Selective sites for polyamine binding to rabbit intestinal brush-border membranes.
Selective sites for polyamine binding to rabbit intestinal brush-border membranes.
复制标题
多胺与兔肠刷状缘膜结合的选择性位点。
DOI：
10.1016/s0006-2952(98)00087-2
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发表时间：
1998
影响因子：
5.8
通讯作者：
Siedel,ER
中科院分区：
文献类型：
--
作者：
Brachet,P;Long,JE;Siedel,ER
肠道多胺转运蛋白尚未确定。我们的目的是表征兔肠刷状缘膜（IBBM）中特定的多胺结合位点，作为鉴定多胺转运蛋白的起始步骤。这是在 4° 和低膜浓度下进行研究的。 [3H]腐胺（PUT）结合的饱和等温线表明具有解离平衡常数Kdof 3.8 μM和位点密度Bmax为58 pmol/mg蛋白质的单一位点群（puT）。 [3H]亚精胺 (SPD) 结合也仅涉及一类位点 (spD)，尽管与 puT 相比，其亲和力较低 (Kd= 106 μM) 且丰度较高 (Bmax= 1240 pmol/mg 蛋白质)。相反，[14C]精胺 (SPM) 结合两类位点（spM1 和 spM2），其亲和力（Kd= 2.5 和 31.4 μM）和丰度（Bmax= 467 和 1617 pmol/mg 蛋白质）不同。 4° 时 SPM 的膜缔合比 SPD 和 PUT 的膜缔合快得多，两者的进展速度相似。与 PUT 和 SPD 解离相反，23° 时的 SPM 解离不遵循一级反应。与[3H]SPD和[14C]SPM不同，特异性结合的[3H]PUT在23°解离，与非放射性多胺的添加无关。甲基乙二醛-双-(脒腙) 是 PUT 结合的极有效抑制剂 (Ki= 3.2 ± 1.5 nM)，但与 PUT 和尸胺 (CAD) 一样，它不会显着改变 [3H]SPD 和 [14C]SPM 结合。肠刷状缘膜可包含至少三个对多胺结合具有特异性并表现出不同配体选择性的位点。位点 pUT 可能与已经描述的用于肠道摄取 PUT 的运输系统有关。
The intestinal polyamine transporters have not yet been identified. Our aim was to characterize specific polyamine binding sites in rabbit intestinal brush-border membranes (IBBM) as a starting step for identification of polyamine transporters. This was investigated at 4° and at low membrane concentration. Saturation isotherms for [3H]putrescine (PUT) binding indicated a single population of sites (puT) with a dissociation equilibrium constant Kdof 3.8 μM and a density of sites Bmaxof 58 pmol/mg of protein. [3H]spermidine (SPD) binding also involved only one class of sites (spD), albeit with a lower affinity (Kd= 106 μM) and higher abundance (Bmax= 1240 pmol/mg of protein) than puT. On the contrary, [14C]spermine (SPM) bound two classes of sites (spM1and spM2) differing in their affinity (Kd= 2.5 and 31.4 μM) and abundance (Bmax= 467 and 1617 pmol/mg of protein, respectively). Membrane association of SPM at 4° was much faster than that of SPD and PUT, both of which proceeded at a similar rate. In contrast to PUT and SPD dissociation, SPM dissociation at 23° did not follow a first-order reaction. Specifically bound [3H]PUT, unlike [3H]SPD and [14C]SPM, dissociated at 23° independently of the addition of nonradioactive polyamine. Methylglyoxal-bis-(guanylhydrazone) was an extremely potent inhibitor of PUT binding (Ki= 3.2 ± 1.5 nM), but as with PUT and cadaverine (CAD), it did not alter [3H]SPD and [14C]SPM binding substantially. The intestinal brush-border membrane may contain at least three sites specific for polyamine binding and exhibiting different ligand selectivity. Site puT might be associated with the transport system already described for intestinal uptake of PUT.