Binding of cAMP Derivatives to Dictyostelium discoideum Cells
Binding of cAMP Derivatives to Dictyostelium discoideum Cells
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cAMP 衍生物与盘基网柄菌细胞的结合
DOI：
10.1016/s0021-9258(18)90745-3
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发表时间：
2001
期刊：
影响因子：
--
通讯作者：
E. Kien
中科院分区：
文献类型：
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作者：
Peter;M. V. Haastert;E. Kien
分析了 16 种 cAMP 衍生物与 ~ic ~y os t ~ ~ u ~ d~sco~deum 细胞表面的结合。如果 cAMP 衍生物不再能够在 N'H2 或 03' 处形成氢键，则结合亲和力会大大降低（超过 14.5 kJ/mol）。碱基部分极性的降低与 cAMP 受体结合亲和力的增加密切相关（r = 0.98，p < 0.1%）。基于这些结果，我们提出 cAMP 通过 N6Hz 和 03' 处的氢键与受体结合，并且腺嘌呤部分结合在受体的疏水裂口中。尚未观察到受体和 cAMP 磷酸盐部分之间的立体特异性相互作用。第一个 D. 细胞衍生物（20 pl，终浓度在 IO-' 和 M 之间）。十秒后，通过添加 100 μl 3.5% 高氯酸 (v/v) 裂解细胞。用 50 μl KHCOB（20℃下 50% 饱和）中和裂解物，并在 8000 X g 下离心 2 分钟。通过放射免疫学方法测量 100 μl 上清液中的 cGMP 含量。在对照实验中研究了 cAMP 衍生物与 cGMP 抗体的相互作用，其中在添加 CAMP 衍生物之前裂解细胞。
The binding of 16 derivatives of cAMP to the surface of ~ i c ~ y o s t ~ ~ u ~ d~sco~deum cells was analyzed. The binding affinity is strongly reduced (more than 14.5 kJ/mol) if a cAMP derivative is no longer able to form a hydrogen bond at N‘H2, or at 03’. Decreasing polarity of the base moiety is closely correlated to increasing binding affinity to the cAMP receptor (r = 0.98, p < 0.1%). Based on these results we propose that cAMP is bound to the receptor via hydrogen bonds at N6Hz and 03‘, and that the adenine moiety is bound in a hydrophobic cleft of the receptor. A stereospecific interac- tion between the receptor and the phosphate moiety of cAMP has not been observed. The first D. cells to derivatives (20 pl, final concentrations between IO-’ and M). Ten s later, cells were lysed by the addition of 100 pl of 3.5% perchloric acid (v/v). Lysates were neutralized with 50 pl of KHCOB (50% saturated at 20 “C) and centrifuged at 8000 X g for 2 min. The cGMP content in 100 pl of the supernatant was measured radioimmunologically. The interaction of the cAMP derivatives with the cGMP antibody was investigated in a control experiment in which cells were lysed before the addition of the CAMP derivatives.