Lorenzen A, Grossekatthöfer B, Kerst B, Vogt H, Fein T, Schwabe U
Pharmakologisches Institut der Universität Heidelberg, Germany.
Biochem Pharmacol. 1996 Nov 8;52(9):1375-85. doi: 10.1016/s0006-2952(96)00465-0.
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.
在大鼠前脑中富集的一种新型腺苷受体激动剂5'-N-乙基羧基酰胺腺苷（NECA）结合位点，在胞质溶胶和微粒体制剂中进行了表征。该位点显示出与其他[3H]NECA结合蛋白不同的药理学特征，并被命名为腺嘌呤素2。在100 microM 2-氯腺苷存在下，[3H]NECA在胞质溶胶中的结合Kd为45.4 nM，Bmax为4711 fmol/mg，在粗膜部分中的Kd为72.4 nM，Bmax为4844 fmol/mg。动力学实验表明腺嘌呤素2上存在两个不同的[3H]NECA结合位点。该蛋白在两种亚细胞制剂中显示出相同的药理学特征。嘌呤类似物以NECA>3'5'环磷酸腺苷（cAMP）>5'-脱氧-5'-氯腺苷>S-腺苷同型半胱氨酸≈5'-脱氧-5'-甲硫基腺苷（MeSA）>腺苷≈腺嘌呤的效力顺序取代[3H]NECA。cAMP变构抑制[3H]NECA结合，而腺嘌呤和MeSA起竞争性作用。蛋白激酶抑制剂和激活剂，如N-（2-氨基乙基）-5-异喹啉磺酰胺、Sp-腺苷环一磷酸硫代酯和（8R*，9S*，11S*）-（-）-9-羟基-9-甲氧基-羰基-8-甲基-2,3,9,10-四氢-8,11-环氧-1H，8H，11H-2,7b，11a-三氮杂二苯并-（a,g）环辛（cde）-三茚-1-酮（K 252a）以纳摩尔浓度与[3H]NECA与腺嘌呤素2的结合相互作用。腺苷-5'-O-（3-硫代三磷酸）（100 microM）将[3H]NECA的亲和力提高到Kd为9 nM，并降低了cAMP的亲和力。这个新型[3H]NECA结合位点的药理学特征类似于腺苷及其衍生物对心脏和平滑肌中磷酸化过程的抑制作用，但与已知的腺苷受体、腺苷结合蛋白和蛋白激酶不同。
