Rangel-Aldao R, Opperdoes F R
Mol Biochem Parasitol. 1984 Feb;10(2):231-41. doi: 10.1016/0166-6851(84)90010-0.
We assayed the cyclic AMP-binding activity of Trypanosoma brucei by two well established methods, such as the one of Gilman (Proc. Natl. Acad. Sci. U.S.A. 67, 305-312, 1970) and Ueland & Doskeland (Biochem. J., 157, 117-126, 1976). The results indicate that the former technique underestimated the total amount of cyclic AMP bound by T. brucei homogenates by up to 7.5 fold. Similar results were obtained with other Trypanosomatidae such as Leishmania tropica, and Crithidia luciliae. The bulk of the cyclic AMP-binding proteins of T. brucei appeared soluble after centrifugation of post-large-granule extracts in isopycnic sucrose-gradients. Upon fractionation by DEAE-Sephacel column chromatography, two peaks of activity eluted which were responsible for all the specific cyclic AMP-binding activity present in the cytosol of T. brucei. These two activities, which we denominated as Peak 'a' and Peak 'b' respectively, differed in a number of properties such as sensitivity to proteases, stability to storage at -20 degrees C, displacement of cyclic AMP bound by adenine analogues, and coefficients of sedimentation.
我们采用了两种成熟的方法来检测布氏锥虫的环磷酸腺苷（cAMP）结合活性，比如吉尔曼（《美国国家科学院院刊》67卷，305 - 312页，1970年）以及厄兰德和多斯克兰德（《生物化学杂志》，157卷，117 - 126页，1976年）所使用的方法。结果表明，前一种技术对布氏锥虫匀浆结合的cAMP总量的低估幅度高达7.5倍。在其他锥虫科寄生虫如热带利什曼原虫和鲁氏克氏锥虫中也获得了类似结果。在等密度蔗糖梯度中对大颗粒后提取物进行离心后，布氏锥虫的大部分cAMP结合蛋白呈现出可溶性。通过DEAE - 葡聚糖凝胶柱色谱法进行分级分离时，洗脱得到了两个活性峰，它们构成了布氏锥虫细胞质中所有特异性cAMP结合活性。我们分别将这两种活性命名为峰“a”和峰“b”，它们在许多特性上存在差异，例如对蛋白酶的敏感性、在-20℃储存的稳定性、被腺嘌呤类似物结合的cAMP的置换情况以及沉降系数。
