Wibo M, Morel N, Godfraind T
Biochim Biophys Acta. 1981 Dec 21;649(3):651-60. doi: 10.1016/0005-2736(81)90170-x.
ATP promotes 45Ca uptake by the microsomal fraction from the longitudinal smooth muscle of guinea-pig ileum and this uptake is stimulated by oxalate. As the microsomal fraction is made up of various subcellular entities, we examined the localization of the Ca2+-transport activity by density gradient centrifugation, taking advantage of the selective effect of digitonin (at low concentration) on the density of plasmalemmal elements. When the 45Ca-uptake activity was measured in the absence of oxalate, its behavior in subfractionation experiments closely paralleled that of the plasmalemmal marker 5'-nucleotidase. In contrast, the additional Ca2+-transport activity elicited by oxalate behaved like NADH-cytochrome c reductase, a putative endoplasmic reticulum marker. The endoplasmic reticulum vesicles constituted only a small part of the membranes in the microsomal fraction, which explains that their Ca2+-storage capacity was not detectable in the absence of Ca2+-trapping agent. Low digitonin concentrations selectively increased the Ca2+ permeability of the plasmalemmal vesicles. The two Ca2+-transport activities were further differentiated by their distinct sensitivity of K+, vanadate and calmodulin. In this respect, the oxalate-insensitive and oxalate-stimulated Ca2+-transport systems resembled, respectively, the sarcolemmal and sarcoplasmic reticulum Ca2+ pumps in cardiac and skeletal muscle, in accordance with the subcellular locations established by density gradient centrifugation.
ATP可促进豚鼠回肠纵行平滑肌微粒体部分摄取45Ca，草酸盐可刺激这种摄取。由于微粒体部分由各种亚细胞实体组成，我们利用洋地黄皂苷（低浓度时）对质膜成分密度的选择性作用，通过密度梯度离心法研究了Ca2+转运活性的定位。在无草酸盐的情况下测定45Ca摄取活性时，其在亚分级实验中的行为与质膜标志物5'-核苷酸酶的行为密切平行。相反，草酸盐引发的额外Ca2+转运活性表现得像NADH-细胞色素c还原酶，一种假定的内质网标志物。内质网小泡仅占微粒体部分膜的一小部分，这就解释了在没有Ca2+捕获剂的情况下无法检测到其Ca2+储存能力。低浓度洋地黄皂苷选择性地增加了质膜小泡的Ca2+通透性。两种Ca2+转运活性因其对K+、钒酸盐和钙调蛋白的不同敏感性而进一步区分。在这方面，草酸盐不敏感和草酸盐刺激的Ca2+转运系统分别类似于心肌和骨骼肌中的肌膜和肌浆网Ca2+泵，这与密度梯度离心法确定的亚细胞定位一致。
