Comparative localization of inositol 1,4,5-trisphosphate and ryanodine receptors in intestinal smooth muscle: an analytical subfractionation study.
Comparative localization of inositol 1,4,5-trisphosphate and ryanodine receptors in intestinal smooth muscle: an analytical subfractionation study.
复制标题
肠平滑肌中肌醇 1,4,5-三磷酸和兰尼定受体的比较定位：一项分析亚分级研究。
作者：
M. Wibo;T. Godfraind
[3H] in (1,4,5)P3-和[3H]ryanodine结合位点在豚鼠肠平滑肌（纵层）的膜组分中被表征，并通过分析细胞分离技术研究了它们的亚细胞定位。在低离心场下收集的片段（N和M部分）主要含有低亲和力[3H]Ins(1,4,5) p3结合位点（KD为80 nM），而微粒体(P)部分只含有高亲和力结合位点（KD为5 nM）。[3H]Ins(1,4,5)P3可沉淀的高亲和力结合位点数量是[3H]ryanodine的9-10倍。这两种高亲和力结合位点均在微粒体中纯化，经等密度梯度离心后，其亚微粒体分布模式与内质网（ER）成分相似，表明Ins(1,4,5)P3和ryanodine受体主要定位于内质网，可能与粗糙内质网和光滑内质网有关。然而，Ins(1,4,5)P3与ryanodine受体的化学量比在高密度rna丰富亚组中明显高于低密度rna贫乏亚组，这表明Ins(1,4,5)P3受体在一定程度上集中在内质网的核糖体包被部分。肠平滑肌中ryanodine与Ins(1,4,5)P3受体的总化学量比较低（1:9-10），可能至少部分解释了在皂素渗透平滑肌细胞中存在缺乏ryanodine敏感的Ca2+通道，但具有Ins(1,4,5)P3敏感通道的Ca（2+）存储室的存在[Iino， Kobayashi和Endo (1988) Biochem]。Biophys。参考文献。152,417-422]。
[3H]Ins(1,4,5)P3- and [3H]ryanodine-binding sites were characterized in membrane fractions from guinea-pig intestinal smooth muscle (longitudinal layer) and their subcellular localization was investigated by analytical cell-fractionation techniques. Fractions collected at low centrifugal fields (N and M fractions) contained predominantly low-affinity [3H]Ins(1,4,5)P3-binding sites (KD 80 nM), whereas microsomal (P) fractions contained only high-affinity binding sites (KD 5 nM). Total sedimentable high-affinity binding sites of [3H]Ins(1,4,5)P3 were 9-10-fold more numerous than those of [3H]ryanodine. Both high-affinity binding sites were purified in microsomal fractions, and their sub-microsomal distribution patterns after isopycnic density-gradient centrifugation were similar to those of presumed endoplasmic reticulum (ER) constituents, indicating that Ins(1,4,5)P3 and ryanodine receptors were localized primarily in ER and probably associated with rough as well as smooth ER. However, the stoichiometric ratio of Ins(1,4,5)P3 to ryanodine receptors was distinctly higher in high-density RNA-rich subfractions than in low-density RNA-poor subfractions, suggesting that Ins(1,4,5)P3 receptors were somewhat concentrated in the ribosome-coated portions of ER. The low overall stoichiometric ratio of ryanodine to Ins(1,4,5)P3 receptors in intestinal smooth muscle (1:9-10) might explain, at least partly, the existence of a Ca(2+)-storage compartment devoid of ryanodine-sensitive Ca2+ channels, but equipped with Ins(1,4,5)P3-sensitive channels, in saponin-permeabilized smooth-muscle cells [Iino, Kobayashi and Endo (1988) Biochem. Biophys. Res. Commun. 152, 417-422].