Phosphoinositide binding specificity among phospholipase C isozymes as determined by photo-cross-linking to novel substrate and product analogs.
Phosphoinositide binding specificity among phospholipase C isozymes as determined by photo-cross-linking to novel substrate and product analogs.
复制标题
通过与新底物和产物类似物的光交联测定磷脂酶 C 同工酶之间的磷酸肌醇结合特异性。
DOI：
10.1021/bi9702288
复制
发表时间：
1997
期刊：
影响因子：
--
通讯作者：
Rebecchi,MJ
中科院分区：
文献类型：
--
作者：
Tall,E;Dorman,G;Garcia,P;Runnels,L;Shah,S;Chen,J;Profit,A;Gu,QM;Chaudhary,A;Prestwich,GD;Rebecchi,MJ
我们检测了四种磷脂酶C（PLC）同工酶中高亲和力磷脂酰肌醇4，5-二磷酸[PI（4，5）P2]和PI（3，4，5）P3结合位点的存在（δ1，β1，β2和β3），通过用肌醇磷酸类似物d-Ins（1，4，5）P3，d-Ins（1，3，4，5）P4和InsP 6以及多磷酸肌醇PI（4，5）P2和PI（3，4，5）P3，其含有可光活化的苯甲酰基二氢肉桂酰胺部分。只有PLC-δ 1被特异性标记。超过90%的标签被发现在胰蛋白酶和胰凝乳蛋白酶的片段与抗血清的pleckstrin同源性（PH）结构域反应，而不到5%的回收片段，包括催化核心。在单独的实验中，分离的δ1-PH结构域也被特异性标记。d-Ins（1，4，5）P3与PLC-δ 1的平衡结合表明存在一个单一的高亲和力结合位点，未检测到d-Ins（1，4，5）P3与PLC-β1、-β2和-β 3的结合。产物d-Ins（1，4，5）P3对PLC-δ 1的催化活性有抑制作用，而对PLC-β1、-β2和-β 3的催化活性无抑制作用。这些结果表明，PH结构域是PLC-δ 1的唯一高亲和力PI（4，5）P2结合位点，而PLC-β1、-β2或-β3中不存在类似位点。这些数据与PLC-δ1的PH结构域而不是β同工酶将催化核心导向富含PI（4，5）P2的膜并受到产物抑制的想法一致。
We tested for the presence of high-affinity phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] and PI(3,4,5)P3binding sites in four phospholipase C (PLC) isozymes (δ1, β1, β2, and β3), by probing these proteins with analogs of inositol phosphates, d-Ins(1,4,5)P3,d-Ins(1,3,4,5)P4, and InsP6, and polyphosphoinositides PI(4,5)P2and PI(3,4,5)P3, which contain a photoactivatable benzoyldihydrocinnamide moiety. Only PLC-δ1was specifically radiolabeled. More than 90% of the label was found in tryptic and chymotryptic fragments which reacted with antisera against the pleckstrin homology (PH) domain, whereas less than 5% was recovered in fragments that encompassed the catalytic core. In separate experiments, the isolated δ1-PH domain was also specifically labeled. Equilibrium binding of d-Ins(1,4,5)P3to PLC-δ1indicated the presence of a single, high-affinity binding site; binding of d-Ins(1,4,5)P3to PLC-β1, -β2, or -β3was not detected. The catalytic activity of PLC-δ1was inhibited by the product d-Ins(1,4,5)P3, whereas no inhibition of PLC-β1, -β2, or -β3activity was observed. These results demonstrate that the PH domain is the sole high-affinity PI(4,5)P2binding site of PLC-δ1and that a similar site is not present in PLC-β1, -β2, or -β3. The data are consistent with the idea that the PH domain of PLC-δ1, but not the β isozymes, directs the catalytic core to membranes enriched in PI(4,5)P2and is subject to product inhibition.