Burgess G M, McKinney J S, Fabiato A, Leslie B A, Putney J W
J Biol Chem. 1983 Dec 25;258(24):15336-45.
The plasma membranes of isolated guinea pig hepatocytes were made permeable with saponin. The cells were then suspended in a medium resembling cytosol in which the level of ATP was kept constant with an ATP-regenerating system. Intracellular ATP-dependent 45Ca and 40Ca sequestration was then followed at various concentrations of Ca2+ in the medium. It was found that ATP-dependent Ca uptake could be divided into two mechanisms: a low affinity high capacity uptake sensitive to 2,4-dinitrophenol (DNP) and oligomycin, thought to be mitochondrial, and a low capacity high affinity uptake, which was insensitive to DNP and oligomycin, thought to be mainly endoplasmic reticulum (ER). The threshold for ATP-dependent Ca uptake by the latter pool was about 20 nM Ca2+. The process had an EC50 value of 0.3 microM (for 45Ca) and a capacity of 2.7 nmol/45Ca/mg of protein. The "ER" mechanism also had a high affinity for ATP (EC50, about 43 microM). There was no significant accumulation of Ca by the postulated mitochondrial pool until the [Ca2+] of the medium was greater than 1 microM. The concentration of Ca2+ in the cytosol of normal unstimulated hepatocytes was estimated from measurements of phosphorylase a activity to be about 0.18 microM. At this [Ca2+], the ER pool of the saponin-treated hepatocytes accumulated Ca but there was no evidence of any Ca uptake into the "mitochondrial" pool. This suggests that most of the exchangeable Ca in a normal cell may be in DNP and oligomycin-insensitive pools (presumably the ER or possibly the plasma membrane) and suggests that these pools are likely to be involved in the increase in cytosolic [Ca2+] which occurs after stimulation by Ca-mobilizing hormones.
用皂角苷使分离出的豚鼠肝细胞的质膜具有通透性。然后将细胞悬浮于类似胞质溶胶的培养基中，通过ATP再生系统使ATP水平保持恒定。接着在培养基中不同浓度的Ca2+条件下，追踪细胞内ATP依赖的45Ca和40Ca螯合情况。结果发现，ATP依赖的钙摄取可分为两种机制：一种是对2,4-二硝基苯酚（DNP）和寡霉素敏感的低亲和力高容量摄取，被认为是线粒体摄取；另一种是对DNP和寡霉素不敏感的低容量高亲和力摄取，被认为主要是内质网（ER）摄取。后一种摄取池依赖ATP摄取钙的阈值约为20 nM Ca2+。该过程的EC50值为0.3 microM（对于45Ca），摄取能力为2.7 nmol/45Ca/mg蛋白质。“ER”机制对ATP也具有高亲和力（EC50约为43 microM）。直到培养基中的[Ca2+]大于1 microM时，推测的线粒体摄取池才会有明显的钙积累。根据磷酸化酶a活性的测量，正常未受刺激的肝细胞胞质溶胶中的Ca2+浓度估计约为0.18 microM。在这个[Ca2+]水平下，经皂角苷处理的肝细胞的ER摄取池会积累钙，但没有证据表明有任何钙摄取进入“线粒体”摄取池。这表明正常细胞中大部分可交换的钙可能存在于对DNP和寡霉素不敏感的摄取池（可能是内质网或可能是质膜）中，并表明这些摄取池可能参与了钙动员激素刺激后胞质溶胶中[Ca2+]的升高。
