Willcocks A L, Cooke A M, Potter B V, Nahorski S R
Biochem Biophys Res Commun. 1987 Aug 14;146(3):1071-8. doi: 10.1016/0006-291x(87)90756-x.
A very high density of stereospecific binding sites for inositol-(1,4,5)P3 have been identified in rat cerebellar membranes using [3H]inositol-(1,4,5)P3 and a rapid centrifugation step to separate free and bound ligand. Binding was shown to be rapid and reversible and of relatively high affinity (KD 23 nM). Incubations were carried out at 4 degrees and under these conditions HPLC analysis demonstrated that there was no significant metabolism of [3H]-(1,4,5)P3 in the presence or absence of ATP over 15 min. The specificity of the site has been carefully evaluated using both natural and novel synthetic inositol phosphates. The stereospecificity is very marked with the D-, DL- and L-isomers of Ins(1,4,5)P3 showing a 1:4:2000 ratio of affinity for the binding site. D-Ins(2,4,5)P3 was the only other phosphate to show relatively high affinity (KD 1500 nM). HPLC-pure Ins(1,3,4)P3 and Ins(1,3,4,5)P4 were substantially weaker and Ins(1,4)P2, Ins-2-P1, Ins-1-P1, Ins(1,2)-cyclic P1 and inositol were totally inactive at concentrations less than 50 microM. These data are discussed in relation to a putative receptor on the endoplasmic reticulum by which Ins(1,4,5)P3 can initiate the release of bound Ca2+.
利用[3H]肌醇-(1,4,5)P3以及快速离心步骤来分离游离和结合的配体，已在大鼠小脑膜中鉴定出非常高浓度的肌醇-(1,4,5)P3立体特异性结合位点。结合表现为快速且可逆，具有相对较高的亲和力（KD为23 nM）。孵育在4℃下进行，在这些条件下，HPLC分析表明，在有或无ATP的情况下，15分钟内[3H]-(1,4,5)P3均无明显代谢。已使用天然和新型合成肌醇磷酸仔细评估了该位点的特异性。立体特异性非常显著，Ins(1,4,5)P3的D-、DL-和L-异构体对结合位点的亲和力之比为1:4:2000。D-Ins(2,4,5)P3是唯一显示出相对较高亲和力（KD为1500 nM）的其他磷酸盐。HPLC纯的Ins(1,3,4)P3和Ins(1,3,4,5)P4亲和力明显较弱，而Ins(1,4)P2、Ins-2-P1、Ins-1-P1、Ins(1,2)-环P1和肌醇在浓度低于50 microM时完全无活性。结合内质网上一个假定的受体对这些数据进行了讨论，通过该受体Ins(1,4,5)P3可引发结合的Ca2+释放。
