Van Haastert P J, De Wit R J
J Biol Chem. 1984 Nov 10;259(21):13321-8.
The binding of [3H]cAMP to Dictyostelium discoideum cells was analyzed on a seconds time scale under both equilibrium and nonequilibrium conditions. The binding of [3H]cAMP increases rapidly to a maximum obtained at about 6 s, which is followed by a decrease to an equilibrium value reached at about 45 s. This decrease of [3H]cAMP binding is not the result of ligand degradation or isotope dilution by cAMP secretion but is due to a transition of high-affinity binding to low-affinity binding. Analysis of the dissociation rate of [3H]cAMP from the binding sites indicates that these high- and low-affinity binding sites are both fast dissociating with a half-life of about 1 s. In addition, these dissociation experiments reveal a third binding type which is slowly dissociating with a half-life of about 15 s. The number and affinity of these slowly dissociating sites does not change during the incubation with [3H]cAMP. The drugs caffeine and chlorpromazine do not change the total number of binding sites, but they change the ratio of the three binding types. In the presence of 10 mM caffeine almost all binding sites are in the low affinity conformation, while in the presence of 0.1 mM chlorpromazine the ratio is shifted to both the high-affinity type and slowly dissociating type. The results indicate that the cAMP-binding activity of D. discoideum cells is heterogeneous. In the absence of cAMP about 4% of the sites are slowly dissociating with Kd = 12.5 nM, about 40% are fast dissociating with high affinity (Kd = 60 nM), and about 60% are fast dissociating with low affinity (Kd = 450 nM). During the binding reaction the number of slowly dissociating sites does not change. The number of high-affinity sites decreases to a minimum of about 10% with a concomitant increase of low-affinity sites to about 90%. This transition of binding types shows first-order kinetics with a half-life of about 9 s. A half-maximal transition is induced by 12.5 nM cAMP.
在平衡和非平衡条件下，以秒为时间尺度分析了[3H]环磷酸腺苷（cAMP）与盘基网柄菌细胞的结合情况。[3H]cAMP的结合迅速增加，在约6秒时达到最大值，随后下降至约45秒时达到的平衡值。[3H]cAMP结合的这种下降不是配体降解或cAMP分泌导致的同位素稀释的结果，而是由于高亲和力结合向低亲和力结合的转变。对[3H]cAMP从结合位点的解离速率分析表明，这些高亲和力和低亲和力结合位点的解离速度都很快，半衰期约为1秒。此外，这些解离实验揭示了第三种结合类型，其解离速度较慢，半衰期约为15秒。在与[3H]cAMP孵育期间，这些缓慢解离位点的数量和亲和力不变。咖啡因和氯丙嗪这两种药物不会改变结合位点的总数，但会改变三种结合类型的比例。在10 mM咖啡因存在的情况下，几乎所有结合位点都处于低亲和力构象，而在0.1 mM氯丙嗪存在的情况下，比例向高亲和力类型和缓慢解离类型转变。结果表明，盘基网柄菌细胞的cAMP结合活性是异质的。在没有cAMP的情况下，约4%的位点缓慢解离，解离常数（Kd）= 12.5 nM，约40%的位点快速解离且具有高亲和力（Kd = 60 nM），约60%的位点快速解离且具有低亲和力（Kd = 450 nM）。在结合反应过程中，缓慢解离位点的数量不变。高亲和力位点的数量减少至约10%的最小值，同时低亲和力位点增加至约90%。这种结合类型的转变呈现一级动力学，半衰期约为9秒。12.5 nM cAMP可诱导半数最大转变。
