Yegutkin G G
Institute of Radiobiology, Belorussian Academy of Sciences, Minsk, Republic of Belarus.
Membr Cell Biol. 1997;11(4):441-7.
We evaluated the binding of [3H]AMP to rat adipose plasma membranes and studied the substrate specificity of the process. The [3H]AMP binding was investigated by high-speed filtration under conditions of virtually complete inhibition of the 5'-nucleotidase activity by EDTA/Na. The Scatchard plot revealed the existence of a single class of AMP-binding sites on the membrane surface with Kd of 2.14 +/- 0.210 microM and the binding capacity (Bmax) of 26.0 +/- 0.68 pmol per mg protein. Addition of ATP (12.5 microM) or ADP (3.5 microM) to the incubation medium resulted in a two-fold increase of Kd, whereas in the presence of adenosine (400 microM) or its pharmacological antagonist theophylline (200 microM), the number of AMP-binding sites decreased. Therefore, ATP and ADP but not adenosine compete with AMP for the same nucleotide-binding site. It is suggested that the observed [3H]AMP binding may be primarily caused by the interaction of AMP with a specific substrate-binding active centre of the membrane ectoenzyme 5'-nucleotidase.
我们评估了[3H]AMP与大鼠脂肪细胞质膜的结合情况，并研究了该过程的底物特异性。通过在EDTA/Na几乎完全抑制5'-核苷酸酶活性的条件下进行高速过滤来研究[3H]AMP的结合。Scatchard图显示膜表面存在一类单一的AMP结合位点，其解离常数（Kd）为2.14±0.210微摩尔，结合容量（Bmax）为每毫克蛋白质26.0±0.68皮摩尔。向孵育培养基中添加ATP（12.5微摩尔）或ADP（3.5微摩尔）会导致Kd增加两倍，而在存在腺苷（400微摩尔）或其药理学拮抗剂茶碱（200微摩尔）的情况下，AMP结合位点的数量会减少。因此，ATP和ADP而非腺苷与AMP竞争相同的核苷酸结合位点。有人提出，观察到的[3H]AMP结合可能主要是由于AMP与膜外切酶5'-核苷酸酶的特定底物结合活性中心相互作用所致。
