Isambert M F, Henry J P
Biochimie. 1981 Mar;63(3):211-9. doi: 10.1016/s0300-9084(81)80194-0.
The ATP-dependent catecholamine uptake system of chromaffin granule membrane has been solubilized and reconstituted in phospholipidic vesicles. The activity of the vesicles implies that both the ATP-dependent H+-translocase and the noradrenaline carrier have been successfully reconstituted. The membrane was solubilized by sodium cholate in presence of asolectin and the asolectin to cholate ratio appeared to be critical. Omission of asolectin resulted in reconstitution of vesicles with an active H+-pump and an inactive transport system. The detergent was removed from the solubilized membranes by filtration on Sephadex G-50 and it has been verified that the residual detergent of the reconstituted preparation was below the concentration inhibitory to the ghost H+-pump. The pH-dependence, Km for ATP and Km for noradrenaline of the reconstituted vesicles were similar to those of the ghosts, but their specific activity and reserpine-resistance were somehow variable. Vesicle activity was limited by transporter reconstitution, thus suggesting that reconstitution of the complete system might be used as an assay for the transporter. The noradrenaline carrier is not physically linked to dopamine beta-hydroxylase and bears no wheat germ agglutinin binding sites.
嗜铬粒细胞膜上依赖ATP的儿茶酚胺摄取系统已被溶解并在磷脂囊泡中重构。囊泡的活性表明依赖ATP的H⁺转运酶和去甲肾上腺素载体均已成功重构。在大豆卵磷脂存在的情况下，用胆酸钠溶解膜，大豆卵磷脂与胆酸钠的比例似乎至关重要。省略大豆卵磷脂会导致重构出具有活性H⁺泵但转运系统无活性的囊泡。通过在葡聚糖G - 50上过滤从溶解的膜中去除去污剂，并且已证实重构制剂中残留的去污剂浓度低于对血影H⁺泵有抑制作用的浓度。重构囊泡的pH依赖性、对ATP的Km值和对去甲肾上腺素的Km值与血影相似，但其比活性和利血平抗性在某种程度上有所变化。囊泡活性受转运体重构的限制，因此表明完整系统的重构可作为转运体的一种检测方法。去甲肾上腺素载体与多巴胺β - 羟化酶没有物理连接，也没有麦胚凝集素结合位点。
