Characterization of a novel adenosine binding protein sensitive to cyclic AMP in rat brain cytosolic and particulate fractions.
A novel binding site for the adenosine receptor agonist 5'-N-ethylcarboxamidoadenosine (NECA), which was enriched in rat forebrain, was characterized in cytosolic and particulate preparations. The site showed a pharmacological profile different from other [3H]NECA binding proteins and was named adenotin 2. [3H]NECA was bound in the presence of 100 microM 2-chloroadenosine with a Kd of 45.4 nM and a Bmax of 4711 fmol/mg in the cytosol and a Kd of 72.4 nM and a Bmax of 4844 fmol/mg in the crude membrane fraction. The presence of two different binding sites on adenotin 2 for [3H]NECA was shown in kinetic experiments. This protein showed identical pharmacological profiles in both subcellular preparations. [3H]NECA was displaced by purine analogues with a rank order of potency of NECA > 3'5' cyclic AMP (cAMP) > 5'-deoxy-5'-chloroadenosine > S-adenosylhomocysteine approximately 5'-deoxy-5'-methylthioadenosine (MeSA) > adenosine approximately adenine. cAMP inhibited [3H]NECA binding allosterically, whereas adenine and MeSA acted competitively. Inhibitors and activators of protein kinases such as N-(2-aminoethyl)-5-isoquinolinesulfonamide, Sp-adenosine cyclic monophophothioate and (8R*, 9S*, 11S*)-(-)-9-hydroxy-9-methoxy -carbonyl-8-methyl-2,3,9, 10-tetrahydro-8,11-epoxy-1H, 8H, 11H-2, 7b, 11a-triazadibenzo-(a,g)cycloocta(cde)-trinden-1-one (K 252a) interacted with [3H]NECA binding to adenotin 2 in nanomolar concentrations. Adenosine-5'-O-(3-thiotriphosphate) (100 microM) increased the affinity of [3H]NECA to a Kd of 9 nM and diminished the affinity of cAMP. The pharmacological characteristics of this novel binding site for [3H]NECA resemble those of the inhibition of phosphorylation processes by adenosine and its derivatives in heart and smooth muscle but are distinct from known adenosine receptors, adenosine binding proteins and protein kinases.
Lorenzen
A
A
Pharmakologisches Institut der Universität Heidelberg, Germany.
Grossekatthöfer
B
B
Kerst
B
B
Vogt
H
H
Fein
T
T
Schwabe
U
U
D000241:Adenosine / Q000031:analogs & derivatives / Q000378:metabolism*; D019830:Adenosine-5'-(N-ethylcarboxamide); D000495:Allosteric Site; D000818:Animals; D001667:Binding, Competitive; D001921:Brain / Q000378:metabolism*; D002352:Carrier Proteins / Q000302:isolation & purification / Q000378:metabolism*; D000242:Cyclic AMP / Q000378:metabolism / Q000494:pharmacology*; D003600:Cytosol / Q000378:metabolism; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D066298:In Vitro Techniques; D007700:Kinetics; D008297:Male; D047428:Protein Kinase Inhibitors; D011494:Protein Kinases / Q000378:metabolism; D058906:Purinergic P1 Receptor Agonists; D011869:Radioligand Assay; D051381:Rats; D017208:Rats, Wistar
D002352:Carrier Proteins; D004791:Enzyme Inhibitors; D047428:Protein Kinase Inhibitors; D058906:Purinergic P1 Receptor Agonists; C064982:adenotin; D019830:Adenosine-5'-(N-ethylcarboxamide); D000242:Cyclic AMP; D011494:Protein Kinases; D000241:Adenosine