Hutchison K A, Prasad M, Work C, Fox I H
Department of Internal Medicine, University Hospital, Ann Arbor, Michigan 48109-0108.
Am J Med Sci. 1991 Jan;301(1):1-8. doi: 10.1097/00000441-199101000-00001.
Adenotin is a low affinity adenosine binding protein that has amino terminal homology with mammalian and avian stress proteins. Human placental adenotin was solubilized and reconstituted into phospholipid vesicles with an overall yield of 30%. The properties of adenotin in vesicles were similar to the native membranes as follows: association has a Kobs of 0.61 +/- 0.03 minute-1; equilibrium is reached in approximately 15 minutes; and the first order dissociation constant is 5.0 +/- 0.3 minute-1. Displacement analysis reveals an agonist potency order and Ki values as follows: N-ethylcarboxamidoadenosine, 0.35 microM; 2-chloroadenosine, 1.5 microM; R-phenylisopropyladenosine, greater than 1000 microM. The addition of 100 microM 5'-guanylylimidodiphosphate did not decrease binding of 5'-N-ethylcarboxamidoadenosine (NECA) at 37 degrees C or 4 degrees C but did decrease the IC50 for PC12 and JAR cell membrane agonist binding from 9.9 to 3.3 microM and increase the binding to 150-211% of the control value at 37 degrees C. The latter studies at 37 degrees C showed high variability. Using binding sites reconstituted into vesicles and gel filtration chromatography and agonist related guanine nucleotide release, the authors investigated whether these changes were related to an interaction between adenotin and a guanine nucleotide regulatory protein. No evidence for such an interaction was found. These data suggest that adenotin retains its binding properties when reconstituted into phospholipid vesicles. The function of this low affinity adenosine binding site remains to be discovered. However, the reconstitution of adenotin into phospholipid vesicles provides a method to study its function.
腺嘌呤核苷蛋白是一种低亲和力的腺苷结合蛋白，其氨基末端与哺乳动物和禽类应激蛋白具有同源性。人胎盘腺嘌呤核苷蛋白被溶解并重新构建到磷脂囊泡中，总产率为30%。囊泡中腺嘌呤核苷蛋白的性质与天然膜相似，如下所示：缔合的观测速率常数（Kobs）为0.61±0.03分钟-1；约15分钟达到平衡；一级解离常数为5.0±0.3分钟-1。置换分析揭示了激动剂效力顺序和抑制常数（Ki）值如下：N-乙基羧基酰胺腺苷，0.35微摩尔；2-氯腺苷，1.5微摩尔；R-苯异丙基腺苷，大于1000微摩尔。添加100微摩尔的5'-鸟苷酰亚胺二磷酸在37℃或4℃时不会降低5'-N-乙基羧基酰胺腺苷（NECA）的结合，但会将PC12和JAR细胞膜激动剂结合的半数抑制浓度（IC50）从9.9微摩尔降至3.3微摩尔，并在37℃时将结合增加至对照值的150%-211%。后者在37℃的研究显示出高度变异性。作者使用重构到囊泡中的结合位点、凝胶过滤色谱法和与激动剂相关的鸟嘌呤核苷酸释放，研究了这些变化是否与腺嘌呤核苷蛋白和鸟嘌呤核苷酸调节蛋白之间的相互作用有关。未发现这种相互作用的证据。这些数据表明，腺嘌呤核苷蛋白重构到磷脂囊泡中时保留了其结合特性。这种低亲和力腺苷结合位点的功能仍有待发现。然而，将腺嘌呤核苷蛋白重构到磷脂囊泡中提供了一种研究其功能的方法。
