Heterogeneous forms of adenotin-1 of different subcellular localization.
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 ([3H]NECA) 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 approximately spleen approximately lung > forebrain approximately 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 (KD 227 nM vs. 105 nM) and a sevenfold higher affinity for 2-chloroadenosine than the cytosolic protein (Ki 1.48 microM vs. 9.25 microM). 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 (Ki 45.8 nM vs. 4.76 microM). 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 hand 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.
Lorenzen
A
A
Universitat Heidelberg, Pharmakologisches Institut, Germany. anna.lorenzen@urz.uni-heidelberg.de
Engelhardt
J
J
Kerst
B
B
Schwabe
U
U
D019830:Adenosine-5'-(N-ethylcarboxamide) / Q000378:metabolism; D000818:Animals; D001665:Binding Sites; D002352:Carrier Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D003600:Cytosol / Q000378:metabolism; D004721:Endoplasmic Reticulum / Q000378:metabolism; D015151:Immunoblotting; D066298:In Vitro Techniques; D007700:Kinetics; D008099:Liver / Q000378:metabolism; D008297:Male; D008970:Molecular Weight; D051381:Rats; D017207:Rats, Sprague-Dawley; D013347:Subcellular Fractions / Q000378:metabolism; D014018:Tissue Distribution
D002352:Carrier Proteins; C064982:adenotin; D019830:Adenosine-5'-(N-ethylcarboxamide)