from rat liver were prepared by differential and density gradient centrifugation. Like the homologous proteins endoplasmin or gp96, adenotin-1 is enriched in the endoplasmic reticulum. Cytosolic and membrane-bound adenotin-1 species are pharmacologically distinct, because in the liver particulate fraction adenotin-1 showed a more rapid binding kinetics, a twofold lower affinity for [ 3H]NECA ( K D 227 nM vs. 105 nM) and a sevenfold higher affinity for 2-chloroadenosine than the cytosolic protein ( K i 1.48 μM vs. 9.25 μM). In rat liver cytosol, two different binding sites were found, which differed in [ 3H]NECA binding kinetics and displayed a hundredfold difference in their affinity for 2-chloro-5′- N-methylcarboxamidoadenosine ( K i 45.8 nM vs. 4.76 μM). The presence of adenotin-1 in subcellular fractions, as determined by radioligand binding, was confirmed by Western blotting. Adenotin-1 was detected as a 98-kDa band in all rat liver subcellular fractions, which agrees with the molecular mass determined for the purified protein. In the cytosol, a 65-kDa band was labeled more intensely than the 98-kDa band. This additional band probably represents the pharmacologically distinct species of adenotin-1 found in the cytosol.