Enyedi P, Szabadkai G, Krause K H, Lew D P, Spät A
Department of Physiology, Semmelweis University of Medicine, Budapest, Hungary.
Cell Calcium. 1993 Jun;14(6):485-92. doi: 10.1016/0143-4160(93)90007-s.
Rat liver was homogenized and subjected to differential centrifugation. When the low speed nuclear pellet was processed on a Percoll gradient, plasma membrane markers and Ins(1,4,5)P3 binding activity purified together. The high speed (microsomal) fraction was subfractionated by sucrose density gradient centrifugation, resulting in 10-fold enrichment of [32P]-Ins(1,4,5)P3 binding. In the sucrose density gradient fractions there was an inverse relationship between the enrichment of plasma membrane markers and Ins(1,4,5)P3 binding sites. Endoplasmic reticulum markers showed a moderate enrichment in the fractions displaying high Ins(1,4,5)P3 binding activity. Calcium binding proteins in the homogenate and in the microsomal subfractions were separated by SDS/PAGE. A 60 kD protein, stained metachromatically with Stains-All was identified as calreticulin with immunoblotting. Its enrichment pattern was similar to that of Ins(1,4,5)P3 binding sites, indicating the co-existence of these two elements of Ca(2+)-metabolism in the same intracellular compartment in the liver.
将大鼠肝脏匀浆并进行差速离心。当低速核沉淀在Percoll梯度上处理时，质膜标志物和肌醇-1,4,5-三磷酸（Ins(1,4,5)P3）结合活性一起得到纯化。高速（微粒体）部分通过蔗糖密度梯度离心进行亚分级分离，使[32P]-Ins(1,4,5)P3结合富集了10倍。在蔗糖密度梯度级分中，质膜标志物的富集与Ins(1,4,5)P3结合位点之间存在反比关系。内质网标志物在显示高Ins(1,4,5)P3结合活性的级分中呈现适度富集。通过十二烷基硫酸钠/聚丙烯酰胺凝胶电泳（SDS/PAGE）分离匀浆和微粒体亚分级分中的钙结合蛋白。用“全染剂”进行异染性染色的一种60kD蛋白质通过免疫印迹鉴定为钙网蛋白。其富集模式与Ins(1,4,5)P3结合位点的相似，表明这两种钙代谢元素在肝脏的同一细胞内区室中共存。
