Sharma R V, Butters C A, McEldoon J P, Bhalla R C
Cell Calcium. 1987 Feb;8(1):65-77. doi: 10.1016/0143-4160(87)90037-6.
We have characterized ATP-dependent Ca2+ transport into highly purified plasma membrane fraction isolated from guinea pig ileum smooth muscle. The membrane fraction contained inside-out sealed vesicles and was enriched 30-40-fold in 5'-nucleotidase and phosphodiesterase I activity as compared to post nuclear supernatant. Plasma membrane vesicles showed high rate (76 nmol/mg/min) and high capacity for ATP dependent Ca2+ transport which was inhibited by addition of Ca2+ ionophore A23187. The inhibitors of mitochondrial Ca2+ transport, i.e., sodium azide, oligomycin and ruthenium red did not inhibit ATP-dependent Ca2+ uptake into plasma membrane vesicles. The energy dependent Ca2+ uptake into plasma membranes showed very high specificity for ATP as energy source and other nucleotide triphosphates were ineffective in supporting Ca2+ transport. Phosphate was significantly better as Ca2+ trapping anion to potentiate ATP-dependent Ca2+ uptake into plasma membrane fraction as compared to oxalate. Orthovanadate, an inhibitor of cell membrane (Ca2+-Mg2+)-ATPase activity, completely inhibited ATP-dependent Ca2+ transport and the Ki was approximately 0.6 microM. ATP-dependent Ca2+ transport and formation of alkali labile phosphorylated intermediate of (Ca2+-Mg2+)-ATPase increased with increasing concentrations of free Ca2+ in the incubation mixture and the Km value for Ca2+ was approximately 0.6-0.7 microM for both the reactions.
我们已经对从豚鼠回肠平滑肌中分离出的高度纯化的质膜组分进行了ATP依赖性Ca2+转运的特性分析。该膜组分包含内翻式封闭囊泡，与核后上清液相比，其5'-核苷酸酶和磷酸二酯酶I活性富集了30-40倍。质膜囊泡显示出高速率（76 nmol/mg/min）和对ATP依赖性Ca2+转运的高容量，添加Ca2+离子载体A23187可抑制该转运。线粒体Ca2+转运抑制剂，即叠氮化钠、寡霉素和钌红，并不抑制ATP依赖性Ca2+摄入质膜囊泡。质膜中能量依赖性Ca2+摄入对作为能量来源的ATP表现出非常高的特异性，其他核苷三磷酸在支持Ca2+转运方面无效。与草酸盐相比，磷酸盐作为Ca2+捕获阴离子能显著增强ATP依赖性Ca2+摄入质膜组分。正钒酸盐是细胞膜（Ca2+-Mg2+）-ATP酶活性的抑制剂，它完全抑制了ATP依赖性Ca2+转运，其Ki约为0.6 microM。ATP依赖性Ca2+转运以及（Ca2+-Mg2+）-ATP酶的碱不稳定磷酸化中间体的形成随着孵育混合物中游离Ca2+浓度的增加而增加，并且这两个反应的Ca2+ Km值约为0.6-0.7 microM。
