Olah M E, Jacobson K A, Stiles G L
Department of Medicine (Cardiology), Duke University Medical Center, Durham, North Carolina 27710.
Arch Biochem Biophys. 1990 Dec;283(2):440-6. doi: 10.1016/0003-9861(90)90665-l.
A1 adenosine receptors (A1AR) acting via the inhibitory guanine nucleotide binding protein inhibit adenylate cyclase activity in brain, cardiac, and adipose tissue. We now report the purification of the A1AR from bovine cerebral cortex. This A1AR is distinct from other A1ARs in that it displays an agonist potency series of N6-R-phenylisopropyladenosine (R-PIA) greater than N6-S-phenylisopropyladenosine greater than (S-PIA) greater than 5'-N-ethylcarboxamidoadenosine (NECA) compared to the traditional potency series of R-PIA greater than NECA greater than S-PIA. The A1AR was solubilized in 1% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (Chaps) and then purified by chromatography on an antagonist [xanthine amine congener (XAC)]-coupled Affi-Gel 10 followed by hydroxylapatite chromatography. Following purification, sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a single protein of Mr 36,000 by silver staining, Na125I iodination with chloramine T and photoaffinity labeling with [125I]8-[4-[[[[2-(4-aminophenyl acetylamino) ethyl] carbonyl] methyl] oxy]-phenyl]-1,3- dipropylxanthine. This single protein displayed all the characteristics of the A1AR, including binding an antagonist radioligand [( 3H]XAC) with high affinity (Kd = 0.7 nM) and in a saturable manner (Bmax greater than 4500 pmol/mg). Agonist competition curves demonstrated the expected bovine brain A1AR pharmacology: R-PIA greater than S-PIA greater than NECA. The overall yield from soluble preparation was 7%. The glycoprotein nature of the purified A1AR was determined with endo- and exoglycosidases. Deglycosylation with endoglycosidase F increased the mobility of the A1AR from Mr 36,000 to Mr 32,000 in a single step. The A1AR was sensitive to neuraminidase but resistant to alpha-mannosidase, suggesting the single carbohydrate chain was of the complex type. This makes the bovine brain A1AR similar to rat brain and fat A1AR in terms of its carbohydrate chains yet the purified A1AR retains its unique agonist potency series observed in membranes.
通过抑制性鸟嘌呤核苷酸结合蛋白起作用的A1腺苷受体（A1AR）可抑制脑、心脏和脂肪组织中的腺苷酸环化酶活性。我们现在报告从牛大脑皮层中纯化出A1AR。这种A1AR与其他A1AR不同，与传统的R-PIA＞NECA＞S-PIA的效价顺序相比，它显示出的激动剂效价顺序为N6-R-苯基异丙基腺苷（R-PIA）＞N6-S-苯基异丙基腺苷（S-PIA）＞5'-N-乙基羧酰胺腺苷（NECA）。A1AR在1%的3-[(3-胆酰胺丙基)二甲基铵]-1-丙烷磺酸盐（Chaps）中溶解，然后通过在拮抗剂[黄嘌呤胺同类物（XAC）]偶联的Affi-Gel 10上进行色谱分离，随后进行羟基磷灰石色谱纯化。纯化后，十二烷基硫酸钠-聚丙烯酰胺凝胶电泳经银染显示出一条分子量为36,000的单一蛋白带，用氯胺T进行Na125I碘化并用[125I]8-[4-[[[[2-(4-氨基苯基乙酰氨基)乙基]羰基]甲基]氧基]-苯基]-1,3-二丙基黄嘌呤进行光亲和标记。这条单一蛋白带显示出A1AR的所有特性，包括以高亲和力（Kd = 0.7 nM）且以可饱和方式（Bmax＞4500 pmol/mg）结合拮抗剂放射性配体[（3H）XAC]。激动剂竞争曲线证明了预期的牛脑A1AR药理学特性：R-PIA＞S-PIA＞NECA。可溶性制剂的总产率为7%。用内切糖苷酶和外切糖苷酶确定了纯化的A1AR的糖蛋白性质。用内切糖苷酶F进行去糖基化使A1AR的迁移率在一步中从分子量36,000增加到分子量32,000。A1AR对神经氨酸酶敏感，但对α-甘露糖苷酶有抗性，这表明单一糖链为复合型。这使得牛脑A1AR在糖链方面与大鼠脑和脂肪A1AR相似，但纯化的A1AR保留了在膜中观察到的独特激动剂效价顺序。
