Fein T, Schulze E, Bär J, Schwabe U
Pharmakologisches Institut, Universität Heidelberg, Germany.
Naunyn Schmiedebergs Arch Pharmacol. 1994 Apr;349(4):374-80. doi: 10.1007/BF00170883.
A low-affinity adenosine binding protein (adenotin) was purified from human platelet membranes by a four-step procedure. Purification was achieved after extraction from human platelet membranes with 0.3% 3-[3-(cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS). Further purification included Sepharose CL6B gel filtration, DEAE-Sepharose CL6B, and hydroxylapatite chromatography. The protein was purified 884-fold to homogeneity with a 25% yield of binding activity from the membranes. 5'-[8(n)-3H]-N-ethylcarboxamidoadenosine ([3H]NECA) binds to the purified protein with a KD of 155 (144-167) nmol/l and a Bmax of 1.85 +/- 0.10 nmol/mg of protein. Sodium dodecylsulfate polyacrylamide gel electrophoresis of purified protein revealed a single band at 98 kDa. The 2-chloro-substituted adenosine analogs 2-chloro-5'-N-methylcarboxamidoadenosine (CIMECA) and 2-chloro-5'-N-ethylcarboxamidoadenosine (CINECA) were identified as new high affinity ligands of the purified protein showing Ki values of 18 nmol/l and 28 nmol/l, respectively. The low-affinity adenosine binding protein showed a pharmacological profile as follows: CIMECA > 5'-N-ethylcarboxamidoadenosine (NECA) > 2-chloroadenosine (CIA) > 2-[4-(2-carboxyethyl)phenethylamino]-5'-N-ethylcarboxamidoadenosin e (CGS 21,680) > R-N6-phenylisopropyl-adenosine (R-PIA). Amino-terminal sequence analysis revealed homologies to endoplasmin, glucose regulated protein (GRP94), tumor rejection antigen precursor (GP96), and some stress related proteins.
采用四步法从人血小板膜中纯化出一种低亲和力腺苷结合蛋白（腺嘌呤素）。用0.3%的3-[3-(胆酰胺丙基)二甲基铵]-1-丙烷磺酸盐（CHAPS）从人血小板膜中提取后实现了纯化。进一步的纯化包括Sepharose CL6B凝胶过滤、DEAE-Sepharose CL6B和羟基磷灰石层析。该蛋白被纯化了884倍达到同质，膜结合活性的产率为25%。5'-[8(n)-3H]-N-乙基羧酰胺腺苷（[3H]NECA）以155（144 - 167）nmol/l的KD和1.85±0.10 nmol/mg蛋白的Bmax与纯化后的蛋白结合。纯化蛋白的十二烷基硫酸钠聚丙烯酰胺凝胶电泳显示在98 kDa处有一条单一的条带。2-氯取代的腺苷类似物2-氯-5'-N-甲基羧酰胺腺苷（CIMECA）和2-氯-5'-N-乙基羧酰胺腺苷（CINECA）被鉴定为该纯化蛋白的新的高亲和力配体，其Ki值分别为18 nmol/l和28 nmol/l。低亲和力腺苷结合蛋白呈现出如下药理学特征：CIMECA > 5'-N-乙基羧酰胺腺苷（NECA）> 2-氯腺苷（CIA）> 2-[4-(2-羧乙基)苯乙氨基]-5'-N-乙基羧酰胺腺苷（CGS 21,680）> R-N6-苯异丙基腺苷（R-PIA）。氨基末端序列分析显示与内质溶素、葡萄糖调节蛋白（GRP94）、肿瘤排斥抗原前体（GP96）以及一些应激相关蛋白具有同源性。
