van Corven E J, van Os C H, Mircheff A K
Biochim Biophys Acta. 1986 Oct 9;861(2):267-76. doi: 10.1016/0005-2736(86)90429-3.
Subcellular fractionation studies were performed to delineate plasma membrane and intracellular membrane populations which might be involved in intracellular Ca2+-homeostasis of rat small intestinal epithelial cells. After a low-speed supernatant fraction had been suspended in 5% sorbitol and subjected to equilibrium centrifugation in a zonal rotor, the Golgi and endoplasmic reticulum markers, galactosyltransferase and NADPH-cytochrome -c reductase, were concentrated in a density region designated Window II. The basal-lateral membrane marker (Na+-K+)-ATPase was concentrated in a higher-density region designated Window III. ATP-dependent Ca2+ transport was equally distributed between the two windows. Several membrane populations could be resolved from each window with good recovery of Ca2+-transport activity by a second density gradient centrifugation step. Second density gradient fractions were subjected to counter-current partitioning in an aqueous polymer two-phase system. Basal-lateral membranes, characterized by an 11-fold enrichment of (Na+-K+)-ATPase, contained ATP-dependent Ca2+-transport activity with Vmax = 3.7 nmol/mg per min and Km = 0.5 microM. A major Golgi-derived population exhibited Ca2+-transport activity with Vmax and Km values similar to those of the basal-lateral membranes. One membrane population, presumed to have been derived from the endoplasmic reticulum, contained Ca2+-transport activity with Vmax = 4 nmol/mg per min and Km = 0.5 microM. In addition to demonstrating that ATP-dependent Ca2+-transport activity has a complex distribution within enterocytes, this study raises the possibility that the basolateral plasma membranes might account for a relatively minor portion of the cell's Ca2+-pumping ability.
进行亚细胞分级分离研究，以描绘可能参与大鼠小肠上皮细胞细胞内钙稳态的质膜和细胞内膜群体。将低速上清液部分悬浮在5%山梨醇中，并在区带转子中进行平衡离心后，高尔基体和内质网标志物半乳糖基转移酶和NADPH - 细胞色素c还原酶集中在指定为窗口II的密度区域。基底外侧膜标志物（Na⁺ - K⁺）-ATP酶集中在密度更高的指定为窗口III的区域。ATP依赖的钙转运在两个窗口之间平均分布。通过第二次密度梯度离心步骤，可以从每个窗口中分离出几个膜群体，钙转运活性有良好的回收率。将第二次密度梯度分离物在水性聚合物两相系统中进行逆流分配。以（Na⁺ - K⁺）-ATP酶富集11倍为特征的基底外侧膜含有ATP依赖的钙转运活性，Vmax = 3.7 nmol/mg每分钟，Km = 0.5 microM。一个主要来源于高尔基体的群体表现出与基底外侧膜相似的Vmax和Km值的钙转运活性。一个推测来源于内质网的膜群体含有钙转运活性，Vmax = 4 nmol/mg每分钟，Km = 0.5 microM。除了证明ATP依赖的钙转运活性在肠细胞内有复杂的分布外，这项研究还提出了基底外侧质膜可能仅占细胞钙泵能力相对较小部分的可能性。
